A circular engineered sortase for interrogating h3 histone in chromatin

Abstract

Discussed herein are novel engineered polypeptides which are effective at cutting and tagging H3 histone tails from endogenous histones, facilitating multiplex "cut-and-paste" middle down proteomics with tandem mass tags. This cut-and-paste proteomics approach permits the quantitative analysis of H3 histone modification crosstalk after treatment with different histone deacetylase inhibitors.

Description

Atty. Dkt. No. 043214-000103WO PTA CIRCULAR ENGINEERED SORTASE FOR INTERROGATING H3 HISTONE IN CHROMATINCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit under 35 U.S.C. § 119 of U.S. Provisional Application 63 / 687,866 filed on August 28, 2024, the content of which is incorporated herein by reference in its entirety.SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on July 30, 2025, is named “043214-000103 WOPT SL.xml” and is 81,920 bytes in size.TECHNICAL FIELD

[0003] The technology described herein includes compositions and methods relating to modification of nucleosomes, specifically, the ligation, modification, and cleavage and of H3 histone tails through the employment of an engineered sortase polypeptide.GOVERNMENT SUPPORT

[0004] This invention was made with government support under Grant. No. 5R35GM149229- 02 awarded by the National Institute of General Medical Sciences, Grant. No. 2127882 awarded by the National Science Foundation, Grant. No. 5P01CA196539-10 awarded by the National Cancer Institute, and Grant. No. 5R01 HD 106051-03 awarded by the Eunice Kennedy Shriver National Institute of Child Health and Human Development. The government has certain rights in the invention.BACKGROUND

[0005] DNA in cells is organized and condensed with the help of histone proteins. These histone proteins consist of a globular domain and flexible tail. Two sets of the four core histone proteins - H2A, H2B, H3 and H4 - assemble to form an octamer, around which the DNA is wrapped. This structure is called a nucleosome.

[0006] Histones also play a critical role in epigenetic regulation. Epigenetics refers to changes in gene expression that are not the result of alterations to the DNA sequence itself, but, e.g., instead modifications of the molecules which join with the DNA to form chromosomes. Post-translational modifications on histones are one of the key regulators of cellular epigenetics. The flexible histone tails are particularly susceptible to modification. Different combinations of histone modifications determine whether genes are activated / expressed or repressed / silenced. Enzymes that add or remove these4934-8116-8174.8Atty. Dkt. No. 043214-000103WO PT modifications, as well as proteins that recognize and interpret these modifications, collectively contribute to DNA accessibility and by extension, gene expression.

[0007] Understanding the causes of and cross-talk between histone modifications remains a central challenge in epigenetics. Disclosed herein are compositions and methods relating to the controlled modification of histones, in particular, H3 histone tails.SUMMARY

[0008] The inventors have developed engineered polypeptides that permit H3 histone tails to be added or removed from H3 histones. This permits detailed studies of the effects of different histone tail structures (e.g., with or without certain modifications) and can permit and improve therapeutic development.

[0009] In one aspect of any of the embodiments, described herein is an engineered polypeptide comprising a sortase domain comprising the sequence MQAKPQIPKDKSKVAGYIEIPDADIKEPVYPGPATX1EQLNRGVSFAX2X3NX4SLDX5QNIX6IAG HTFIDRPNYQFTNLKAAKX7GSMVYFKVGNETRKYKMTSIRX8VKPX9XioXiii2Xi3X14Xi5X16X17KGKDKQLTLITCDDYNEX18TGVWEX19RKIFVATEVK (SEQ ID NO: 14), wherein X is any amino acids and X1112 is one or two amino acids.

[0010] In some embodiments of any of the aspects, the engineered polypeptide further comprises SEQ ID NO: 36. In some embodiments of any of the aspects, the engineered polypeptide further comprises at least one split intein sequence. In some embodiments of any of the aspects, the engineered polypeptide further comprises a pair of split intein sequences.

[0011] In some embodiments of any of the aspects, the split intein sequences flank the sortase domain. In some embodiments of any of the aspects, the engineered polypeptide further comprises linker domains between each of the pair of split intein sequences and the sortase domain. In some embodiments of any of the aspects, the engineered polypeptide further comprises, from N-terminus to C-terminus, an N-terminus split intein comprising the sequence of SEQ ID NO: 15, an N-terminus linker comprising the sequence of SEQ ID NO: 16, the sortase domain, a C-terminus linker comprising the sequence of SEQ ID NO: 17, and a C-terminus split intein comprising the sequence of SEQ ID NO: 18.

[0012] In some embodiments of any of the aspects, the engineered polypeptide is circularized. In some embodiments of any of the aspects, a linker can be located C-terminal or N-terminal of the of the sortase domain, or the linker can be internal in the polypeptide. In some embodiments of any of the aspects, e.g., for a polypeptide that will be circularized, a linker can be located at the C-terminal or N- terminal of the engineered polypeptide before circularization.

[0013] One aspect of any of the embodiments is a method of producing a circularized engineered polypeptide comprising a sortase domain, the method comprising: expressing an engineered4934-8116-8174.8Atty. Dkt. No. 043214-000103WO PT polypeptide described herein, thereby excising the intein sequences and producing the circularized engineered polypeptide comprising the sortase domain.

[0014] One aspect of any of the embodiments is a method of producing a tailless H3 histone, the method comprising contacting an engineered polypeptide described herein with a composition comprising at least one tailed H3 histone; and separating at least one cleaved histone tail peptide from at least one tailless H3 histone.

[0015] One aspect of any of the embodiments is a method of producing an intact histone octamer, the method comprising producing core histones comprising tailless H3 histones using a method described herein and assembling the intact histone octamer via octamer assembly.

[0016] One aspect of any of the embodiments is a method of producing a modified nucleosome, the method comprising producing an intact histone octamer using a method described herein, and wrapping the intact histone octamer with DNA.

[0017] One aspect of any of the embodiments is a method for producing a tailless H3 nucleosome, the method comprising producing an intact histone octamer comprising tailless H3 histones using a method described herein, and wrapping the intact histone octamer with DNA to form a tailless H3 nucleosome.

[0018] One aspect of any of the embodiments is a method for histone tail isolation, the method comprising contacting at least one isolated protein with an engineered polypeptide described herein and an oligoglycine tandem mass tag (TMT) peptide, under conditions comprising at least one of: a temperature of between 37 and 42 °C; a pH of between 7.0 and 7.5; an ionic strength of between 30 and 50 mM; a histone protein concentration of between 10 and 20 pM; an engineered polypeptide concentration of between 100 and 400 pM; an oligoglycine TMT peptide concentration of between 0.25 and 1 mM; a phenylmethylsulfonyl fluoride (PMSF) concentration of between 0 and 2.5 mM; and a dithiothreitol (DTT) concentration of between 1 and 10 mM; thereby cleaving at least one histone tail from the at least one histone protein; and isolating the at least one cleaved histone tail.

[0019] One aspect of any of the embodiments is a method of attaching a tail to a tailless nucleosome, the method comprising producing a tailless H3 nucleosome using a method described herein and contacting the tailless H3 nucleosome with a histone tail and a engineered polypeptide described herein, under conditions comprising at least one of: a temperature of 37 °C; an ionic strength of 185 mM; a tailless nucleosome concentration of 1.65 pM; an engineered polypeptide concentration of 200 pM; and a histone tail peptide concentration of between 16.5 and 99 pM; thereby ligating at least the histone tail to the tailless H3 nucleosome.

[0020] One aspect of any of the embodiments is a method for late-stage tail modification of a nucleosome, or a method of chemo-enzymatic H3 histone tagging, the method comprising producing a modified nucleosome using a method described herein, and modifying at least one residue of at least one histone tail. One aspect of any of the embodiments is a method for late-stage tail modification of 4934-8116-8174.8Atty. Dkt. No. 043214-000103WO PT nucleosomes, or a method of chemo-enzymatic H3 histone tagging, the method comprising, producing a modified nucleosome using a method described herein, and modifying at least one residue of each of a first histone tail and at least one residues of a second histone tail, wherein the modification of at least one residue of the first histone is the same modification as the modification of at least one residue of the second histone tail. One aspect of any of the embodiments is a method for late-stage tail modification of nucleosomes, the method comprising, producing a modified nucleosome using a method described herein, and modifying at least one residue of at least one of a first histone tail or at least one residue of a second histone tail.

[0021] One aspect of any of the embodiments is a method of histone tagging, the method comprising, producing a tailless H3 histone according to a method described herein, and contacting the tailless H3 histone with a histone tail comprising a tag and the engineered polypeptide described herein to provide a tagged H3 histone.

[0022] One aspect of any of the embodiments is a method of chemo-enzymatic tagged mass spectrometry, the method comprising, producing a chemo-enzymatic tagged H3 histone using a method described herein, and performing mass spectrometry on the chemo-enzymatic tagged H3 histone.

[0023] One aspect of any of the embodiments is a method of middle-down proteomics of histone tails, the method comprising, producing at least one isolated cleaved histone tail according to a method described herein, and performing mass spectrometry on the at least one isolated cleaved histone tail.

[0024] In one aspect of any of the embodiments, described herein is a nucleic acid encoding a polypeptide as described herein. In one aspect of any of the embodiments, described herein is a vector comprising nucleic acid encoding a polypeptide as described herein.

[0025] In one aspect of any of the embodiments described herein, there is provided a cell comprising an engineered polypeptide or vector as described herein. In some embodiments of any of the aspects, the cell is a eukaryotic cell, optionally a mammalian cell, optionally a human cell. In one aspect of any of the embodiments, the cell is able to express any of the engineered polypeptides provided herein.

[0026] In another aspect provided herein is a kit. The kit can comprise any of the engineered polypeptides or compositions comprising same provided herein and packaging and materials. Accordingly, in some embodiments the kit comprises an engineered polypeptide as provided herein.BRIEF DESCRIPTION OF THE DRAWINGS

[0027] FIGs. 1A.-1H depict ‘cut-and-paste’ isolation of H3 histone tail peptides with cWll sortase enables quantitative middle-down proteomics. FIG. 1A depicts a workflow for isolating tandem mass tagged H3 histone tails with cWll sortase. Figure discloses “GGGKX” as SEQ ID NO: 58. FIG. IB depicts SDS-PAGE of 14-hour sortase reaction in a nuclear acid extract. FIG. 1C depicts Tris-4934-8116-8174.8Atty. Dkt. No. 043214-000103WO PTTricine PAGE of H3 histone(l-34) synthetic standards (1: 140 ng; 2: 420 ng) and TCA precipitated H3(l-32)-TMT from the acid extract reaction; bands between 25 and 37 kDa are histone Hl. FIG. ID depicts the structure of the TMT-labelled oligoglycine peptide illustrating sortase reactive GGG, charge carrying K and carboxamide, and TMT labelled aminoalanine. Figure discloses SEQ ID NO: 58. FIG. IE depicts unique proteoforms detected in each 6plex sample and shared between the two samples. FIG. IF depicts quantifiable proteoforms detected in each 6plex sample and shared between the two samples. FIG. 1G depicts a volcano plot of significant (p<0.05) log2-fold change and overall abundance of H3 proteoforms in HEK293T cells following treatment with LSD 1 / HDAC1 / CoREST complexspecific inhibitor Corin. FIG. 1H depicts a volcano plot of significant (p<0.05) log2-fold change and overall abundance of H3 proteoforms in HEK293T cells following treatment with pan class one HD AC inhibitor MS275.

[0028] FIG. 2. Designer nucleosome synthesis by sortase ligation. Single, combinatorial and asymmetric modifications prepared by sortase nucleosome ligation (ubiquitin structure PDBID: 1UBQ).63Isolated yields (bold) and yields estimated by area under the curve (parenthetical) are reported as a value (n = 1), range (n = 2) or mean with standard deviation (n > 3).

[0029] FIG. 3. Asymmetric nucleosome synthesis by sortase ligation. (Top) Asymmetric modifications prepared by sortase nucleosome ligation. Isolated yields (bold) and yields estimated by area under the curve (parenthetical) are reported as a value (n = 1), range (n = 2) or mean with standard deviation (n > 3). (Bottom left) Western blot characterization of 147 bp nucleosomes: (1) H3 (aa33- 135) starting material; (2) asymmetric H3K27ac & H3 (aa33-135) intermediate; (3) asymmetric H3K27ac & H3K9ac / K14ac / Kl 8ac / K23ac product. (Bottom right) Deconvoluted mass spectrum of 147 bp asymmetric H3K27ac & H3K9ac / K14ac / K18ac / K23ac nucleosome.

[0030] FIGs. 4A-4C depict trends in histone deacylase activity toward metabolic acylations. FIG. 4A depicts LoglO transformed V / [E] (min1) of Sirtuins 1, 2 and 6 and MiDAC toward 147 bp nucleosomes with one to eight carbon acylations of H3K9. FIG. 4B depicts LoglO transformed V / [E] (min1) of class one and class three HDACs toward 147 bp nucleosomes with four carbon metabolism- linked acylations of H3K9.FIG. 4C depicts LoglO transformed V / [E] (min1) of class one and class three HDACs toward 147 bp nucleosomes with H3K9 succinylation.

[0031] FIGs. 5A-5C depict symmetric and Asymmetric effects of hyperacetylation on deacylase and demethylase activity. Fig. 5A depicts Sirt2 site-specific deacetylation rates with symmetrical mono-acetylated (lac / lac; left, dark grey) and penta-acetylated (5ac / 5ac; right, light grey) 147 bp nucleosomes. FIG. 5B depicts Sirt6 site-specific deacetylation rates with symmetrical monoacetylated (lac / lac; left, dark grey) and penta-acetylated (5ac / 5ac; right, light grey) 147 bp nucleosomes. FIG. 5C depicts MiDAC site-specific deacetylation rates with symmetrical monoacetylated (lac / lac; left, dark grey) and penta-acetylated (5ac / 5ac; right, light grey) 147 bp nucleosomes. FIG. 5D depicts Sirt2 site-specific deacetylation rates with asymmetric 147 bp4934-8116-8174.8Atty. Dkt. No. 043214-000103WO PT nucleosomes isolating the specified acetylation site on a single tail; the second H3 tail was modified with either four acetylations (lac / 4ac; right, dark grey) at the other predominant acetylation sites, or zero acetylations (lac / Oac; left, light grey). FIG. 5E depicts Sirt6 site-specific deacetylation rates with asymmetric 147 bpnucleosomes isolating the specified acetylation site on a single tail; the second H3 tail was modified with either four acetylations (lac / 4ac; right, dark grey) at the other predominant acetylation sites, or zero acetylations (lac / Oac; left, light grey). FIG. 5F depicts MiDAC site-specific deacetylation rates with asymmetric 147 bpnucleosomes isolating the specified acetylation site on a single tail; the second H3 tail was modified with either four acetylations (lac / 4ac; right, dark grey) at the other predominant acetylation sites, or zero acetylations (lac / Oac; left, light grey). FIG. 5G depicts an illustration of in cis and in trans PTM interactions with a regulatory enzyme acting on a nucleosome substrate. FIG. 5H depicts an illustration of an asymmetric nucleosome used to test for an in trans effect on a PTM regulatory enzyme. FIG. 51 depicts LSD 1 -CoREST 1 (LC) demethylation rates with asymmetric nucleosomes containing H3K14ac concurrently on the same H3 histone with H3K4me2 (left, dark grey), H3K4me2 and H3K14ac separately on different H3 histone (middle, light grey), and without H3K14ac (right, white) (* indicates p < 0.05, ** indicates p < 0.01,*** indicates p < 0.001, **** indicates p < 0.0001).

[0032] FIG. 6. Combinatorial effect of H3K9me3 and K18Ub / K23Ub on DNMT1 RFTS domain binding. RFTS-sfGFP EMSA following titration with asymmetric H3K9me3 / K18Ub / K23Ub & unmodified H3 nucleosome (left), asymmetric H3K18Ub / K23Ub & H3K9me3 nucleosome (middle), asymmetric H3K18Ub / K23Ub & unmodified H3 nucleosome (right).

[0033] FIGs. 7A-7C. Cleavage of purified, recombinant H3 histone by sortase mutants. FIG. 7A depicts sites of mutations found in Wil sortase, with origin indicated by shade: F40 (light gray); enhanced sortase (medium gray); FireProt (dark gray). Calcium (dark gray, circle); binding residues are depicted in in the dashed circle, and bound substrate is depicted in the solid line circle. Mutations introduced in pymol (PDBID: 2KID), followed by Rosetta energy minimization. FIG. 7B depicts SDS- PAGE of sortase cleavage reaction with recombinant, unmodified H3 histone: i & ii - sortase mutant standards; iii & iv - H3 tail peptide standards; v - H3 protein standard; 0 - F40 sortase; 6-16 - F40- derived mutants. FIG. 7C depicts specific enhanced sortase mutations introduced to F40 in mutants W6 through W16 and resultant H3 tail cleavage yields.

[0034] FIGs. 8A-8F. Cleavage of semisynthetic, modified H3 histone by W11. Fig. 8A depicts rates of H3 cleavage by W11 sortase in the presence of post-translational modifications near the sortase motif (A29-G33). Rates were determined by densitometry (ImageJ) from SDS-PAGE analysis of starting material, H3(l-135), disappearance and product, H3(33-135), formation over time. Data was fitted to a non-linear one-phase decay using GRAPHPAD PRISM™ 10.2.3. FIG. 8B depicts representative SDS-PAGE gels used for evaluating Wil sortase cleavage rate: unmodified, heterologously expressed H3. FIG. 8C depicts representative SDS-PAGE gels used for evaluating W114934-8116-8174.8Atty. Dkt. No. 043214-000103WO PT sortase cleavage rate: unmodified, semisynthetic H3. FIG. 8D depicts representative SDS-PAGE gels used for evaluating Wil sortase cleavage rate: semisynthetic H3K27me3. FIG. 8E depicts representative SDS-PAGE gels used for evaluating Wil sortase cleavage rate: semisynthetic H3K27ac. FIG. 8F depicts representative SDS-PAGE gels used for evaluating Wil sortase cleavage rate: semisynthetic H3S28phos.

[0035] FIG. 9A. SDS-PAGE analysis of sortase Wil & cWll reactions in nuclear acid extracts. Wil reaction with standards: lane 1 - Wil; lane 2 - H3(l-135); lane 3 - H3(33-135); lane 4 & 5 - H3(l-32) standard; lane 6 - 0 hr acid extract reaction; lane 7 - 16 hr acid extract reaction; lane 8- TCA pelleted protein from 16 hr acid extract reaction; lane 9 - buffer exchanged supernatant from TCA precipitation. FIG. 9B. SDS-PAGE analysis of sortase Wil & cWll reactions in nuclear acid extracts. Representative replicate acid extract reactions with cWll: lane 1 - 0 hr acid extract reaction replicate 1; lane 2 - 16 hr acid extract reaction replicate 1; lane 3 - 0 hr acid extract reaction replicate 2; lane 4 - 16 hr acid extract reaction replicate 2; lane 5 - 0 hr acid extract reaction replicate 3; lane 6- 16 hr acid extract reaction replicate 3; lane 7 - 0 hr acid extract reaction replicate 4; lane 8 - 16 hr acid extract reaction replicate 4; lane 9 -0 hr acid extract reaction replicate 5; lane 10 - 16 hr acid extract reaction replicate 5; lane 11 - 0 hr acid extract reaction replicate 6; lane 12 - 16 hr acid extract reaction replicate 6. Lanes 1 & 2 reprinted from FIGs. 1B-1C.

[0036] FIGs. 10A-10E. Sortase Wil mutant activity in the presence of co-solvent, detergent or chaotrope. FIGs 10A depicts endpoint SDS-PAGE analysis of 25 °C sortase W11 reaction run with variant buffer conditions including increased salt, added detergent, organic co-solvent, or chaotrope. In all cases cleavage of H3 was evaluated by densitometry (Image!), and percent conversion was calculated as the sum of H3(33-135) and H3(l-32) over the cumulative intensity of all H3-derived bands. FIG. 10B depicts Endpoint SDS-PAGE analysis of duplicate 25 °C sortase reaction with Wil. In all cases cleavage of H3 was evaluated by densitometry (ImageJ), and percent conversion was calculated as the sum of H3(33-135) and H3(l-32) over the cumulative intensity of all H3-derived bands. FIG. 10C depicts endpoint SDS-PAGE analysis of duplicate 25 °C sortase reaction with FireProt mutant W1 l(E108Q, SI 16V). In all cases cleavage of H3 was evaluated by densitometry (ImageJ), and percent conversion was calculated as the sum of H3(33-135) and H3(l-32) over the cumulative intensity of all H3-derived bands. FIG. 10D depicts endpoint SDS-PAGE analysis of duplicate 37 °C sortase reaction with Wil. In all cases cleavage of H3 was evaluated by densitometry (ImageJ), and percent conversion was calculated as the sum of H3(33-135) and H3(l-32) over the cumulative intensity of all H3-derived bands. FIG. 10E depicts endpoint SDS-PAGE analysis of duplicate 37 °C sortase reaction with FireProt mutant W1 l(E108Q, SI 16V). In all cases cleavage of H3 was evaluated by densitometry (ImageJ), and percent conversion was calculated as the sum of H3(33-135) and H3(l-32) over the cumulative intensity of all H3-derived bands.4934-8116-8174.8Atty. Dkt. No. 043214-000103WO PT

[0037] FIG. 11. Histone tails isolated by trichloroacetic acid precipitation of reaction protein components. Representative histone tails isolated by TCA precipitation from the sortase reaction conducted in nuclear acid extracts. Samples are derived from reactions shown in FIG. 9B, and a portion (lanes 1-3) of this gel is re-printed in FIG. 1. Representative TCA products from replicate acid extract reactions with cWl 1: lane 1 & 2 - H3(l-32) peptide standards for quantification; lane 3 & 4 - replicateI product; lane 5 & 6 - replicate 2 product; lane 7 & 8 - replicate 3 product; lane 9 & 10 - replicate 4 product; lane 11 & 12 - replicate 5 product; lane 13 & 14 - replicate 6 product.

[0038] FIGs. 12A-12C. Characterization of H3(33-135) octamer overexpression and purification. FIG. 12A depicts SDS-PAGE evaluation of fractions from IMAC purification of cooverexpressed His-TEV-H2A, H2B, H3(33-135) andH4. Lanes: 1 - lysate pellet; 2 -lysate supernatant;3 - IMAC column flow through; 4 - lysis buffer wash; 5 - 20 mM imidazole wash 1 ; 6 - 20 mM imidazole wash 2; 7 - 200 mM imidazole elution; 8 - ladder; 9 - lysate pellet; 10 - lysate supernatant;II - IMAC column flow through; 12 - lysis buffer wash; 13 - 20 mM imidazole wash 1; 14 - 20 mM imidazole wash 2; 15 - 200 mM imidazole elution. CAT - chloramphenicol acetyltransferase. FIG. 12B depicts SDS-PAGE evaluation of TEV cleavage of His-TEV-H2A octamer, and superdex200 fractions from post-TEV purification. Lanes: 1-8 - sequential superdex200 fractions (see FIG. 12C); 9 - TEV cleavage endpoint; 10 - TEV cleavage starting point; 11 - IMAC 20 mM imidazole wash 1 ; 12 - IMAC 20 mM imidazole wash 2. FIG. 12C depicts Superdex200 chromatogram from purification following TEV cleavage of His-TEV-H2A octamer. Numbering (top) reflects corresponds to gel lanes in FIG.12B.

[0039] FIG. 13 A. Western blot analysis of cWll nucleosome ligation. Anti-H3 western blot (1 minute exposure) of cWll sortase ligation time-course replicates and DEAE fractions from an unoptimized gradient. Lanes: 1 - H3(33-135) standard; 2 - 0.5 hours; 3 - 1 hour; 4 - 4 hours; 5 - DEAE first peak; 6 - DEAE second peak; 7 - H3(l-135) standard; 8 - ladder; 9- H3(33-135) standard; 10 - 0.5 hours; 11 - 1 hour; 12 - 4 hours; 13 - DEAE first peak; 14 - DEAE second peak.

[0040] FIG. 13B. Western blot analysis of cWll nucleosome ligation. Long exposure (10 minutes) of western blot from FIG. 13A. Percent conversion was assessed by densitometry using ImageJ.

[0041] FIG. 14. cWll nucleosome ligation time course chromatograms. Change in product distribution over the course of the cWll sortase ligation using either 5 equivalents of H3 tail peptide (left) or 15 equivalents (right). Peaks correspond to nucleosome with two copies of full length H3 (left), one copy of full length H3 and one copy of N-terminally truncated H3 (aa33-135) (middle) and starting material nucleosome with two copies of truncated H3 (aa33-135) (right).

[0042] FIGs. 15A-15M. Mass spectrometric characterization of cWll nucleosome ligation products. Raw spectra deconvoluted with UniDec.5FIG. 15A. 147 bp unmodified nucleosome. (Left) Deconvoluted mass spectrum: H4 (circle) calculated mass 11236.15 Da, found: 11235.6 Da; H2B 4934-8116-8174.8Atty. Dkt. No. 043214-000103WO PT(downward pointed triangle) calculated mass 13493.68 Da, found: 13493.2 Da; H2A (upward pointed triangle) calculated mass 13950.2 Da, found: 13949.6 Da; unmodified H3 (rightward pointed triangle) calculated mass 15238.61 Da, found: 15238.2 Da. (Right) Raw mass spectrum: H4 (circle); H2B (downward pointed triangle); H2A (upward pointed triangle; H3 (rightward pointed triangle). FIG. 15B. Raw spectra deconvoluted with UniDec.5147 bp H3K4-monomethyl nucleosome. (Left) Deconvoluted mass spectrum: H4 (circle) calculated mass 11236.15 Da, found: 11235.6 Da; H2B (downward pointed triangle) calculated mass 13493.68 Da, found: 13493.2 Da; H2A (upward pointed triangle) calculated mass 13950.2 Da, found: 13949.6 Da; H3K4mel (rightward pointed triangle) calculated mass 15251.63 Da, found: 15252.1 Da. (Right) Raw mass spectrum: H4 (circle); H2B (downward pointed triangle); H2A (upward pointed triangle; H3K4mel (rightward pointed triangle). FIG. 15C. Raw spectra deconvoluted with UniDec. 147 bp H3K4-dimethyl 1 nucleosome. (Left) Deconvoluted mass spectrum: H4 (circle) calculated mass 11236.15 Da, found: 11235.4 Da; H2B (downward pointed triangle) calculated mass 13493.68 Da, found: 13493.0 Da; H2A (upward pointed triangle) calculated mass 13950.2 Da, found: 13949.5 Da; H3K4me2 (rightward pointed triangle) calculated mass 15266.66 Da, found: 15266.4 Da. (Right) Raw mass spectrum: H4 (circle); H2B (downward pointed triangle); H2A (upward pointed triangle; H3K4me2 (rightward pointed triangle). FIG. 15D. Raw spectra deconvoluted with UniDec. 47 bp H3K4-trimethyl nucleosome. (Left) Deconvoluted mass spectrum: H4 (circle) calculated mass 11236.15 Da, found: 11235.4 Da; H2B (downward pointed triangle) calculated mass 13493.68 Da, found: 13493.0 Da; H2A (upward pointed triangle) calculated mass 13950.2 Da, found: 13949.5 Da; H3K4me3 (rightward pointed triangle) calculated mass 15308.70 Da, found: 15308.2 Da. (Right) Raw mass spectrum: H4 (circle); H2B (downward pointed triangle); H2A (upward pointed triangle; H3K4me3 (rightward pointed triangle). FIG. 15E. Raw spectra deconvoluted with UniDec. 147 bp H3K9-propionyl nucleosome. (Left) Deconvoluted mass spectrum: H4 (circle) calculated mass 11236.15 Da, found: 11235.5 Da; H2B (downward pointed triangle) calculated mass 13493.68 Da, found: 13493.1 Da; H2A (upward pointed triangle) calculated mass 13950.2 Da, found: 13949.6 Da; H3K9pr (rightward pointed triangle) calculated mass 15294.67 Da, found: 15294.2 Da. (Right) Raw mass spectrum: H4 (circle); H2B (downward pointed triangle); H2A (upward pointed triangle; H3K9pr (rightward pointed triangle). FIG. 15F. Raw spectra deconvoluted with UniDec. 147 bp H3K9-butyryl nucleosome. (Left) Deconvoluted mass spectrum: H4 (circle) calculated mass 11236.15 Da, found: 11235.4 Da; H2B (downward pointed triangle) calculated mass 13493.68 Da, found: 13493.0 Da; H2A (upward pointed triangle) calculated mass 13950.2 Da, found: 13949.5 Da; H3K9bu (red rightward pointed triangle) calculated mass 15308.70 Da, found: 15308.2 Da. (Right) Raw mass spectrum: H4 (circle); H2B (downward pointed triangle); H2A (upward pointed triangle; H3K9bu (rightward pointed triangle). FIG. 15G. Raw spectra deconvoluted with UniDec. 147 bp H3K9-crotonyl nucleosome. (Left) Deconvoluted mass spectrum: H4 (circle) calculated mass 11236.15 Da, found: 11235.4 Da; H2B (downward pointed triangle) calculated mass 13493.68 Da, found: 13493.0 Da; H2A (upward pointed triangle) calculated mass4934-8116-8174.8Atty. Dkt. No. 043214-000103WO PT13950.2 Da, found: 13949.5 Da; H3K9cro (rightward pointed triangle) calculated mass 15306.68, found: 15306.4. (Right) Raw mass spectrum: H4 (circle); H2B (downward pointed triangle); H2A (upward pointed triangle); H3K9cro (rightward pointed triangle). FIG. 15H. Raw spectra deconvoluted with UniDec. 147 bp H3K9-octanoyl nucleosome. (Left) Deconvoluted mass spectrum: H4 (circle) calculated mass 11236.15 Da, found: 11235.2 Da; H2B (downward pointed triangle) calculated mass 13493.68 Da, found: 13493.3 Da; H2A (upward pointed triangle) calculated mass 13950.2 Da, found: 13948.7 Da; H3K9oct (rightward pointed triangle) calculated mass 15363.80, found: 15364.8. (Right) Raw mass spectrum: H4 (circle); H2B (downward pointed triangle); H2A (upward pointed triangle); H3K9oct (rightward pointed triangle). FIG. 151. Raw spectra deconvoluted with UniDec.147 bp H3K9- succinyl nucleosome. (Left) Deconvoluted mass spectrum: H4 (circle) calculated mass 11236.15 Da, found: 11234.9 Da; H2B (downward pointed triangle) calculated mass 13493.68 Da, found: 13942.6 Da; H2A (upward pointed triangle) calculated mass 13950.2 Da, found: 13949.7 Da; H3K9suc (rightward pointed triangle) calculated mass 15337.67, found: 15338.6. (Right) Raw mass spectrum: H4 (circle); H2B (downward pointed triangle); H2A (upward pointed triangle); H3K9suc (rightward pointed triangle). FIG. 15J. Raw spectra deconvoluted with UniDec. 147 bp H3K9-anti- hydroxyisobutyryl nucleosome. (Left) Deconvoluted mass spectrum: H4 (circle) calculated mass 11236.15 Da, found: 11235.5 Da; H2B (downward pointed triangle) calculated mass 13493.68 Da, found: 13493.0 Da; H2A (upward pointed triangle) calculated mass 13950.2 Da, found: 13949.5 Da; H3K9hib (rightward pointed triangle) calculated mass 15324.70, found: 15324.2. (Right) Raw mass spectrum: H4 (circle); H2B (downward pointed triangle); H2A (upward pointed triangle); H3K9hib (rightward pointed triangle). FIG. 15K. Raw spectra deconvoluted with UniDec. 147 bp H3K9-lactyl nucleosome. (Left) Deconvoluted mass spectrum: H4 (circle) calculated mass 11236.15 Da, found: 11235.5 Da; H2B (downward pointed triangle) calculated mass 13493.68 Da, found: 13493.0 Da; H2A (upward pointed triangle) calculated mass 13950.2 Da, found: 13949.5 Da; H3K91ac (rightward pointed triangle) calculated mass 15310.67, found: 15310.2. (Right) Raw mass spectrum: H4 (circle); H2B (downward pointed triangle); H2A (upward pointed triangle); H3K91ac (rightward pointed triangle). FIG. 15L. Raw spectra deconvoluted with UniDec. 147 bp H3K9-P-hydroxybutyryl nucleosome. (Left) Deconvoluted mass spectrum: H4 (circle) calculated mass 11236.15 Da, found: 11235.5 Da; H2B (tdownward pointed triangle) calculated mass 13493.68 Da, found: 13493.0 Da; H2A (upward pointed triangle) calculated mass 13950.2 Da, found: 13949.5 Da; H3K9bhb (rightward pointed triangle) calculated mass 15324.70, found: 15324.6. (Right) Raw mass spectrum: H4 (circle); H2B (downward pointed triangle); H2A (upward pointed triangle); H3K9bhb (rightward pointed triangle). FIG. 15M. Raw spectra deconvoluted with UniDec. 147 bp H3K9-N-methyl thiourea nucleosome. (Left) Deconvoluted mass spectrum: H4 (circle) calculated mass 11236.15 Da, found: 11235.5 Da; H2B (downward pointed triangle) calculated mass 13493.68 Da, found: 13493.1 Da; H2A (upward pointed triangle) calculated mass 13950.2 Da, found: 13949.6 Da; H3K9mtu (rightward pointed triangle) 4934-8116-8174.8Atty. Dkt. No. 043214-000103WO PT calculated mass 15311.72, found: 15311.2 Da. (Right) Raw mass spectrum: H4 (circle); H2B (downward pointed triangle); H2A (upward pointed triangle); H3K9mtu (rightward pointed triangle).

[0043] FIGs. 16A-16D. SDS-PAGE and TBE native gel characterization of cWl 1 nucleosome ligation products. FIG. 16A. Representative SDS-PAGE of 147 bp H3(aa33-135) nucleosome starting material, products of the cWll sortase ligation, and purified H3(aa33-135) octamer prior to TEV cleavage. FIG. 16B. Representative TBE native gel of 147 bp H3(aa33-135) nucleosome starting material, products of the cWll sortase ligation. FIG. 16C. Representative TBE native gel of 147 bp products of the cWll sortase ligation, and an unmodified 147 bp nucleosome prepared by traditional nucleosome reconstitution. FIG. 16D. Representative TBE native gel of 147 bp products of the cWll sortase ligation.

[0044] FIGs. 17A-17E. Characterization of asymmetric H3K4me2 H3K14ac ligation intermediate and final products. FIG. 17A. Anti-H3 (left), anti-H3K4me2 (middle), and anti-H3K14ac (right) western blot visualization of asymmetric K4me2 nucleosome synthesis starting material, intermediates, and final products: H3 (aa33-135) starting material; (2) asymmetric H3K4me2 & H3 (aa33-135) intermediate; (3) asymmetric H3K4me2 & H3 (aa33-135) intermediate; (4) asymmetric H3K4me2 & H3K14ac product; (5) asymmetric H3K4me2 & unmodified H3 product. FIG. 17B. Anti- 113 (left), anti-H3K4me2 (middle), and anti-H3K14ac (right) western blot visualization of asymmetric K4me2 nucleosome synthesis starting material, intermediates, and final products: H3 (aa33-135) starting material; (2) asymmetric H3K4me2 & H3 (aa33-135) intermediate; (3) asymmetric H3K4me2 & unmodified H3 product (4) asymmetric H3K4me2 & H3K14ac product. FIG. 17C. Deconvoluted mass spectrum (top) and raw mass spectrum (bottom) of 185 bp asymmetric H3K4me2 & H3K14ac product: H4 (circle) calculated mass 11236.15 Da, found: 11235.4 Da; H2B (downward pointed triangle) calculated mass 13493.68 Da, found: 13493.1 Da; H2A (upward pointed triangle) calculated mass 13950.2 Da, found: 13949.6 Da; H3K4me2 (rightward pointed triangle) calculated mass 15267.62 Da, found: 15266.1 Da; H3K14ac (square) calculated mass 15281.61 Da, found: 15280.3 Da. Raw spectra deconvoluted with UniDec. FIG. 17D. Deconvoluted mass spectrum (top) and raw mass spectrum (bottom) of 185 bp asymmetric unmodified H3 & H3K4me2 product: H4 (circle) calculated mass 11236.15 Da, found: 11235.5 Da; H2B (downward pointed triangle) calculated mass 13493.68 Da, found: 13493.1 Da; H2A (upward pointed triangle) calculated mass 13950.2 Da, found: 13949.4 Da; H3 (rightward pointed triangle) calculated mass 15238.61 Da, found: 15238.1 Da; H3K4me2(square) calculated mass 15266.66 Da, found: 15266.2 Da. Raw spectra deconvoluted with UniDec. FIG. 17E. Deconvoluted mass spectrum (top) and raw mass spectrum (bottom) of 185 bp asymmetric unmodified H3 & H3K4me2K14ac product: H4 (circle) calculated mass 11236.15 Da, found: 11235.6 Da; H2B (downward pointed triangle) calculated mass 13493.68 Da, found: 13492.9 Da; H2A (upward pointed triangle) calculated mass 13950.2 Da, found: 13949.5 Da; H3 (rightward pointed triangle) calculated mass 15238.61 Da, found: 15238.2 Da; H3K4me2K14ac (square) calculated mass 15309.71 Da, found: 15308.5 Da. Raw spectra deconvoluted with UniDec.4934-8116-8174.8Atty. Dkt. No. 043214-000103WO PT

[0045] FIGs. 18A-18C. Cryo electron microscopy characterization of nucleosomes prepared by cWll nucleosome ligation. FIG. 18A. Raw cryo-EM images of cWll nucleosome. FIG. 18B. Representative 2D Class averages from 14300 particles. FIG. 18C. 4.7-A cryo-EM reconstruction map of cWll nucleosome displayed in two separate views related by 90°. A structural model of canonical X. laevis nucleosome (PDBID: 1KX5) is docked in to the EM density. DNA, H2A, H2B, H3 and H4 labeled in the reconstruction.

[0046] FIG. 19. Western blot characterization of asymmetric mono / tetra-acetylated nucleosomes. Anti-H3 (bottom), anti-H3K9ac (middle) and either anti-H3K18ac (top left), anti- H3K23ac (top center), or anti-H3K27ac (top right). Lanes: 1 -tailless starting material; 2 - intermediate single H3 tail product; 3 - final asymmetric mono-acetylated / tetra-acetylated nucleosomes.

[0047] FIGs. 20A-20F. Mass spectrometric characterization of asymmetric mono / tetra- acetylated nucleosomes and asymmetric mono-acetylated / unmodified nucleosomes. FIG. 20A. Deconvoluted mass spectra of asymmetric H3K18ac & H3K9 / 14 / 23 / 27ac nucleosome. Raw spectra deconvoluted with UniDec. FIG. 20B. Deconvoluted mass spectra of asymmetric H3K23ac & H3K9 / 14 / 18 / 27ac nucleosome. Raw spectra deconvoluted with UniDec. FIG. 20C. Deconvoluted mass spectra of asymmetric H3K27ac & H3K9 / 14 / 18 / 23ac nucleosome: Mono-acetyl H3 calc’d for C672H1133N215O187S2 [M]+: 15280.64 Da, Found: 15280 Da; Tetra-acetyl H3 calc’d for C678H1139N215O190S2 [M]+: 15406.75 Da, Found: 15406 Da. Raw spectra deconvoluted with UniDec. FIG. 20D. Deconvoluted mass spectra of asymmetric H3K18ac & unmodified H3 nucleosome. Raw spectra deconvoluted with UniDec. FIG. 20E. Deconvoluted mass spectra of asymmetric H3K27ac & unmodified H3 nucleosome. Raw spectra deconvoluted with UniDec. FIG. 20F. Deconvoluted mass spectra of asymmetric H3K23ac & unmodified H3 nucleosome: Mono-acetyl H3 calc’d for C672H1133N215O187S2 [M]+: 15280.64 Da, Found: 15280 Da; unmodified H3 Calc’d for C670H1131N215O186S2 [M]+: 15238.61 Da, Found: 15238 Da. Raw spectra deconvoluted with UniDec.

[0048] FIGs. 21A-21F. Mass spectrometric characterization of ubiquitinated H3 peptides and ubiquitinated nucleosome. FIG. 21A. Deconvoluted mass spectra of asymmetric 185 bp H3K9me3 / K18Ub(G76A) / K23Ub(G76A) & unmodified H3 nucleosome: H4 (circle) calculated mass 11236.15 Da, found: 11235.4 Da; H2B (downward pointed triangle) calculated mass 13493.68 Da, found: 13493.1 Da; H2A (upward pointed triangle) calculated mass 13950.2 Da, found: 13949.7 Da; H3 (rightward pointed triangle) calculated mass 15238.61 Da, found: 15239.0 Da; H3K9me3 / K18Ub(G76A) / K23Ub(G76A) (square) calculated mass 32402.18 Da, found: 32402.4 Da. Raw spectra deconvoluted with UniDec. FIG. 21B. Raw mass spectra of asymmetric 185 bp H3K9me3 / K18Ub(G76A) / K23Ub(G76A) & unmodified H3 nucleosome: H4 (circle) calculated mass 11236.15 Da, found: 11235.4 Da; H2B (downward pointed triangle) calculated mass 13493.68 Da, found: 13493.1 Da; H2A (upward pointed triangle) calculated mass 13950.2 Da, found: 13949.7 Da; 4934-8116-8174.8Atty. Dkt. No. 043214-000103WO PTH3 (rightward pointed triangle) calculated mass 15238.61 Da, found: 15239.0 Da; H3K9me3 / K18Ub(G76A) / K23Ub(G76A) (square) calculated mass 32402.18 Da, found: 32402.4 Da. FIG. 21C. Deconvoluted mass spectra of asymmetric 185 bp H3K18Ub(G76A) / K23Ub(G76A) & H3K9me3 nucleosome: H4 (circle) calculated mass 11236.15 Da, found: 11235.4 Da; H2B (downward pointed triangle) calculated mass 13493.68 Da, found: 13493.3 Da; H2A (upward pointed triangle) calculated mass 13950.2 Da, found: 13949.8 Da; H3K9me3(rightward pointed triangle) calculated mass15279.68 Da, found: 15279.8 Da; H3K18Ub(G76A) / K23Ub(G76A) (square) calculated mass 32360.51 Da, found: 32359.3 Da. Raw spectra deconvoluted with UniDec. FIG. 21D. Raw mass spectra of asymmetric 185 bp H3K18Ub(G76A) / K23Ub(G76A) & H3K9me3 nucleosome: H4 (circle) calculated mass 11236.15 Da, found: 11235.4 Da; H2B (downward pointed triangle) calculated mass 13493.68 Da, found: 13493.3 Da; H2A (upward pointed triangle) calculated mass 13950.2 Da, found: 13949.8 Da; H3K9me3 (rightward pointed triangle) calculated mass 15279.68 Da, found: 15279.8 Da; H3K18Ub(G76A) / K23Ub(G76A) (square) calculated mass 32360.51 Da, found: 32359.3 Da. FIG. 21E. Deconvoluted mass spectra of asymmetric 185 bp H3K18Ub(G76A) / K23Ub(G76A) & unmodified H3 nucleosome: H4 (circle) calculated mass 11236.15 Da, found: 11235.4 Da; H2B (downward pointed triangle) calculated mass 13493.68 Da, found: 13493.1 Da; H2A (upward pointed triangle) calculated mass 13950.2 Da, found: 13949.7 Da; H3 (rightward pointed triangle) calculated mass 15238.61 Da, found: 15239.0 Da; H3K18Ub(G76A) / K23Ub(G76A) (square) calculated mass 32360.51 Da, found: 32359.6 Da. Raw spectra deconvoluted with UniDec. FIG. 21F. Raw mass spectra of asymmetric 185 bp H3K18Ub(G76A) / K23Ub(G76A) & unmodified H3 nucleosome: H4 (circle) calculated mass 11236.15 Da, found: 11235.4 Da; H2B (downward pointed triangle) calculated mass13493.68 Da, found: 13493.1 Da; H2A (upward pointed triangle) calculated mass 13950.2 Da, found: 13949.7 Da; H3 (rightward pointed triangle) calculated mass 15238.61 Da, found: 15239.0 Da; H3K18Ub(G76A) / K23Ub(G76A) (square) calculated mass 32360.51 Da, found: 32359.6 Da.

[0049] FIGs. 22A-22C. Characterization of asymmetric unmodified / ubiquitinated nucleosome. FIG. 22A. Anti-H3 blot of tailless nucleosome starting material, intermediate single tail ligation product, asymmetrically modified products, and intermediate fractions from weak anion exchange purification. Lanes: 1 - tailless starting material & ladder; 2 -final asymmetric H3K18Ub / K23Ub and unmodified H3 nucleosomes; 3-6 - impure weak anion exchange fractions in elution order; 7 -intermediate single H3 tail product; 8 - ladder; 9 -final asymmetric H3K18Ub / K23Ub and H3K9me3 nucleosomes; 10-14 -impure weak anion exchange fractions in elution order 14 - intermediate single H3 tail product; 15 - ladder. FIG. 22B. Anti-H3 blot of tailless nucleosome starting material, intermediate single tail ligation product, asymmetrically modified products, and intermediate fractions from weak anion exchange purification. Lanes: 1 -ladder; 2 -final asymmetric H3K9me3 / K18Ub / K23Ub and unmodified H3 nucleosomes; 3-8 - impure weak anion exchange fractions in elution order. FIG. 22C. TBE native gel of 185 bp starting material symmetric ubiquitinated4934-8116-8174.8Atty. Dkt. No. 043214-000103WO PT products of the cWll sortase ligation, and asymmetric ubiquitinated products of the cWll sortase ligation.

[0050] FIGs. 23A-23X. Mass spectrometric characterization of peptide substrates used in cWll nucleosome ligation. FIG. 23A. Raw spectra deconvoluted with UniDec. H3(l-34) unmodified T32-G33 amide linkage. Left: Analytical RP-HPLC chromatogram (Cl 8, 0-30% B, 23 min). Middle: Intact peptide ESI-MS. Right: Deconvoluted peptide ESI-MS (Calculated exact mass for C144H261N55O43 [M]+: 3448.99 Da; Observed: 3448.8 Da). FIG. 23B. Raw spectra deconvoluted with UniDec. H3(l-34) K4mel T32-G33 amide linkage. Left: Analytical RP-HPLC chromatogram (C18, 0-30% B, 23 min). Middle: Intact peptide ESI-MS. Right: Deconvoluted peptide ESI-MS (Calculated exact mass for C145H263N55O43 [M]+: 3463.01 Da; Observed: 3463.8 Da). FIG. 23C. Raw spectra deconvoluted with UniDec. H3(l-34) K4me2 T32-G33 amide linkage. Left: Analytical RP-HPLC chromatogram (C18, 0-30% B, 23 min). Middle: Intact peptide ESI-MS. Right: Deconvoluted peptide ESI-MS (Calculated exact mass for C146H265N55O43 [M]+: 3477.02 Da; Observed: 3476.9 Da). FIG. 23D. Raw spectra deconvoluted with UniDec. H3(l-34) K4me3 T32-G33 amide linkage. Left: Analytical RP-HPLC chromatogram (Cl 8, 7-30% B, 30 min). Middle: Intact peptide ESI-MS. Right: Deconvoluted peptide ESI-MS (Calculated exact mass for C148H266N54O46 [M]+: 3492.05 Da; Observed: 3491.1 Da). FIG. 23E. Raw spectra deconvoluted with UniDec. H3(l- 34) K9-propionyl T32-G33 depsipeptide. Left: Analytical RP-HPLC chromatogram (Cl 8, 7-30% B, 30 min). Middle: Intact peptide ESI-MS. Right: Deconvoluted peptide ESI-MS (Calculated exact mass for C148H266N54O45 [M]+: 3520.02 Da; Observed: 3519.7 Da). FIG. 23F. Raw spectra deconvoluted with UniDec. H3(l-34) K9-butyryl T32-G33 depsipeptide. Left: Analytical RP-HPLC chromatogram (C18, 7-30% B, 30 min). Middle: Intact peptide ESI-MS. Right: Deconvoluted peptide ESI-MS (Calculated exact mass for C148H266N54O45 [M]+: 3520.02 Da; Observed: 3519.7 Da). FIG. 23G. Raw spectra deconvoluted with UniDec. H3(l-34) K9-crotonyl T32-G33 depsipeptide. Left: Analytical RP-HPLC chromatogram (Cl 8, 7-30% B, 30 min). Middle: Intact peptide ESI-MS. Right: Deconvoluted peptide ESI-MS (Calculated exact mass for C148H264N54O45 [M]+: 3518.00 Da; Observed: 3518.7 Da). FIG. 23H. Raw spectra deconvoluted with UniDec. H3(l-34) K9-octanoyl T32- G33 depsipeptide. Left: Analytical RP-HPLC chromatogram (Cl 8, 0-30% B, 23 min). Middle: Intact peptide ESI-MS. Right: Deconvoluted peptide ESI-MS (Calculated exact mass for C152H274N54O45 [M]+: 3576.08 Da; Observed: 3575.7 Da). FIG. 231. Raw spectra deconvoluted with UniDec. H3(l-34) K9-lactyl T32-G33 depsipeptide. Left: Analytical RP-HPLC chromatogram (Cl 8, 0-30% B, 23 min). Middle: Intact peptide ESI-MS. Right: Deconvoluted peptide ESI-MS (Calculated exact mass for C147H264N54O46 [M]+: 3522.00 Da; Observed: 3521.5 Da). FIG. 23J. Raw spectra deconvoluted with UniDec. H3(l-34) K9-succinyl T32-G33 depsipeptide. Left: Analytical RP-HPLC chromatogram (C18, 0-30% B, 23 min). Middle: Intact peptide ESI-MS. Right: Deconvoluted peptide ESI-MS (Calculated exact mass for C148H264N54O47 [M]+: 3549.99 Da; Observed: 3549.4 Da). FIG. 23K. Raw spectra deconvoluted with UniDec. H3(l-34) K9-anti-hydroxyisbutyryl T32-G33 depsipeptide.4934-8116-8174.8Atty. Dkt. No. 043214-000103WO PTLeft: Analytical RP-HPLC chromatogram (Cl 8, 7-30% B, 30 min). Middle: Intact peptide ESI-MS. Right: Deconvoluted peptide ESI-MS (Calculated exact mass for C148H266N54O46 [M]+: 3536.01 Da; Observed: 3535.8 Da). FIG. 23L. Raw spectra deconvoluted with UniDec. H3(l-34) K9- - hydroxybutyryl T32-G33 depsipeptide. Left: Analytical RP-HPLC chromatogram (Cl 8, 730% B, 30 min). Middle: Intact peptide ESI-MS. Right: Deconvoluted peptide ESI-MS (Calculated exact mass for C148H266N54O46 [M]+: 3536.01 Da; Observed: 3536.3 Da). FIG. 23M. Raw spectra deconvoluted with UniDec. H3(l-34) K9-N-methylthiourea T32-G33 depsipeptide. Left: Analytical RP-HPLC chromatogram (C18, 7-30% B, 30 min). Middle: Intact peptide ESI-MS. Right: Deconvoluted peptide ESI-MS (Calculated exact mass for C146H263N55O44S [M]+: 3522.98 Da; Observed: 3523.7 Da). FIG. 23N. Raw spectra deconvoluted with UniDec. H3(l-34) K9norleucine T32-G33 depsipeptide. Left: Analytical RP-HPLC chromatogram (Cl 8, 7-30% B, 30 min). Middle: Intact peptide ESI-MS. Right: Deconvoluted peptide ESI-MS (Calculated exact mass for C144H259N53O44 [M]+: 3433.98 Da; Observed: 3433.9 Da). FIG. 230. Raw spectra deconvoluted with UniDec. H3(l-34) K9-myristoyl T32-G33 depsipeptide. Left: Analytical RP-HPLC chromatogram (Cl 8, 7-30% B, 30 min). Middle: Intact peptide ESI-MS. Right: Deconvoluted peptide ESI-MS (Calculated exact mass for C158H286N54O45 [M]+: 3660.18 Da; Observed: 3659.8 Da). FIG. 23P. Raw spectra deconvoluted with UniDec. H3(l-34) S28-phosphoryl T32-G33 depsipeptide. Left: Analytical RP-HPLC chromatogram (C18, 0-65% B, 30 min). Middle: Intact peptide ESI-MS. Right: Deconvoluted peptide ESI-MS (Calculated exact mass for C144H260N54O47P [M]+: 3528.94 Da; Observed: 3529.0 Da). FIG. 23Q. Raw spectra deconvoluted with UniDec. H3(l-34) S31 -phosphoryl T32-G33 depsipeptide. Left: Analytical RP-HPLC chromatogram (Cl 8, 7-30% B, 30 min). Middle: Intact peptide ESI-MS. Right: Deconvoluted peptide ESI-MS (Calculated exact mass for C144H260N54O48P [M]+: 3544.93 Da; Observed: 3546.1 Da). FIG. 23R. Raw spectra deconvoluted with UniDec. H3(l-34) K4-trimethyl K9-acetyl T32-G33 depsipeptide. Left: Analytical RP-HPLC chromatogram (Cl 8, 7-30% B, 30 min). Middle: Intact peptide ESI-MS. Right: Deconvoluted peptide ESI-MS (Calculated exact mass for C144H260N54O48P [M]+: 3544.93 Da; Observed: 3546.1 Da). FIG. 23S. Raw spectra deconvoluted with UniDec. H3(l-34) K9-acetyl K14-acetyl K18-acetyl K27-acetyl T32-G33 depsipeptide (aa35-37) KRK. Left: Analytical RP-HPLC chromatogram (Cl 8, 7-30% B, 30 min). Middle: Intact peptide ESI- MS. Right: Deconvoluted peptide ESI-MS (Calculated exact mass for C170H305N62O51 [M]+: 4030.31 Da; Observed: 4029.1 Da). FIG. 23T. Raw spectra deconvoluted with UniDec. H3(l-34) K9- acetyl K14-acetyl K18-acetyl K23-acetyl T32-G33 depsipeptide (aa35-38) KKKK (SEQ ID NO: 50). Left: Analytical RP-HPLC chromatogram (Cl 8, 7-30% B, 30 min). Middle: Intact peptide ESI-MS. Right: Deconvoluted peptide ESI-MS (Calculated exact mass C176H316N62O52 [M]+: 4130.40 Da; Observed: 4130.3 Da). FIG. 23U. Raw spectra deconvoluted with UniDec. H3(l-34) K9-acetyl K14- acetyl K23-acetyl K27-acetyl T32-G33 depsipeptide (aa35-38) KKKK (SEQ ID NO: 50). Left: Analytical RP-HPLC chromatogram (Cl 8, 7-30% B, 30 min). Middle: Intact peptide ESI-MS. Right: Deconvoluted peptide ESI-MS (Calculated exact mass C176H316N62O52 [M]+: 4130.40 Da; 4934-8116-8174.8Atty. Dkt. No. 043214-000103WO PTObserved: 4130.0 Da). FIG. 23V. Raw spectra deconvoluted with UniDec. H3(l-34) K9-acetyl K14- acetyl K18-acetyl K23-acetyl K27-acetyl T32-G33 depsipeptide (aa35-38) KKKK (SEQ ID NO: 50). Left: Analytical RP-HPLC chromatogram (Cl 8, 7-30% B, 30 min). Middle: Intact peptide ESI-MS. Right: Deconvoluted peptide ESI-MS (Calculated exact mass C178H318N62O53 [M]+: 4172.41 Da; Observed: 4172.0 Da) FIG. 23W. Raw spectra deconvoluted with UniDec. H3(l-34) K18- Ubiquitin(G76A) K23-Ubiquitin(G76A) T32-G33 amide. Left: Deconvoluted peptide ESI-MS (Calculated exact mass C902H1519N265O277S2 [M]+: 20572.45 Da; Observed: 20572.1 Da). Right: Intact peptide ESI-MS. FIG. 23X. Raw spectra deconvoluted with UniDec. H3(l-34) K9-trimethyl K18-Ubiquitin(G76A) K23-Ubiquitin(G76A) T32-G33 amide. Left: Deconvoluted peptide ESI-MS (Calculated exact mass for C905H1525N265O277S2 [M]+: 20614.53 Da; Observed: 20614.0 Da). Right: Intact peptide ESI-MS.

[0051] FIGs. 24A-24E. Western blot measurement of Sirt2 activity toward mono-acetylated nucleosomes. FIG. 24A. Western blots (top; n=4) and native TBE gels (bottom) of Sirt2 deacetylation assays with 147 bp H3K9acetyl (H3K9ac) nucleosomes. FIG. 24B. Western blots (top; n=2) and native TBE gels (bottom) of Sirt2 deacetylation assays with 147 bp H3K14acetyl (H3K14ac) nucleosomes. FIG. 24C. Western blots (top; n=4) and native TBE gels (bottom) of Sirt2 deacetylation assays with 147 bp H3K18acetyl (H3K8ac) nucleosomes. FIG. 24D. Western blots (top; n=4) and native TBE gels (bottom) of Sirt2 deacetylation assays with 147 bp H3K13acetyl (H3K13ac) nucleosomes. FIG. 24E. Western blots (left; n=4) and native TBE gels (right) of Sirt2 deacetylation assays with 147 bp H3K27acetyl (H3K27ac) nucleosomes.

[0052] FIGs. 25A-25C. Western blot measurement of MiDAC activity toward mono- and penta-acetylated nucleosomes. Fig. 25A. Western blots (top; anti-H3K9ac, anti-H3K14ac, anti- H3K18ac, anti-H3K23ac, anti-H3K27ac) and native TBE gels (bottom) of MiDAC deacetylation assays with 147 bp H3K9ac / K14ac / K18ac / K23ac / K27ac (H3Kpenta-ac) nucleosomes (n=4). FIG. 25B. Western blots (top; anti-H3K23ac) and native TBE gels (bottom) of MiDAC deacetylation assays with 147 bp H3K23acetyl (H3K23ac) nucleosomes (n=4). FIG. 25C. Western blots (top; anti-H3K23ac) and native TBE gels (bottom) of MiDAC deacetylation assays with 147 bp H3K9ac / K14ac / K18ac / K23ac / K27ac (H3Kpenta-ac) nucleosomes (top left; n=4) and 147 bp H3K27acetyl (H3K27ac) nucleosomes (top right; n=4).

[0053] FIGs. 26A-26E. Comparison of Sirt6 activity toward nucleosomes prepared by conventional reconstitution and cWll nucleosome ligation. FIG. 2 A. Representative western blots (top; n=12 of n=16) and native TBE gels (bottom) of Sirt6 deacetylation assays with 147 bp H3K9ac nucleosome prepared by sortase ligation. FIG. 26B. Western blots (top; n=4) and native TBE gels (bottom) of Sirt6 deacetylation assays with 147 bp H3K9ac nucleosome prepared by traditional nucleosome reconstitution. FIG. 26C.. Deacylation fitting for sortase nucleosomes (left; GRAPHPAD PRISM™ 10.2.3; one-phase decay) and nucleosomes prepared by literature protocols (left; 4934-8116-8174.8Atty. Dkt. No. 043214-000103WO PTGRAPHPAD PRISM™ 10.2.3; one-phase decay) from FIG. 26A and FIG. 26B respectively. FIG. 26D. Sirt6 deacetylation rates (V / [E]) for H3K9ac nucleosomes prepared by the cWll sortase ligation (Sortase) and nucleosomes prepared by literature protocols (Traditional); error bars indicate standard deviation. FIG. 26E. Average V / [E] ± SEM values for Sirt6 deacetylation of H3K9ac nucleosomes prepared by either cWl 1 sortase ligation (n=16) or literature protocols (n=4).

[0054] FIGs. 27A-27C. Comparison of LSD 1 / HD AC 1 / CoREST activity toward nucleosomes prepared by conventional reconstitution and cWl 1 nucleosome ligation. FIG. 27A. Western blots (top), deacylation fitting (right; GRAPHPAD PRISM™ 10.2.3; one-phase decay), and native TBE gels (bottom) of LSD 1 / HDACl / CoREST (LHC) deacetylation assays with 147 bp H3K9ac nucleosome prepared by the sortase ligation (top left; n=2) or traditional nucleosome reconstitution (top right; n=4). FIG. 27B. LHC complex deacetylation rates (V / [E]) for H3K9ac nucleosomes prepared by the cWll sortase ligation (Sortase) and nucleosomes prepared by literature protocols (Traditional); error bars indicate standard deviation. FIG. 27C. Average V / [E] ± SEM values for LHC deacetylation of H3K9ac nucleosomes prepared by either cWl 1 sortase ligation or literature protocols.

[0055] FIGs. 28A-28D. Western blot measurement of Sirtl activity by length of H3K9 acylation carbon chain. FIG. 28A. Western blots (top left; anti-H3K9ac; n=4), deacylation fitting (bottom; GRAPHPAD PRISM™ 10.2.3; one-phase decay), and native TBE gels (top right) of Sirtl deacetylation assays with 147bp H3K9acetyl (H3K9ac) nucleosomes. FIG. 28B. Western blots (top left; anti-H3K9ac antibody; n=2), deacylation fitting (bottom; GRAPHPAD PRISM™ 10.2.3; one- phase decay), and native TBE gels (top right) of Sirtl deacetylation assays with 147 bp H3K9propionyl (H3K9pr) nucleosomes. FIG. 28C. Western blots (top left; anti-H3K9bu; n=2), deacylation fitting (top right; GRAPHPAD PRISM™ 10.2.3; one-phase decay), and native TBE gels (bottom) of Sirtl deacetylation assays with 147 bp H3K9butyryl (H3K9bu) nucleosomes. FIG. 28D. Western blots (top left; anti-H3K9but; n=2), deacylation fitting (bottom; GRAPHPAD PRISM™ 10.2.3; one-phase decay), and native TBE gels (top right) of Sirtl deacetylation assays with 147 bp H3K9octanoyl (H3K9oct) nucleosomes.

[0056] FIGs. 29A-29C. Western blot measurement of Sirt2 activity by length of H3K9 acylation carbon chain. FIG. 29A. Western blots (top; anti-H3K9ac antibody; n=2), deacylation fitting (right; GRAPHPAD PRISM™ 10.2.3; one-phase decay), and native TBE gels (bottom) of Sirt2 deacetylation assays with H3K9propionyl (H3K9pr) nucleosomes. FIG. 29B. Western blots (top; anti- H3K9bu; n=2), deacylation fitting (right; GRAPHPAD PRISM™ 10.2.3; one-phase decay), and native TBE gels (bottom) of Sirt2 deacetylation assays with H3K9butyryl (H3K9bu) nucleosomes. FIG. 29C. Western blots (top; anti-H3K9bu; n=4), deacylation fitting (right; GRAPHPAD PRISM™ 10.2.3; one- phase decay), and native TBE gels (bottom) of Sirt2 deacetylation assays with H3K9octanoyl (H3K9oct) nucleosomes. Deacylation with 30 nM enzyme (leftmost) was too fast to fit, and is not included in subsequent V / [E] calculation.4934-8116-8174.8Atty. Dkt. No. 043214-000103WO PT

[0057] FIGs. 30A-30C. Western blot measurement of Sirt6 activity by length of H3K9 acylation carbon chain. FIG. 30A. Western blots (top left; anti-H3K9ac antibody; n=5), deacylation fitting (bottom left; GRAPHPAD PRISM™ 10.2.3; one-phase decay), and native TBE gels (right) of Sirt6 deacetylation assays with 147 bp H3K9propionyl (H3K9pr) nucleosomes. FIG. 30B. Western blots (top left, anti-H3K9bu; n=6), deacylation fitting (bottom left; GRAPHPAD PRISM™ 10.2.3; one- phase decay), and native TBE gels (right) of Sirt6 deacetylation assays with 147 bp H3K9butyryl (H3K9bu) nucleosomes. FIG. 30C. Western blots (top left; anti-H3K9bu; n=4), deacylation fitting (bottom left; GRAPHPAD PRISM™ 10.2.3; one-phase decay), and native TBE gels (right) of Sirt6 deacetylation assays with 147 bp H3K9octanoyl (H3K9oct) nucleosomes.

[0058] FIGs. 31A-31D. Western blot measurement of MiDAC activity by length of H3K9 acylation carbon chain. FIG. 31A. Western blots (top left; anti-H3K9ac; n=4), deacylation fitting (bottom left; GRAPHPAD PRISM™ 10.2.3; one-phase decay), and native TBE gels (right) of MiDAC deacetylation assays with 147 bp H3K9acetyl (H3K9ac) nucleosomes. FIG. 31B. Western (top left; anti-H3K9ac antibody; n=5), deacylation fitting (bottom left; GRAPHPAD PRISM™ 10.2.3; one-phase decay), and native TBE gels (right) of MiDAC deacetylation assays with 147 bp H3K9propionyl (H3K9pr) nucleosomes. FIG. 31C. Western blots (top left; anti-H3K9bu; n=4), deacylation fitting (bottom left; GRAPHPAD PRISM™ 10.2.3; one-phase decay), and native TBE gels (right) of MiDAC deacetylation assays with 147 bp H3K9butyryl (H3K9bu) nucleosomes. FIG. 31D. Western blots ((top left; anti-H3K9bu antibody; n=4), deacylation fitting (bottom left; GRAPHPAD PRISM™ 10.2.3; one- phase decay), and native TBE gels (right) of MiDAC deacetylation assays with 147 bp H3K9octanoyl (H3K9oct) nucleosomes.

[0059] FIGs. 32A-32C. Western blot measurement of Sirtl activity toward four carbon acylations of H3K9. FIG. 32A. Western blots (top left; anti-H3K9cro; n=2), deacylation fitting (top right; GRAPHPAD PRISM™ 10.2.3; one-phase decay), and native TBE gels (bottom) of Sirtl deacetylation assays with 147 bp H3K9crotonyl (H3K9cro) nucleosomes. FIG. 32B. Western blots (top left; anti-H3K9hib; n=2), deacylation fitting (top right; GRAPHPAD PRISM™ 10.2.3; one-phase decay), and native TBE gels (bottom) of Sirtl deacetylation assays with 147 bp H3K9anti- hydroxyisobutyryl nucleosomes. FIG. 32C. Western blots (top left; anti-H3K9succ; n=2), deacylation fitting (top right; GRAPHPAD PRISM™ 10.2.3; one-phase decay), and native TBE gels (bottom) of Sirtl deacetylation assays with 147 bp H3K9succinyl nucleosomes.

[0060] FIGs. 33A-33C. Western blot measurement of Sirt2 activity toward four carbon acylations of H3K9. FIG. 33A. Western blots (top; anti-H3K9cro; n=2), deacylation fitting (right; GRAPHPAD PRISM™ 10.2.3; one-phase decay), and native TBE gels (bottom) of Sirt2 deacetylation assays with 147 bp H3K9crotonyl (H3K9cro) nucleosomes. FIG. 33B. Western blots (top; anti- H3K9hib; n=2), deacylation fitting (right; GRAPHPAD PRISM™ 10.2.3; one-phase decay), and native TBE gels (bottom) of Sirt2 deacetylation assays with 147 bp H3K9anti-hydroxyisobutyryl 4934-8116-8174.8Atty. Dkt. No. 043214-000103WO PT nucleosomes. FIG. 33C. Western blots (top; anti-H3K9succ; n=2), deacylation fitting (right; GRAPHPAD PRISM™ 10.2.3; one-phase decay), and native TBE gels (bottom) of Sirt6 deacetylation assays with 147 bp H3K9succinyl nucleosomes.

[0061] FIGs. 34A-34C. Western blot measurement of Sirt6 activity toward four carbon acylations of H3K9. FIG. 34A. Western blots (top left; anti-H3K9cro; n=6), deacylation fitting (bottom left; GRAPHPAD PRISM™ 10.2.3; one-phase decay), and native TBE gels (right) of Sirt6 deacetylation assays with 147 bp H3K9crotonyl (H3K9cro) nucleosomes. FIG. 34B. Western blots (top; anti-H3K9hib; n=8), deacylation fitting (bottom; GRAPHPAD PRISM™ 10.2.3; one-phase decay), and native TBE gels (middle) of Sirt6 deacetylation assays with 147 bp H3K9anti-hydroxyisobutyryl nucleosomes. FIG. 34C. Western blots (left; anti-H3K9succ; n=3), deacylation fitting (middle; GRAPHPAD PRISM™ 10.2.3; one-phase decay), and native TBE gels (right) of Sirt6 deacetylation assays with 147 bp H3K9succinyl nucleosomes.

[0062] FIGs. 35A-35C. Western blot measurement of MiDAC activity toward four carbon acylations of H3K9. FIG. 35A. Western blots (top left; anti-H3K9cro; n=6), deacylation fitting (bottom left; GRAPHPAD PRISM™ 10.2.3; one-phase decay), and native TBE gels (right) of MiDAC deacetylation assays with 147 bp H3K9crotonyl (H3K9cro) nucleosomes. FIG. 35B. Western blots (top left; anti-H3K9hib; n=3), deacylation fitting (bottom left; GRAPHPAD PRISM™ 10.2.3; one-phase decay), and native TBE gels (bottom) of MiDAC deacetylation assays with 147 bp H3K9anti- hydroxyisobutyryl (H3K9hib) nucleosomes. FIG. 35C. Western blots (left; anti-H3K9succ; n=4), deacylation fitting (middle; GRAPHPAD PRISM™ 10.2.3; one-phase decay), and native TBE gels (right) of MiDAC deacetylation assays with 147 bp H3K9succinyl (H3K9succ) nucleosomes.

[0063] FIGs. 36A-36D. Western blot measurement of LHC activity toward four carbon acylations of H3K9. FIG. 36A. Western blots (top left; anti-H3K9bu; n=4), deacylation fitting (bottom left; GRAPHPAD PRISM™ 10.2.3; one-phase decay), and native TBE gels (right) of LHC deacetylation assays with 147 bp H3K9butyryl (H3K9bu) nucleosomes. FIG. 36B. Western blots (top left; anti-H3K9cro; n=4), deacylation fitting (bottom left; GRAPHPAD PRISM™ 10.2.3; one-phase decay), and native TBE gels (right) of LHC deacetylation assays with 147 bp H3K9crotonyl (H3K9cro) nucleosomes. FIG. 36C. Western blots (top left; anti-H3K9hib; n=2), deacylation fitting (top right; GRAPHPAD PRISM™ 10.2.3; one-phase decay), and native TBE gels (bottom) of LHC deacetylation assays with 147 bp H3K9anti-hydroxyisobutyryl (H3K9hib) nucleosomes. FIG. 36D. Western blots (top left; anti-H3K9succ; n=2), deacylation fitting (top right; GRAPHPAD PRISM™ 10.2.3; one-phase decay), and native TBE gels (bottom) of LHC deacetylation assays with 147 bp H3K9succinyl (H3K9succ) nucleosomes.

[0064] FIGs. 37A-37D. Western blot measurement of free HDAC1 activity toward four carbon acylations of H3K9. FIG. 37A. Western blots (top left; anti-H3K9bu; n=2), deacylation fitting (top right; GRAPHPAD PRISM™ 10.2.3; one-phase decay), and native TBE gels (bottom) of HD AC 4934-8116-8174.8Atty. Dkt. No. 043214-000103WO PT deacetylation assays with 147 bp H3K9butyryl (H3K9bu) nucleosomes. FIG. 37B. Western blots (top left; anti-H3K9cro; n=2), deacylation fitting (top right; GRAPHPAD PRISM™ 10.2.3; one-phase decay), and native TBE gels (bottom) of HD AC deacetylation assays with 147 bp H3K9crotonyl (H3K9cro) nucleosomes. FIG. 37C. Western blots (top left; anti-H3K9hib; n=2), deacylation fitting (top right; GRAPHPAD PRISM™ 10.2.3; one-phase decay), and native TBE gels (bottom) of HD AC deacetylation assays with 147 bp H3K9anti-hydroxyisobutyryl (H3K9hib) nucleosomes. FIG. 37D. Western blots (top left; anti-H3K9hib; n=2), deacylation fitting (top right; GRAPHPAD PRISM™ 10.2.3; one-phase decay), and native TBE gels (bottom) of HDAC deacetylation assays with 147 bp H3K9succinyl (H3K9succ) nucleosomes.

[0065] FIGs. 38A-38B. Western blot measurement of Sirtuin5 activity toward acetylated and succinylated H3K9. FIG. 38A. Western blots (top; anti-H3K9ac; n=2), deacylation fitting (right; GRAPHPAD PRISM™ 10.2.3; one-phase decay), and native TBE gels (bottom) of Sirt5 deacetylation assays with 147 bp H3K9acetul (H3K9ac) nucleosomes. FIG. 38B. Western blots (top left; anti- H3K9succ; n=5), deacylation fitting (center left; GRAPHPAD PRISM™ 10.2.3; one-phase decay), and native TBE gels (right) of Sirt5 deacetylation assays with 147 bp H3K9succinyl (H3K9succ) nucleosomes. Deacylation with 5 and 10 nM enzyme (bottom left) was too fast to fit, and is not included in subsequent V / [E] calculation.

[0066] FIG. 39. Validation of H3Kac single site antibody specificity. Western blot analysis of synthetic nucleosomes with site-specific acetylations at (left-to-right) H3K9, H3K14, H3K18, H3K23, H3K27, or all five positions (H3penta-ac) with site-specific antibodies toward (top-to-bottom) H3K9ac, H3K14ac, H3K18ac, H3K23ac or H3K27ac. Molecular weight markers are visible in blots for H3K14ac, H3K18ac, H3K23ac and H3K27ac.

[0067] FIGs. 40A-40E. Western blot measurement of Sirt2 activity toward mono-acetylated nucleosomes. FIG. 40A. Western blots (top; n=4) and native TBE gels (bottom) of Sirt2 deacetylation assays with 147 bp H3K9acetyl (H3K9ac) nucleosomes. FIG. 40B. Western blots (top; n=2) and native TBE gels (bottom) of Sirt2 deacetylation assays with 147 bp H3K14acetyl (H3K14ac) nucleosomes. FIG. 40C. Western blots (top; n=4) and native TBE gels (bottom) of Sirt2 deacetylation assays with 147 bp H3K18acetyl (H3K8ac) nucleosomes. FIG. 40D. Western blots (top; n=4) and native TBE gels (bottom) of Sirt2 deacetylation assays with 147 bp H3K13acetyl (H3K13ac) nucleosomes. FIG. 40E. Western blots (left; n=4) and native TBE gels (right) of Sirt2 deacetylation assays with 147 bp H3K27acetyl (H3K27ac) nucleosomes.

[0068] FIGs. 41A-41C. Western blot measurement of Sirt2 activity toward penta-acetylated nucleosomes. FIG. 41 A. Western blots (top; anti-H3K9ac, anti-H3K18ac, anti-H3K27ac; n=4) and native TBE gels (bottom) of Sirt2 deacetylation assays with 147 bp H3K9ac / K14ac / K18ac / K23ac / K27ac (H3Kpenta-ac) nucleosomes. FIG. 41B. Western blots (top; anti- H3K14ac; n=2) and native TBE gels (bottom) of Sirt2 deacetylation assays with 147 bp4934-8116-8174.8Atty. Dkt. No. 043214-000103WO PTH3K9ac / K14ac / K18ac / K23ac / K27ac (H3Kpenta-ac) nucleosomes. FIG. 41C. Western blots (top; anti- H3K23ac, anti-H3K23ac; n=4) and native TBE gels (bottom) of Sirt2 deacetylation assays with 147 bp H3K9ac / K14ac / K18ac / K23ac / K27ac (H3Kpenta-ac) nucleosomes.

[0069] FIGs. 42A-42C. Western blot measurement of Sirt6 activity toward mono- and pentaacetylated nucleosomes. FIG. 42A. Western blots (top; anti-H3K9ac, anti-H3K14ac, anti-H3K18ac, anti-H3K23ac, anti-H3K27ac; n=4) and native TBE gels (bottom) of Sirt6 deacetylation assays with 147 bp H3K9ac / K14ac / K18ac / K23ac / K27ac (H3Kpenta-ac) nucleosomes. FIG. 42B. Western blots (top; anti-H3K14ac) and native TBE gels (bottom) of parallel Sirt6 deacetylation assays with 147 bp H3K9ac / K14ac / K18ac / K23ac / K27ac (H3Kpenta-ac) nucleosomes (top left; n=2) and 147 bp H3K14acetyl (H3K14ac) nucleosomes (top right; n=2). FIG. 42C. Western blots (top; anti-H3K27ac) and native TBE gels (bottom) of parallel Sirt6 deacetylation assays with 147 bp H3K9ac / K14ac / K18ac / K23ac / K27ac (H3Kpenta-ac) nucleosomes (top left; n=4) and 147 bp H3K27acetyl (H3K27ac) nucleosomes (top right; n=9).

[0070] FIGs. 43A-43C. Western blot measurement of MiDAC activity toward mono- and penta-acetylated nucleosomes. FIG. 43A. Western blots (top; anti-H3K9ac, anti-H3K14ac, anti- H3K18ac, anti-H3K23ac, anti-H3K27ac) and native TBE gels (bottom) of MiDAC deacetylation assays with 147 bp H3K9ac / K14ac / K18ac / K23ac / K27ac (H3Kpenta-ac) nucleosomes (n=4). FIG. 43B. Western blots (top; anti-H3K23ac) and native TBE gels (bottom) of MiDAC deacetylation assays with 147 bp H3K23acetyl (H3K23ac) nucleosomes (n=4). FIG. 43C. Western blots (top; anti-H3K23ac) and native TBE gels (bottom) of MiDAC deacetylation assays with 147 bp H3K9ac / K14ac / K18ac / K23ac / K27ac (H3Kpenta-ac) nucleosomes (top left; n=4) and 147 bp H3K27acetyl (H3K27ac) nucleosomes (top right; n=4).

[0071] FIGs. 44A-44F. Western blot measurement of Sirt2 activity toward asymmetrically acetylated nucleosomes. FIG. 44A. Western blots (top; anti-H3K18ac; n=4) and native TBE gels (bottom) of Sirt2 deacetylation assays with 147 bp asymmetric unmodified H3 & H3K18ac nucleosomes. FIG. 44B. Western blots (top; anti-H3K18ac; n=4) and native TBE gels (bottom) of Sirt2 deacetylation assays with 147 bp asymmetric H3K9ac / K14ac / K23ac / K27ac (H3tetra-ac) & H3K18ac nucleosomes. FIG. 44C. Western blots (top; anti-H3K23ac; n=4) and native TBE gels (bottom) of Sirt2 deacetylation assays with 147 bp asymmetric unmodified H3 & H3K23ac nucleosomes. FIG. 44D. Western blots (top; anti-H3K23ac; n=4) and native TBE gels (bottom) of Sirt2 deacetylation assays with 147 bp asymmetric H3K9ac / K14ac / K18ac / K27ac (H3tetra-ac) & H3K23ac nucleosomes. FIG. 44E. Western blots (top; anti-H3K27ac; n=4) and native TBE gels (bottom) of Sirt2 deacetylation assays with 147 bp asymmetric unmodified H3 & H3K27ac nucleosomes. FIG. 44F. Western blots (top; anti- H3K27ac; n=4) and native TBE gels (bottom) of Sirt2 deacetylation assays with 147 bp asymmetric H3K9ac / K14ac / K18ac / K23ac (H3tetra-ac) & H3K27ac nucleosomes.4934-8116-8174.8Atty. Dkt. No. 043214-000103WO PT

[0072] FIGs. 45A-45B. Western blot measurement of Sirt6 activity toward asymmetrically acetylated nucleosomes. FIG 45A. Western blots (top; anti-H3K27ac; n=4) and native TBE gels (bottom) of Sirt6 deacetylation assays with 147 bp asymmetric unmodified H3 & H3K27ac nucleosomes. FIG. 45B. Western blots (top; anti-H3K27ac; n=4) and native TBE gels (bottom) of Sirt6 deacetylation assays with 147 bp asymmetric H3K9ac / K14ac / K18ac / K23ac (H3tetra-ac) & H3K27ac nucleosomes.

[0073] FIGs. 46A-46D. Western blot measurement of MiDAC activity toward asymmetrically acetylated nucleosomes. FIG. 46A. Western blots (top; anti-H3K23ac; n=4) and native TBE gels (bottom) of MiDAC deacetylation assays with 147 bp asymmetric unmodified H3 & H3K23ac nucleosomes. FIG. 46B. Western blots (top; anti-H3K23ac; n=8) and native TBE gels (bottom) of MiDAC deacetylation assays with 147 bp asymmetric H3K9ac / K14ac / K18ac / K27ac (H3tetra-ac) & H3K23ac nucleosomes. FIG. 46C. Western blots (top; anti-H3K27ac; n=6) and native TBE gels (bottom) of MiDAC deacetylation assays with 147 bp asymmetric unmodified H3 & H3K27ac nucleosomes. FIG. 46D. Western blots (top; anti-H3K27ac; n=6) and native TBE gels (bottom) of MiDAC deacetylation assays with 147 bp asymmetric H3K9ac / K14ac / K18ac / K23ac (H3tetra-ac) & H3K27ac nucleosomes.

[0074] FIGs. 47A-47C. Western blot measurement of LC activity toward nucleosomes with asymmetric methylation and acetylation. FIG. 47 A. Western blots (top; anti-H3K4me2; n=6) and representative TBE native gel (left) of LC demethylation assays with 185 bp asymmetric unmodified H3 & H3K4me2 nucleosomes. FIG. 47B. Western blots (top, right; anti-H3K4me2; n=6) and representative TBE native gel (center) of LC demethylation assays with 185 bp asymmetric unmodified H3K14ac & H3K4me2 nucleosomes. FIG. 47C. Western blots (bottom; anti-H3K4me2; n=4) and representative TBE native gel (right) of LC demethylation assays with 185 bp asymmetric unmodified H3 & H3K4me2K14ac nucleosomes.

[0075] FIGs. 48A-48F. Mass spectrometric characterization of ubiquitinated H3 peptides and ubiquitinated nucleosome. FIG. 48A. Deconvoluted mass spectra of asymmetric 185 bp H3K9me3 / K18Ub(G76A) / K23Ub(G76A) & unmodified H3 nucleosome: H4 (circle) calculated mass 11236.15 Da, found: 11235.4 Da; H2B (downward pointed triangle) calculated mass 13493.68 Da, found: 13493.1 Da; H2A (upward pointed triangle) calculated mass 13950.2 Da, found: 13949.7 Da; H3 (rightward pointed triangle) calculated mass 15238.61 Da, found: 15239.0 Da; H3K9me3 / K18Ub(G76A) / K23Ub(G76A) (square) calculated mass 32402.18 Da, found: 32402.4 Da. Raw spectra deconvoluted with UniDec. FIG. 48B. Raw mass spectra of asymmetric 185 bp H3K9me3 / K18Ub(G76A) / K23Ub(G76A) & unmodified H3 nucleosome: H4 (circle) calculated mass 11236.15 Da, found: 11235.4 Da; H2B (downward pointed triangle) calculated mass 13493.68 Da, found: 13493.1 Da; H2A (upward pointed triangle) calculated mass 13950.2 Da, found: 13949.7 Da; H3 (rightward pointed triangle) calculated mass 15238.61 Da, found: 15239.0 Da;4934-8116-8174.8Atty. Dkt. No. 043214-000103WO PTH3K9me3 / K18Ub(G76A) / K23Ub(G76A) (square) calculated mass 32402.18 Da, found: 32402.4 Da. Raw spectra deconvoluted with UniDec. FIG. 48C. Deconvoluted mass spectra of asymmetric 185 bp H3K18Ub(G76A) / K23Ub(G76A) & H3K9me3 nucleosome: H4 (circle) calculated mass 11236.15 Da, found: 11235.4 Da; H2B (downward pointed triangle) calculated mass 13493.68 Da, found: 13493.3 Da; H2A (upward pointed triangle) calculated mass 13950.2 Da, found: 13949.8 Da; H3K9me3 (rightward pointed triangle) calculated mass 15279.68 Da, found: 15279.8 Da; H3K18Ub(G76A) / K23Ub(G76A) (square) calculated mass 32360.51 Da, found: 32359.3 Da. Raw spectra deconvoluted with UniDec. FIG. 48D. Raw mass spectra of asymmetric 185 bp H3K18Ub(G76A) / K23Ub(G76A) & H3K9me3 nucleosome: H4 (circle) calculated mass 11236.15 Da, found: 11235.4 Da; H2B (downward pointed triangle) calculated mass 13493.68 Da, found: 13493.3 Da; H2A (upward pointed triangle) calculated mass 13950.2 Da, found: 13949.8 Da; H3K9me3 (rightward pointed triangle) calculated mass 15279.68 Da, found: 15279.8 Da; H3K18Ub(G76A) / K23Ub(G76A) (square) calculated mass 32360.51 Da, found: 32359.3 Da. Raw spectra deconvoluted with UniDec. FIG. 48E. Deconvoluted mass spectra of asymmetric 185 bp H3K18Ub(G76A) / K23Ub(G76A) & unmodified H3 nucleosome: H4 (circle) calculated mass 11236.15 Da, found: 11235.4 Da; H2B (downward pointed triangle) calculated mass 13493.68 Da, found: 13493.1 Da; H2A (upward pointed triangle) calculated mass 13950.2 Da, found: 13949.7 Da; H3 (rightward pointed triangle) calculated mass 15238.61 Da, found: 15239.0 Da; H3K18Ub(G76A) / K23Ub(G76A) (square) calculated mass 32360.51 Da, found: 32359.6 Da. Raw spectra deconvoluted with UniDec. FIG. 48F. Raw mass spectra of asymmetric 185 bp H3K18Ub(G76A) / K23Ub(G76A) & unmodified H3 nucleosome: H4 (circle) calculated mass 11236.15 Da, found: 11235.4 Da; H2B (downward pointed triangle) calculated mass 13493.68 Da, found: 13493.1 Da; H2A (upward pointed triangle) calculated mass 13950.2 Da, found: 13949.7 Da; H3 (rightward pointed triangle) calculated mass 15238.61 Da, found: 15239.0 Da; H3K18Ub(G76A) / K23Ub(G76A) (square) calculated mass 32360.51 Da, found: 32359.6 Da. Raw spectra deconvoluted with UniDec.

[0076] FIGs. 49A-49C. Characterization of asymmetric unmodified / ubiquitinated nucleosome. Anti-H3 blot of tailless nucleosome starting material, intermediate single tail ligation product, asymmetrically modified products, and intermediate fractions from weak anion exchange purification. FIG. 49A. Lanes: 1 - tailless starting material & ladder; 2 -final asymmetric H3K18Ub / K23Ub and unmodified H3 nucleosomes; 3-6 - impure weak anion exchange fractions in elution order ; 7 - intermediate single H3 tail product; 8 - ladder; 9 -final asymmetric H3K18Ub / K23Ub and H3K9me3 nucleosomes; 10-14 - impure weak anion exchange fractions in elution order 14 - intermediate single H3 tail product; 15 - ladder. FIG. 49B. Lanes: 1 -ladder; 2 -final asymmetric H3K9me3 / K18Ub / K23Ub and unmodified H3 nucleosomes; 3-8 - impure weak anion exchange fractions in elution order. FIG. 49C. TBE native gel of 185 bp starting material symmetric4934-8116-8174.8Atty. Dkt. No. 043214-000103WO PT ubiquitinated products of the cWl 1 sortase ligation, and asymmetric ubiquitinated products of the cWl 1 sortase ligation.

[0077] FIGs. 50A-50D. Electrophoretic mobility shift assay titrating asymmetric nucleosomes with sfGFP-RFTS fusion. FIG. 50A. Fluorescence visualization of sfGFP-RFTS fusion (titrant) binding to 185 bp nucleosomes with asymmetric H3K9me3 / K18Ub / K23Ub and unmodified H3 (left), asymmetric K18Ub / K23Ub and H3K9me3 (middle), or asymmetric K18Ub / K23Ub and unmodified H3 (right) after 1 hr at 4 °C. FIG. 50B. Ethidium bromide (EtBr) visualization of sfGFP-RFTS fusion (titrant) binding to 185 bp nucleosomes with asymmetric H3K9me3 / K18Ub / K23Ub and unmodified H3 (left), asymmetric K18Ub / K23Ub and H3K9me3 (middle), or asymmetric K18Ub / K23Ub and unmodified H3 (right) after 1 hr at 4 °C. FIG. 50C. Fluorescence visualization of sfGFP-RFTS fusion (titrant) binding to 185 bp nucleosomes with asymmetric H3K9me3 / K18Ub / K23Ub and unmodified H3 (left), asymmetric K18Ub / K23Ub and H3K9me3 (middle), or asymmetric K18Ub / K23Ub and unmodified H3 (right) after 24 hr at 4 °C. FIG. 50D. Ethidium bromide (EtBr) visualization of sfGFP- RFTS fusion (titrant) binding to 185 bp nucleosomes with asymmetric H3K9me3 / K18Ub / K23Ub and unmodified H3 (left), asymmetric K18Ub / K23Ub and H3K9me3 (middle), or asymmetric K18Ub / K23Ub and unmodified H3 (right) after 24 hr at 4 °C.

[0078] FIGs. 51A-51D. Electrophoretic mobility shift assay titrating sfGFP-RFTS fusion with asymmetric nucleosomes. FIG. 51 A. Fluorescence visualization of sfGFP-RFTS fusion binding to 185 bp nucleosomes (titrant) with asymmetric H3K9me3 / K18Ub / K23Ub and unmodified H3 (left), asymmetric K18Ub / K23Ub and H3K9me3 (middle), or asymmetric K18Ub / K23Ub and unmodified H3 (right) after 1 hr at 4 °C. FIG. 51B. Ethidium bromide (EtBr) visualization of sfGFP-RFTS fusion binding to 185 bp nucleosomes (titrant) with asymmetric H3K9me3 / K18Ub / K23Ub and unmodified H3 (left), asymmetric K18Ub / K23Ub and H3K9me3 (middle), or asymmetric K18Ub / K23Ub and unmodified H3 (right) after 1 hr at 4 °C. FIG. 51C. Fluorescence visualization of sfGFP-RFTS fusion binding to 185 bp nucleosomes (titrant) with asymmetric H3K9me3 / K18Ub / K23Ub and unmodified H3 (left), asymmetric K18Ub / K23Ub and H3K9me3 (middle), or asymmetric K18Ub / K23Ub and unmodified H3 (right) after 24 hr at 4 °C. FIG. 51D. Ethidium bromide (EtBr) visualization of sfGFP- RFTS fusion binding to 185 bp nucleosomes (titrant) with asymmetric H3K9me3 / K18Ub / K23Ub and unmodified H3 (left), asymmetric K18Ub / K23Ub and H3K9me3 (middle), or asymmetric K18Ub / K23Ub and unmodified H3 (right) after 24 hr at 4 °C.DETAILED DESCRIPTION

[0079] It should be understood that this invention is not limited to the particular methodology, protocols, and reagents, etc., described herein and as such can vary. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the claims.4934-8116-8174.8Atty. Dkt. No. 043214-000103WO PT

[0080] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. All documents, or portions of documents, cited in this application, including, but not limited to, patents, patent applications, articles, books, and treatises, are hereby expressly incorporated by reference in their entirety for any purpose.

[0081] Described herein are compositions and methods that are useful in understanding the patterns and functional interactions of histone tail posttranslational modifications.

[0082] Modified nucleosomes are useful in dissecting the impact of multiple modifications on eraser enzyme processing and molecular recognition. Disclosed herein are engineered polypeptides that display highly favorable properties for introducing scarless H3 tails onto nucleosomes. Additionally, disclosed herein are methods for histone, histone octamer, and nucleosome modification wherein these engineered polypeptides enable and accelerate modification. Moreover, the methods disclosed herein facilitate multiplex “cut-and-paste” middle down proteomics with tandem mass tags. This cut-and-paste proteomics approach permits the quantitative analysis of H3 histone modification crosstalk after treatment with different histone deacetylase inhibitors. These chemoenzymatic tail isolation and modification strategies made possible with the engineered polypeptides disclosed herein are broadly useful to epigenetics discovery and therapeutic development.

[0083] Overcome herein are two significant challenges in the field: limitations on the ready availability of designer nucleosomes and middle-down mass spectrometric analysis of H3 histone modifications. Middle-down mass spectrometric analysis of H3 histone can provide precise information about the interplay between modifications within individual H3 histone tails by evaluating intact H3 protein tails isolated from cellular histones. Herein limitations on middle-down mass spectrometric analysis of H3 histone are addressed through the application of novel engineered polypeptides. Compositions comprising one or more of the engineered polypeptides described herein can be employed to create asymmetric nucleosomes bearing distinct patterns of modifications on the H3 tails. The availability of such asymmetric nucleosomes has allowed new insights into molecular recognition of nucleosomes by eraser and reader proteins. In addition, efficient isolation of H3 peptide tails from crude extracts of endogenous histones with the engineered polypeptides disclosed herein permits concurrent labeling with tandem mass tags. This ‘cut-and-paste’ approach enhances the middle-down proteomics analysis of H3 histone tails.

[0084] In one aspect of any of the embodiments, described herein is an engineered polypeptide comprising a sortase domain comprising the sequence MQAKPQIPKDKSKVAGYIEIPDADIKEPVYPGPATX1EQLNRGVSFAX2X3NX4SLDX5QNIX6IAG HTFIDRPNYQFTNLKAAKXyGSMVYFKVGNETRKYKMTSIRXsVKPXsX X iXBXhXis XI6XI7KGKDKQLTLITCDDYNEXI8TGVWEXI9RKIFVATEVK (SEQ ID NO: 14), wherein X is any amino acids and X1112 is one or two amino acids.4934-8116-8174.8Atty. Dkt. No. 043214-000103WO PT

[0085] In some embodiments of any of the aspects, the sortase domain consists essentially of the sequence of SEQ ID NO: 14. In some embodiments of any of the aspects, the sortase domain consists of the sequence of SEQ ID NO: 14. In some embodiments of any of the aspects, the engineered polypeptide comprises the sortase domain. In some embodiments of any of the aspects, the engineered polypeptide consists essentially of the sortase domain. In some embodiments of any of the aspects, the engineered polypeptide consists of the sortase domain.

[0086] As used herein, “sortase” refers to a group of enzymes that modify proteins by recognizing a carboxyl-terminal sorting signal and either cleaving or ligating the polypeptide comprising the signal. The sorting signal comprises at least one amino acid that is recognized by the enzyme as a cleavage site. Sortases are capable of cleavage as well as ligation through transpeptidation. The structures and activities of sortases, as well as the sequences of the signals that they recognize are known in the art. For example, sortases are described in, e.g., Amacher et al. Trends Biochem Sci 49(7):596-610 (2024); Nuijens et al. Front Chem 7:829 (2019); and Pishesha et al. Annu Rev Cell Dev Biol 34: 163-188 (2018); each of which is incorporated by reference herein in its entirety. Additionally, proteins can be circularized by the action of a sortase enzyme. The use of sortase enzymes to circularize polypeptides is described in more detail in, e.g., Cowper et al. ChemBioChem 2013 14:809-812; Antos et al., Journal of Biological Chemistry 2009284:16028-36; and Tsukiji et al. ChemBioChem 2009 10:787-798; each of which is incorporated by reference herein in its entirety.

[0087] As used herein, “sortase domain” refers to a portion of a polypeptide which exhibits sortase activity. Exemplary sortase domain sequences are described herein.

[0088] In some embodiments of any of the aspects, the sortase domain comprises the sequence of SEQ ID NO: 14. In some embodiments of any of the aspects, the sortase domain consists of the sequence of SEQ ID NO: 14. In some embodiments of any of the aspects, the sortase domain consists essentially of the sequence of SEQ ID NO: 14. In some embodiments of any of the aspects, the sortase domain does not comprise SEQ ID NO: 3, SEQ ID NO: 24, SEQ ID NO: 19, SEQ ID NO: 23, SEQ ID NO: 20, or SEQ ID NO 25. . In some embodiments of any of the aspects, the sortase domain does not consist of SEQ ID NO: 3, SEQ ID NO: 24, SEQ ID NO: 19, SEQ ID NO: 23, SEQ ID NO: 20, or SEQ ID NO 25. In some embodiments of any of the aspects, the sortase domain does not consist essentially of SEQ ID NO: 3, SEQ ID NO: 24, SEQ ID NO: 19, SEQ ID NO: 23, SEQ ID NO: 20, or SEQ ID NO 25.

[0089] As used herein, “recognition domain” or “recognition motif’ refers to the portion of a polypeptide sequence that a cognate sortase recognizes for cleavage or ligation. Cognate recognition domains or motifs for sortases are known in the art. Exemplary recognition domains and motifs are provided herien4934-8116-8174.8Atty. Dkt. No. 043214-000103WO PT

[0090] In some embodiments, the recognition domain comprises residues 29-33 of the polypeptide sequence that a cognate sortase recognizes for cleavage or ligation. In some embodiments, the recognition domain consists of residues 29-33 of the polypeptide sequence that a cognate sortase recognizes for cleavage or ligation. In some embodiments, the recognition domain consists essentially of residues 29-33 of the polypeptide sequence that a cognate sortase recognizes for cleavage or ligation.

[0091] In some embodiments, the recognition domain comprises the sequence APXTG (SEQ ID NO: 51) of the polypeptide sequence that a cognate sortase recognizes for cleavage or ligation. In some embodiments, the recognition domain consists of the sequence APXTG (SEQ ID NO: 51) of the polypeptide sequence that a cognate sortase recognizes for cleavage or ligation. In some embodiments, the recognition domain consists essentially of the sequence APXTG (SEQ ID NO: 51) of the polypeptide sequence that a cognate sortase recognizes for cleavage or ligation. In some embodiments, the recognition domain comprises the sequence APXTG (SEQ ID NO: 51) at residues 29-33 of the polypeptide sequence that a cognate sortase recognizes for cleavage or ligation. In some embodiments, the recognition domain consists of the sequence APXTG (SEQ ID NO: 51) at residues 29-33 of the polypeptide sequence that a cognate sortase recognizes for cleavage or ligation. In some embodiments, the recognition domain consists essentially of the sequence APXTG (SEQ ID NO: 51) at residues 29-33 of the polypeptide sequence that a cognate sortase recognizes for cleavage or ligation. In some embodiments, the recognition domain comprises the sequence APATG (SEQ ID NO:52) of the polypeptide sequence that a cognate sortase recognizes for cleavage or ligation. In some embodiments, the recognition domain consists of the sequence APATG (SEQ ID NO: 52) of the polypeptide sequence that a cognate sortase recognizes for cleavage or ligation. In some embodiments, the recognition domain consists essentially of the sequence APATG (SEQ ID NO: 52) of the polypeptide sequence that a cognate sortase recognizes for cleavage or ligation. In some embodiments, the recognition domain comprises the sequence APATG (SEQ ID NO: 52) at residues 29-33 of the polypeptide sequence that a cognate sortase recognizes for cleavage or ligation. In some embodiments, the recognition domain consists of the sequence APATG (SEQ ID NO: 52) at residues 29-33 of the polypeptide sequence that a cognate sortase recognizes for cleavage or ligation. In some embodiments, the recognition domain consists essentially of the sequence APATG (SEQ ID NO: 52) at residues 29-33 of the polypeptide sequence that a cognate sortase recognizes for cleavage or ligation. In some embodiments, the recognition domain comprises the sequence APSTG (SEQ ID NO:53) of the polypeptide sequence that a cognate sortase recognizes for cleavage or ligation. In some embodiments, the recognition domain consists of the sequence APSTG (SEQ ID NO: 53) of the polypeptide sequence that a cognate sortase recognizes for cleavage or ligation. In some embodiments, the recognition domain consists essentially of the sequence APSTG (SEQ ID NO: 53) of the polypeptide sequence that a cognate sortase recognizes for cleavage or ligation. In some embodiments, the recognition domain comprises the sequence APSTG (SEQ ID NO: 53) at residues4934-8116-8174.8Atty. Dkt. No. 043214-000103WO PT29-33 of the polypeptide sequence that a cognate sortase recognizes for cleavage or ligation. In some embodiments, the recognition domain consists of the sequence APSTG (SEQ ID NO: 53) at residues 29-33 of the polypeptide sequence that a cognate sortase recognizes for cleavage or ligation. In some embodiments, the recognition domain consists essentially of the sequence APSTG (SEQ ID NO: 53) at residues 29-33 of the polypeptide sequence that a cognate sortase recognizes for cleavage or ligation.

[0092] In some embodiments of any of the aspects, Xi of SEQ ID NO: 14 is P, S, or R. In some embodiments of any of the aspects, Xi of SEQ ID NO: 14 is S or R.

[0093] In some embodiments of any of the aspects, X2 of SEQ ID NO: 14 is E, A, D, Q, or N.In some embodiments of any of the aspects, X2 of SEQ ID NO: 14 is A, D, Q, or N.

[0094] In some embodiments of any of the aspects, X3 of SEQ ID NO: 14 is E, A, D, Q, or N.In some embodiments of any of the aspects, X3 of SEQ ID NO: 14 is A, D, Q, or N.

[0095] In some embodiments of any of the aspects, X4 of SEQ ID NO: 14 is E or Q. In some embodiments of any of the aspects, X4 of SEQ ID NO: 14 is Q.

[0096] In some embodiments of any of the aspects, X5 of SEQ ID NO: 14 is D, A, E, Q, or N.In some embodiments of any of the aspects, X5 of SEQ ID NO: 14 is A, E, Q, or N.

[0097] In some embodiments of any of the aspects, X& of SEQ ID NO: 14 is S or V. In some embodiments of any of the aspects, X& of SEQ ID NO: 14 is V.

[0098] In some embodiments of any of the aspects, X7 of SEQ ID NO: 14 is M or K. In some embodiments of any of the aspects, X7 of SEQ ID NO: 14 is M.

[0099] In some embodiments of any of the aspects, Xg of SEQ ID NO: 14 is D or N. In some embodiments of any of the aspects, Xg of SEQ ID NO: 14 is N.

[0100] In some embodiments of any of the aspects, X9 of SEQ ID NO: 14 is Q or T. In some embodiments of any of the aspects, X9 of SEQ ID NO: 14 is T.

[0101] In some embodiments of any of the aspects, X10 of SEQ ID NO: 14 is D or A. In some embodiments of any of the aspects, X10 of SEQ ID NO: 14 is A.

[0102] In some embodiments of any of the aspects, X1112 of SEQ ID NO: 14 is VG, VE or V.In some embodiments of any of the aspects, X1112 of SEQ ID NO: 14 is V.

[0103] In some embodiments of any of the aspects, X13 of SEQ ID NO: 14 is M or V. In some embodiments of any of the aspects, X13 of SEQ ID NO: 14 is M.

[0104] In some embodiments of any of the aspects, X14 of SEQ ID NO: 14 is H or L. In some embodiments of any of the aspects, X14of SEQ ID NO: 14 is H.

[0105] In some embodiments of any of the aspects, X15 of SEQ ID NO: 14 is L or D. In some embodiments of any of the aspects, X15 of SEQ ID NO: 14 is L.

[0106] In some embodiments of any of the aspects, Xi6 of SEQ ID NO: 14 is A or E. In some embodiments of any of the aspects, Xi6 of SEQ ID NO: 14 is A.4934-8116-8174.8Atty. Dkt. No. 043214-000103WO PT

[0107] In some embodiments of any of the aspects, X17 of SEQ ID NO: 14 is Q, E, or R. In some embodiments of any of the aspects, X17 of SEQ ID NO: 14 is E or R. In some embodiments of any of the aspects, X17 of SEQ ID NO: 14 is R.

[0108] In some embodiments of any of the aspects, Xis of SEQ ID NO: 14 is K or E. In some embodiments of any of the aspects, Xis of SEQ ID NO: 14 is E.

[0109] In some embodiments of any of the aspects, X19 of SEQ ID NO: 14 is K or T. In some embodiments of any of the aspects, X19 of SEQ ID NO: 14 is T.

[0110] In some embodiments of any of the aspects, Xs of SEQ ID NO: 14 is N, Xis of SEQ IDNO: 14 is E, and X19of SEQ ID NO: 14 is T.

[0111] In some embodiments of any of the aspects, the engineered polypeptide comprises at least one of: Xi of SEQ ID NO: 14 is S or R; X2of SEQ ID NO: 14 is A, D, Q, or N; X3of SEQ ID NO: 14 is A, D, Q, or N; X5of SEQ ID NO: 14 is A, E, Q, or N; and X17of SEQ ID NO: 14 is E or R.

[0112] In some embodiments of any of the aspects, in SEQ ID NO: 14: Xi is S or R; X214 is A, D, Q, or N; X3is A, D, Q, or N; X5 is A, E, Q, or N; X17 is E or R.

[0113] In some embodiments of any of the aspects, the sortase domain comprises a sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 14. In some embodiments of any of the aspects, the sortase domain comprises a sequence with at least 85% sequence identity to the sequence of SEQ ID NO: 14. In some embodiments of any of the aspects, the sortase domain comprises a sequence with at least 90% sequence identity to the sequence of SEQ ID NO: 14. In some embodiments of any of the aspects, the sortase domain comprises a sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 14. In some embodiments of any of the aspects, the sortase domain comprises a sequence with at least 98% sequence identity to the sequence of SEQ ID NO: 14. In some embodiments of any of the aspects, the sortase domain comprises a sequence with at least 99% sequence identity to the sequence of SEQ ID NO: 14. In some embodiments of any of the aspects, the sortase domain comprises a sequence which retains the sortase activity of SEQ ID NO: 14.

[0114] In some embodiments of any of the aspects, in SEQ ID NO: 14, Xi is P, X2is E, X3is E, X4 is E, X5 is D, Xe is S, X7 is M, Xs is D, X9 is Q, X10 is D, Xm2is VG, Xi3is M, X14 is H, X15 is L, Xi6 is A, X17 is E, Xis is K, and X19 is K (SEQ ID NO: 3). In some embodiments of any of the aspects, the sortase domain comprises SEQ ID NO: 3. In some embodiments of any of the aspects, the sortase domain consists essentially of SEQ ID NO: 3. In some embodiments of any of the aspects, the sortase domain consists of SEQ ID NO: 3.

[0115] In some embodiments of any of the aspects, the sortase domain comprises a sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 3. In some embodiments of any of the aspects, the sortase domain comprises a sequence with at least 85% sequence identity to the sequence of SEQ ID NO: 3. In some embodiments of any of the aspects, the sortase domain comprises a sequence with at least 90% sequence identity to the sequence of SEQ ID NO: 3. In some embodiments 4934-8116-8174.8Atty. Dkt. No. 043214-000103WO PT of any of the aspects, the sortase domain comprises a sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 3. In some embodiments of any of the aspects, the sortase domain comprises a sequence with at least 98% sequence identity to the sequence of SEQ ID NO: 3. In some embodiments of any of the aspects, the sortase domain comprises a sequence with at least 99% sequence identity to the sequence of SEQ ID NO: 3. In some embodiments of any of the aspects, the sortase domain comprises a sequence which retains the sortase activity of SEQ ID NO: 3.

[0116] In some embodiments of any of the aspects, in SEQ ID NO: 14, Xi is S, X2 is E, X3 is E, X4 is E, X5 is D, Xe is S, X7 is M, Xg is N, X9 is T, X10 is A, X1112 is V, X13 is M, X14 is H, X15 is L, Xi6 is A, X17 is E, Xis is K, and X19 is K (SEQ ID NO: 4). In some embodiments of any of the aspects, the sortase domain comprises SEQ ID NO: 4. In some embodiments of any of the aspects, the sortase domain consists essentially of SEQ ID NO: 4. In some embodiments of any of the aspects, the sortase domain consists of SEQ ID NO: 4.

[0117] In some embodiments of any of the aspects, the sortase domain comprises a sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 4. In some embodiments of any of the aspects, the sortase domain comprises a sequence with at least 85% sequence identity to the sequence of SEQ ID NO: 4. In some embodiments of any of the aspects, the sortase domain comprises a sequence with at least 90% sequence identity to the sequence of SEQ ID NO: 4. In some embodiments of any of the aspects, the sortase domain comprises a sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 4. In some embodiments of any of the aspects, the sortase domain comprises a sequence with at least 98% sequence identity to the sequence of SEQ ID NO: 4. In some embodiments of any of the aspects, the sortase domain comprises a sequence with at least 99% sequence identity to the sequence of SEQ ID NO: 4. In some embodiments of any of the aspects, the sortase domain comprises a sequence which retains the sortase activity of SEQ ID NO: 4.

[0118] In some embodiments of any of the aspects, in SEQ ID NO: 14, Xi is P, X2 is E, X3 is E, X4 is E, X5 is D, Xe is S, X7 is M, Xg is N, X9 is Q, X10 is D, X1112 is V, X13 is M, Xu is H, X15 is L, Xu is A, X17 is E, Xis is E, and X19 is T (SEQ ID NO: 5). In some embodiments of any of the aspects, the sortase domain comprises SEQ ID NO: 5. In some embodiments of any of the aspects, the sortase domain consists essentially of SEQ ID NO: 5. In some embodiments of any of the aspects, the sortase domain consists of SEQ ID NO: 5.

[0119] In some embodiments of any of the aspects, the sortase domain comprises a sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 5. In some embodiments of any of the aspects, the sortase domain comprises a sequence with at least 85% sequence identity to the sequence of SEQ ID NO: 5. In some embodiments of any of the aspects, the sortase domain comprises a sequence with at least 90% sequence identity to the sequence of SEQ ID NO: 5. In some embodiments of any of the aspects, the sortase domain comprises a sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 5. In some embodiments of any of the aspects, the sortase domain4934-8116-8174.8Atty. Dkt. No. 043214-000103WO PT comprises a sequence with at least 98% sequence identity to the sequence of SEQ ID NO: 5. In some embodiments of any of the aspects, the sortase domain comprises a sequence with at least 99% sequence identity to the sequence of SEQ ID NO: 5. In some embodiments of any of the aspects, the sortase domain comprises a sequence which retains the sortase activity of SEQ ID NO: 5.

[0120] In some embodiments of any of the aspects, in SEQ ID NO: 14, Xi is S, X2 is E, X3 is E, X4 is E, X5 is D, Xe is S, X7 is M, Xg is N, X9 is Q, X10 is D, X1112 is V, X13 is M, X14 is H, X15 is L, Xi6 is A, X17 is E, Xis is K, and X19 is T (SEQ ID NO: 6). In some embodiments of any of the aspects, the sortase domain comprises SEQ ID NO: 6. In some embodiments of any of the aspects, the sortase domain consists essentially of SEQ ID NO: 6. In some embodiments of any of the aspects, the sortase domain consists of SEQ ID NO: 6.1n some embodiments of any of the aspects, the sortase domain comprises a sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 6. In some embodiments of any of the aspects, the sortase domain comprises a sequence with at least 85% sequence identity to the sequence of SEQ ID NO: 6. In some embodiments of any of the aspects, the sortase domain comprises a sequence with at least 90% sequence identity to the sequence of SEQ ID NO: 6. In some embodiments of any of the aspects, the sortase domain comprises a sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 6. In some embodiments of any of the aspects, the sortase domain comprises a sequence with at least 98% sequence identity to the sequence of SEQ ID NO: 6. In some embodiments of any of the aspects, the sortase domain comprises a sequence with at least 99% sequence identity to the sequence of SEQ ID NO: 6. In some embodiments of any of the aspects, the sortase domain comprises a sequence which retains the sortase activity of SEQ ID NO: 6.

[0121] In some embodiments of any of the aspects, in SEQ ID NO: 14, Xi is S, X2 is E, X3 is E, X4 is E, X5 is D, Xe is S, X7 is M, Xg is N, X9 is Q, X10 is D, X1112 is V, X13 is M, Xu is H, X15 is L, Xu is A, X17 is E, Xis is E, and X19 is T (SEQ ID NO: 7). In some embodiments of any of the aspects, the sortase domain comprises SEQ ID NO: 7. In some embodiments of any of the aspects, the sortase domain consists essentially of SEQ ID NO: 7. In some embodiments of any of the aspects, the sortase domain consists of SEQ ID NO: 7.

[0122] In some embodiments of any of the aspects, the sortase domain comprises a sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 7. In some embodiments of any of the aspects, the sortase domain comprises a sequence with at least 85% sequence identity to the sequence of SEQ ID NO: 7. In some embodiments of any of the aspects, the sortase domain comprises a sequence with at least 90% sequence identity to the sequence of SEQ ID NO: 7. In some embodiments of any of the aspects, the sortase domain comprises a sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 7. In some embodiments of any of the aspects, the sortase domain comprises a sequence with at least 98% sequence identity to the sequence of SEQ ID NO: 7. In some embodiments of any of the aspects, the sortase domain comprises a sequence with at least 99% sequence4934-8116-8174.8Atty. Dkt. No. 043214-000103WO PT identity to the sequence of SEQ ID NO: 7. In some embodiments of any of the aspects, the sortase domain comprises a sequence which retains the sortase activity of SEQ ID NO: 7.

[0123] In some embodiments of any of the aspects, in SEQ ID NO: 14, Xi is S, X2 is E, X3 is E, X4 is E, X5 is D, Xe is E, X7 is S, Xg is N, X9 is T, X10 is A, X1112 is V, X13 is M, Xu is H, X15 is L, Xu is A, X17 is E, Xu is K, and X19 is T (SEQ ID NO: 8). In some embodiments of any of the aspects, the sortase domain comprises SEQ ID NO: 8. In some embodiments of any of the aspects, the sortase domain consists essentially of SEQ ID NO: 8. In some embodiments of any of the aspects, the sortase domain consists of SEQ ID NO: 8.

[0124] In some embodiments of any of the aspects, the sortase domain comprises a sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 8. In some embodiments of any of the aspects, the sortase domain comprises a sequence with at least 85% sequence identity to the sequence of SEQ ID NO: 8. In some embodiments of any of the aspects, the sortase domain comprises a sequence with at least 90% sequence identity to the sequence of SEQ ID NO: 8. In some embodiments of any of the aspects, the sortase domain comprises a sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 8. In some embodiments of any of the aspects, the sortase domain comprises a sequence with at least 98% sequence identity to the sequence of SEQ ID NO: 8. In some embodiments of any of the aspects, the sortase domain comprises a sequence with at least 99% sequence identity to the sequence of SEQ ID NO: 8. In some embodiments of any of the aspects, the sortase domain comprises a sequence which retains the sortase activity of SEQ ID NO: 8.

[0125] In some embodiments of any of the aspects, in SEQ ID NO: 14, Xi is S, X2 is E, X3 is E, X4 is E, X5 is D, Xe is S, X7 is M, Xg is N, X9 is Q, X10 is A, X1112 is V , X13 is M, X is H, X15 is L, Xu is A, X17 is E, Xis is E, and X19 is T (SEQ ID NO: 9). In some embodiments of any of the aspects, the sortase domain comprises SEQ ID NO: 9. In some embodiments of any of the aspects, the sortase domain consists essentially of SEQ ID NO: 9. In some embodiments of any of the aspects, the sortase domain consists of SEQ ID NO: 9.

[0126] In some embodiments of any of the aspects, the sortase domain comprises a sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 9. In some embodiments of any of the aspects, the sortase domain comprises a sequence with at least 85% sequence identity to the sequence of SEQ ID NO: 9. In some embodiments of any of the aspects, the sortase domain comprises a sequence with at least 90% sequence identity to the sequence of SEQ ID NO: 9. In some embodiments of any of the aspects, the sortase domain comprises a sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 9. In some embodiments of any of the aspects, the sortase domain comprises a sequence with at least 98% sequence identity to the sequence of SEQ ID NO: 9. In some embodiments of any of the aspects, the sortase domain comprises a sequence with at least 99% sequence identity to the sequence of SEQ ID NO: 9. In some embodiments of any of the aspects, the sortase domain comprises a sequence which retains the sortase activity of SEQ ID NO: 9.4934-8116-8174.8Atty. Dkt. No. 043214-000103WO PT

[0127] In some embodiments of any of the aspects, in SEQ ID NO: 14, Xi is R, X2 is E, X3 is E, X4 is E, X5 is D, Xe is S, X7 is M, Xg is N, X9 is Q, X10 is ,D X1112 is V, X13 is M, Xu is H, X15 is L, Xu is A, X17 is E, Xis is E, and X19 is T (SEQ ID NO: 10). In some embodiments of any of the aspects, the sortase domain comprises SEQ ID NO: 10. In some embodiments of any of the aspects, the sortase domain consists essentially of SEQ ID NO: 10. In some embodiments of any of the aspects, the sortase domain consists of SEQ ID NO: 10.

[0128] In some embodiments of any of the aspects, the sortase domain comprises a sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 10. In some embodiments of any of the aspects, the sortase domain comprises a sequence with at least 85% sequence identity to the sequence of SEQ ID NO: 10. In some embodiments of any of the aspects, the sortase domain comprises a sequence with at least 90% sequence identity to the sequence of SEQ ID NO: 10. In some embodiments of any of the aspects, the sortase domain comprises a sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 10. In some embodiments of any of the aspects, the sortase domain comprises a sequence with at least 98% sequence identity to the sequence of SEQ ID NO: 10. In some embodiments of any of the aspects, the sortase domain comprises a sequence with at least 99% sequence identity to the sequence of SEQ ID NO: 10. In some embodiments of any of the aspects, the sortase domain comprises a sequence which retains the sortase activity of SEQ ID NO: 10.

[0129] In some embodiments of any of the aspects, in SEQ ID NO: 14, Xi is R, X2 is E, X3 is E, X4 is E, X5 is D, Xe is S, X7 is M, Xg is N, X9 is Q, X10 is A, X1112 is V, X13 is M, X is H, X15 is L, Xu is A, X17 is E, Xis is K, and X19 is T (SEQ ID NO: 11). In some embodiments of any of the aspects, the sortase domain comprises SEQ ID NO: 11. In some embodiments of any of the aspects, the sortase domain consists essentially of SEQ ID NO: 11. In some embodiments of any of the aspects, the sortase domain consists of SEQ ID NO: 11.

[0130] In some embodiments of any of the aspects, the sortase domain comprises a sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 11. In some embodiments of any of the aspects, the sortase domain comprises a sequence with at least 85% sequence identity to the sequence of SEQ ID NO: 11. In some embodiments of any of the aspects, the sortase domain comprises a sequence with at least 90% sequence identity to the sequence of SEQ ID NO: 11. In some embodiments of any of the aspects, the sortase domain comprises a sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 11. In some embodiments of any of the aspects, the sortase domain comprises a sequence with at least 98% sequence identity to the sequence of SEQ ID NO: 11. In some embodiments of any of the aspects, the sortase domain comprises a sequence with at least 99% sequence identity to the sequence of SEQ ID NO: 11. In some embodiments of any of the aspects, the sortase domain comprises a sequence which retains the sortase activity of SEQ ID NO: 11.

[0131] In some embodiments of any of the aspects, in SEQ ID NO: 14, Xi is S, X2 is E, X3 is E, X4 is Q, X5 is D, Xe is V, X7 is M, Xg is N, X9 is Q, X10 is D, X1112 is V, X13 is M, Xu is H, X15 is L, 4934-8116-8174.8Atty. Dkt. No. 043214-000103WO PTXi6 is A, X17 is E, Xis is E, and X19 is T (SEQ ID NO: 12). In some embodiments of any of the aspects, the sortase domain comprises SEQ ID NO: 12. In some embodiments of any of the aspects, the sortase domain consists essentially of SEQ ID NO: 12. In some embodiments of any of the aspects, the sortase domain consists of SEQ ID NO: 12.

[0132] In some embodiments of any of the aspects, the sortase domain comprises a sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 12. In some embodiments of any of the aspects, the sortase domain comprises a sequence with at least 85% sequence identity to the sequence of SEQ ID NO: 12. In some embodiments of any of the aspects, the sortase domain comprises a sequence with at least 90% sequence identity to the sequence of SEQ ID NO: 12. In some embodiments of any of the aspects, the sortase domain comprises a sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 12. In some embodiments of any of the aspects, the sortase domain comprises a sequence with at least 98% sequence identity to the sequence of SEQ ID NO: 12. In some embodiments of any of the aspects, the sortase domain comprises a sequence with at least 99% sequence identity to the sequence of SEQ ID NO: 12. In some embodiments of any of the aspects, the sortase domain comprises a sequence which retains the sortase activity of SEQ ID NO: 12.

[0133] In some embodiments of any of the aspects, the engineered polypeptide does not comprise SEQ ID NO: 3, SEQ ID NO: 24, SEQ ID NO: 19, SEQ ID NO: 23, SEQ ID NO: 20, or SEQ ID NO 25.

[0134] In some embodiments of any of the aspects, the engineered polypeptide further comprises SEQ ID NO: 36. In some embodiments of any of the aspects, the engineered polypeptide further comprises SEQ ID NO: 36 at the C-terminus of the engineered polypeptide. In some embodiments of any of the aspects, the engineered polypeptide further comprises SEQ ID NO: 36 at the C-terminal of the sortase domain.

[0135] In some embodiments of any of the aspects, the engineered polypeptide further comprises at least one split intein sequence. In some embodiments of any of the aspects, the engineered polypeptide further comprises a pair of split intein sequences.

[0136] As used herein, “intein” refers to one or more sequences, which when present in a polypeptide mediates self-cleaving and self-ligating of the polypeptide. The collective attributes of being both self-cleaving and self ligating are referred to as “self-splicing” or “splicing.” An intein is cleaved from the polypeptide and mediates ligation of the non-intein polypeptide sequences (exteins) to form a splice version of the polypeptide which does not include the intein sequence. Where two separate intein sequences act together to accomplish the splicing, they are referred to as “split inteins.” Various inteins and their use are described in more detail in, e.g. Elleuche et al. Applied Microbiology and Biotechnology. 2010 87:479-489; Cowper et al. ChemBioChem 2013 14:809-812; Ahlmann-Eltze et al. 2015 hdl.handle.net / 1721.1 / 96071; Evans et al. Journal of Biological Chemistry 1999274:18359-4934-8116-8174.8Atty. Dkt. No. 043214-000103WO PT18363; and Evans et al. Biopoly 1999 51: 333-342; each of which is incorporated by reference herein in its entirety.

[0137] An intein may be inserted internally to the polypeptide sequence or fused terminally to the polypeptide. A split intein can flank both ends of a polypeptide, i.e., one portion of the split intein is at the N-terminus and one portion of the split intein is at the C-terminus. An intein insertion in a polypeptide may allow control of a polypetpide by yielding a protein that has one activity when the intein is present and another activity after intein cleavage or splicing. In some cases, the intein splicing reaction can be controlled by one or more of a variety of induction conditions.

[0138] Intein insertion sites can be determined experimentally. To determine if an insertion site will permit intein splicing, the intein-protein fusion gene can be constructed and cloned using known methods in the art, the intein-modified protein can be expressed, and the intein-modified protein tested for its ability to splice either spontaneously or under induction conditions.

[0139] The use of two intein domains can permit protein circularization (e.g., using the pTWIN vectors commercially available from New England Biolabs; Ipswich, Mass, (e.g., Cat. No. N6951S)). When inteins are used to flank a central sequence, the central sequence can be circularized when the inteins excise themselves.

[0140] In some embodiments of any of the aspects, the split intein sequences flank the sortase domain. In some embodiments of any of the aspects, the engineered polypeptide further comprises linker domains between each of the pair of split intein sequences and the sortase domain. In some embodiments of any of the aspects, the engineered polypeptide further comprises, from N-terminus to C-terminus, an N-terminus split intein comprising the sequence of SEQ ID NO: 15, an N-terminus linker comprising the sequence of SEQ ID NO: 16, the sortase domain, a C-terminus linker comprising the sequence of SEQ ID NO: 17, and a C-terminus split intein comprising the sequence of SEQ ID NO: 18.

[0141] In some embodiments of any of the aspects, the intein sequence comprises SEQ ID NO: 15. In some embodiments of any of the aspects, the intein sequence consists of SEQ ID NO: 15. In some embodiments of any of the aspects, the intein sequence consists essentially of SEQ ID NO: 15. In some embodiments of any of the aspects, the intein sequence comprises SEQ ID NO: 18. In some embodiments of any of the aspects, the intein sequence consists of SEQ ID NO: 18. In some embodiments of any of the aspects, the intein sequence consists essentially of SEQ ID NO: 18.

[0142] In some embodiments of any of the aspects, the N-terminus split intein sequence comprises SEQ ID NO: 15. In some embodiments of any of the aspects, the N-terminus split intein sequence consists of SEQ ID NO: 15. In some embodiments of any of the aspects, the N-terminus split intein sequence consists essentially of SEQ ID NO: 15. In some embodiments of any of the aspects, the C-terminus split intein sequence comprises SEQ ID NO: 18. In some embodiments of any of the aspects,4934-8116-8174.8Atty. Dkt. No. 043214-000103WO PT the C-terminus split intern sequence consists of SEQ ID NO: 18. In some embodiments of any of the aspects, the C-terminus split intein sequence consists essentially of SEQ ID NO: 18.

[0143] In some embodiments of any of the aspects, the engineered polypeptide is circularized. As used herein, “circularized” when used in reference to a polypeptide refers to the fact that the peptide sequence is not linear in nature, e.g., it does not have an N-terminus or C-terminus. A polypeptide which is circularized can form any shape, e.g., a circle, an oval, or a polygon. In some embodiments of any of the aspects, the loop is formed through direct amide bonding between the N-terminus and C-terminus of the polypeptide. In some embodiments of any of the aspects, the loop is formed through bonding involving amino acid side chains.

[0144] Intein sequences can be separated from a sortase domain by a linker. As used herein, a linker domain is defined as a sequence of amino acids within a protein that connects distinct protein domains. In some embodiments of any of the aspects, a linker can be located C-terminal or N-terminal of the of the sortase domain, or the linker can be internal in the polypeptide. In some embodiments of any of the aspects, e.g., for a polypeptide that will be circularized, a linker can be located at the C- terminal or N-terminal of the engineered polypeptide before circularization.

[0145] In some embodiments of any of the aspects, the linker domain comprises one or more cysteine, phenylalanine, asparagine, glycine, serine, histidine, alanine, glutamate, or tyrosine residues. In some embodiments of any of the aspects, the linker comprises the sequence CFN. In some embodiments of any of the aspects, the linker comprises the sequence AEY. In some embodiments of any of the aspects, the linker comprises sequences CFN and AEY. In some embodiments of any of the aspects, the linker comprises 6 amino acids. In some embodiments of any of the aspects, the linker comprises the sequence CFN and further comprises 6 amino acids. In some embodiments of any of the aspects, the linker comprises the sequence AEY and further comprises 6 amino acids. In some embodiments of any of the aspects, the linker comprises the sequence CFN and AEY and is 6 amino acids. In some embodiments of any of the aspects, the linker comprises CFNGGSS (SEQ ID NO: 16). In some embodiments of any of the aspects, the linker comprises HHHHHHAEY (SEQ ID NO: 49). In some embodiments of any of the aspects, the linker comprises SEQ ID NOs. 16 and 49. In some embodiments of any of the aspects, the linker comprises SEQ ID NO: 16 and further comprises up to 16 amino acids. In some embodiments of any of the aspects, the linker comprises SEQ ID NO: 49 and further comprises up to 16 amino acids. In some embodiments of any of the aspects, the linker comprises SEQ ID NOs: 16 and 49 and further comprises up to 16 amino acids. In some embodiments of any of the aspects, the linker comprises 3-20 amino acids. In some embodiments of any of the aspects, the linker comprises 14-18 amino acids. In some embodiments of any of the aspects, the linker comprises 3-7 amino acids. In some embodiments of any of the aspects, the linker comprises 3, 4, 5, 6, or 7 amino acids.4934-8116-8174.8Atty. Dkt. No. 043214-000103WO PT

[0146] Illustrative examples of linkers include glycine polymers (G)n; glycine-serine polymers (61-581.5)11 (SEQ ID NO: 37), where n is an integer of at least one, two, three, four, or five; glycinealanine polymers; alanine-serine polymers; and other flexible linkers known in the art. Glycine and glycine-serine polymers are relatively unstructured, and therefore may be able to serve as a neutral tether between domains of the proteins described herein. Glycine accesses significantly more phi-psi space than even alanine, and is much less restricted than residues with longer side chains (see Scheraga, Rev. Computational Chem. 11173-142 (1992)). The ordinarily skilled artisan will recognize that design of a protein in particular embodiments can include linkers that are all or partially flexible, such that the linker can include a flexible linker as well as one or more portions that confer less flexible structure to provide for a desired circularized structure.

[0147] Other exemplary linkers include, but are not limited to the following amino acid sequences: GGG; DGGGS (SEQ ID NO: 54); TGEKP (SEQ ID NO: 55) (see, e.g., Liu et al., PNAS 5525-5530 (1997)); GGRR (SEQ ID NO: 56) (Pomerantz et al. 1995, supra); (GGGGS)nwherein=l, 2, 3, 4 or 5 (SEQ ID NO: 38) (Kim et al., PNAS 93, 1156-1160 (1996); EGKSSGSGSESKVD (SEQ ID NO: 39) (Chaudhary et al., 1990, Proc. Natl. Acad. Sci. U.S.A. 87:1066-1070); KESGSVSSEQLAQFRSLD (SEQ ID NO: 40) (Bird et al., 1988, Science 242:423-426), GGRRGGGS (SEQ ID NO: 41); LRQRDGERP (SEQ ID NO: 42); LRQKDGGGSERP (SEQ ID NO: 43); LRQKD(GGGS)2 ERP (SEQ ID NO: 44). Alternatively, flexible linkers can be rationally designed using a computer program capable of modeling both DNA-binding sites and the peptides themselves (Desjarlais & Berg, PNAS 90:2256-2260 (1993), PNAS 91:11099-11103 (1994) or by phage display methods. In one embodiment, the linker comprises the following amino acid sequence: GSTSGSGKPGSGEGSTKG (SEQ ID NO: 45) (Cooper et al., Blood, 101(4): 1637-1644 (2003)).

[0148] In some embodiments of any of the aspects, the linker comprises SEQ ID NO: 16. In some embodiments of any of the aspects, the linker consists of SEQ ID NO: 16. In some embodiments of any of the aspects, the linker consists essentially of SEQ ID NO: 16. In some embodiments of any of the aspects, the linker comprises SEQ ID NO: 17. In some embodiments of any of the aspects, the linker consists of SEQ ID NO: 17. In some embodiments of any of the aspects, the linker consists essentially of SEQ ID NO: 17. In some embodiments of any of the aspects, the N-terminus linker comprises SEQ ID NO: 16. In some embodiments of any of the aspects, the N-terminus linker consists of SEQ ID NO: 16. In some embodiments of any of the aspects, the N-terminus linker consists essentially of SEQ ID NO: 16. In some embodiments of any of the aspects, the C-terminus linker comprises SEQ ID NO: 17. In some embodiments of any of the aspects, the C-terminus linker consists of SEQ ID NO: 17. In some embodiments of any of the aspects, the C-terminus linker consists essentially of SEQ ID NO: 17.

[0149] One aspect of any of the embodiments is a method of producing a circularized engineered polypeptide comprising a sortase domain, the method comprising: expressing an engineered4934-8116-8174.8Atty. Dkt. No. 043214-000103WO PT polypeptide described herein, thereby excising the intein sequences and producing the circularized engineered polypeptide comprising the sortase domain.

[0150] In some embodiments of any of the aspects, the circularized engineered polypeptide is produced through peptide bond formation between the N-terminus of a N-terminus linker and a C- terminus of the C-terminus linker.

[0151] A histone is a protein that plays a role in organizing, condensing, and regulating access to DNA. Histone proteins make up the core of chromatin, the complex of DNA and proteins found in the nucleus of eukaryotic cells. Histones comprise a globular domain or “histone fold” and a flexible tail. These small, positively charged proteins assemble via octamer assembly to form a histone octamer, which comprises two copies of each of the four core histones - H2A, H2B, H3 and H4. DNA assembles with the histone octamer by wrapping around this octamer to form a nucleosome, the repeating unit of chromatin. The sequences, structure, and function of histones are known in the art, for example, see Torrez-Perez et al., Trends in Pharmacological Sciences, 42(11): 897-911 (2021), Voss et al. Trend Genet 41:506-521 (2025), ; the contents of which are incorporated herein in its entirety.

[0152] As used herein, “histone tail” refers to the flexible region of a histone which protrudes from the globular core, e.g., N-terminal or C-terminal domains which do not form alpha helices. The histone tail may be intact, e.g., attached to the globular core of the histone, or isolated, e.g., cleaved from the globular core of the histone. The sequences and structure of histone tails are known in the art, see e.g, Iwasaki et al. FEBS Open Bio 3:363-369 (2013); the contents of which are incorporated by reference herein in their entirety.

[0153] In some embodiments of any of the aspects, a histone H3 tail is the N-terminal histone H3 tail. In some embodiments of any of the aspects, a histone tail comprises SEQ ID NO: 1. In some embodiments of any of the aspects, a histone tail consists essentially of SEQ ID NO: 1. In some embodiments of any of the aspects, a histone tail consists of SEQ ID NO: 1.

[0154] In some embodiments of any of the aspects, a histone H3 tail is the N-terminal histone H3 tail. In some embodiments of any of the aspects, a histone tail comprises SEQ ID NO: 2. In some embodiments of any of the aspects, a histone tail consists essentially of SEQ ID NO: 2. In some embodiments of any of the aspects, a histone tail consists of SEQ ID NO: 2.

[0155] In some embodiments of any of the aspects, a histone H3 tail is the N-terminal histone H3 tail. In some embodiments of any of the aspects, a histone tail comprises SEQ ID NO: 46. In some embodiments of any of the aspects, a histone tail consists essentially of SEQ ID NO: 46. In some embodiments of any of the aspects, a histone tail consists of SEQ ID NO: 46.

[0156] In some embodiments of any of the aspects, a histone tail comprises SEQ ID NO: 47. In some embodiments of any of the aspects, a histone tail consists essentially of SEQ ID NO: 47. In some embodiments of any of the aspects, a histone tail consists of SEQ ID NO: 47.4934-8116-8174.8Atty. Dkt. No. 043214-000103WO PT

[0157] In some embodiments of any of the aspects, a histone tail comprises SEQ ID NO: 48. In some embodiments of any of the aspects, a histone tail consists essentially of SEQ ID NO: 48. In some embodiments of any of the aspects, a histone tail consists of SEQ ID NO: 48.

[0158] A tailed histone is a histone polypeptide comprising the globular or histone fold domain and at least one histone tail domain. As used herein, “tailed H3 histone” refers to the H3 histone comprising a globular core with an intact protruding N-terminal tail.

[0159] In some embodiments, part or all of the tail of a tailed H3 histone is cleaved by the engineered polypeptide to form a “tailless H3 histone”. A tailless histone is a histone polypeptide i) comprising the globular or histone fold domain and ii) not comprising at least one naturally-occurring histone tail or comprising only a portion of at least one complete, naturally-occurring histone tail domain. A “tailless H3 histone” refers to an H3 histone without an intact N-terminal tail.

[0160] In some embodiments of any of the aspects, a tailless H3 histone does not comprise an N-terminal histone tail. In some embodiments of any of the aspects, a tailless H3 histone does not comprise the sequence of SEQ ID NO: 1. In some embodiments of any of the aspects, a tailless H3 histone does not comprise the sequence of SEQ ID NO: 2. In some embodiments of any of the aspects, a tailless H3 histone does not comprise the sequence of SEQ ID NO: 46. In some embodiments of any of the aspects, a tailless H3 histone does not comprise the sequence of SEQ ID NO: 47. In some embodiments of any of the aspects, a tailless H3 histone does not comprise the sequence of SEQ ID NO: 48.

[0161] A cleaved histone tail peptide is a histone tail which is not covalently attached to a globular or histone fold domain. In some embodiments of any of the aspects, a cleaved histone tail peptide comprises a N-terminal histone tail. In some embodiments of any of the aspects, a cleaved histone tail peptide comprises a N-terminal H3 histone tail. In some embodiments of any of the aspects, a cleaved histone tail peptide consists essentially of an N-terminal H3 histone tail. In some embodiments of any of the aspects, a cleaved histone tail peptide consists of an N-terminal H3 histone tail.

[0162] In some embodiments of any of the aspects, a tailless H3 histone comprises the sequence of SEQ ID NO: 1. In some embodiments of any of the aspects, a tailless H3 histone comprises the sequence of SEQ ID NO: 2. In some embodiments of any of the aspects, a tailless H3 histone comprises the sequence of SEQ ID NO: 46. In some embodiments of any of the aspects, a tailless H3 histone consists essentially of the sequence of SEQ ID NO: 47. In some embodiments of any of the aspects, a tailless H3 histone consists of the sequence of SEQ ID NO: 48.

[0163] In some embodiments of any of the aspects, a tailless H3 histone comprises the sequence of SEQ ID NO: 1. In some embodiments of any of the aspects, a tailless H3 histone comprises the sequence of SEQ ID NO: 2. In some embodiments of any of the aspects, a tailless H3 histone comprises the sequence of SEQ ID NO: 46. In some embodiments of any of the aspects, a tailless H3 4934-8116-8174.8Atty. Dkt. No. 043214-000103WO PT histone consists essentially of the sequence of SEQ ID NO: 47. In some embodiments of any of the aspects, a tailless H3 histone consists of the sequence of SEQ ID NO: 48.

[0164] In some embodiments of any of the aspects, a tailless H3 histone comprises the sequence of SEQ ID NO: 1. In some embodiments of any of the aspects, a tailless H3 histone comprises the sequence of SEQ ID NO: 2. In some embodiments of any of the aspects, a tailless H3 histone comprises the sequence of SEQ ID NO: 46. In some embodiments of any of the aspects, a tailless H3 histone consists essentially of the sequence of SEQ ID NO: 47. In some embodiments of any of the aspects, a tailless H3 histone consists of the sequence of SEQ ID NO: 48.

[0165] Histones can undergo post-translational modification (PTM), a process by which chemical changes occur to proteins after their synthesis. These PTMs can play important roles in epigenetics, or the changes in gene expression or cellular phenotype that do not arise from alterations of the DNA sequence itself. As used herein, the term “post-translational modification” or “PTM” refers to a reaction wherein a chemical moiety is covalently added to or non-covalently binds to protein, e.g., a histone protein.

[0166] Many proteins can be post-translationally modified through the covalent addition or transient non-covalent binding of a chemical after the initial synthesis (i.e., translation) of the polypeptide chain. Such chemical moieties usually are added by an enzyme to an amino acid side chain or to the carboxyl or amino terminal end of the polypeptide chain and may be cleaved off by another enzyme. Single or multiple chemical moieties, either the same or different chemical moieties, can be added to or bound to a single protein molecule. PTM of a protein can alter its biological function, such as its enzyme activity, its binding to or activation of other proteins, or its turnover, and is important in cell signaling events, development of an organism, and disease. Examples of PTM covered by the methods of the invention described herein include, but are not limited to, ubiquitination, phosphorylation, sumoylation, neddylation, ADP-ribosylation, glycosylation, acetylation, S- nitrosylation or nitrosylation, citrullination or deimination, the addition of OClcNAc, methylation, hydroxylation, fattenylation, ufinylation, prenylation, myristoylation, S-palmitoylation, tyrosine sulfation, formylation, and carboxylation. In some embodiments, a PTM can include both a covalent addition and non-covalent binding of a chemical moiety to a protein. Similarly, a PTM can involve removal of a covalently conjugated or a non-covalently bound chemical moiety. Examples of PTM removal of a covalently conjugated or non-covalently bound chemical moiety covered by the methods of the invention described herein include, but are not limited to, deubiquitination (DUB), dephosphorylation, deglycosylation, desumoylation, deacetylation, deS-nitrosylation, denitrosylation, decitrullination or dedeimination, deneddylation, de-ADP-ribosylation, removal of OClcNAc, demethylation, de-hydroxylation, defattenylation, deufinylation, deprenylation, demyristoylation, de- S-palmitoylation, tyrosine desulfation, deformylation, decarboxylation, and deamidation.4934-8116-8174.8Atty. Dkt. No. 043214-000103WO PT

[0167] As used herein, “ubiquitination” or “ubiquitylation” refers to the post- translational modification of a protein by the covalent attachment (via an isopeptide bond) of one or more ubiquitin monomers. The ubiquitylation cascade is started by the El enzyme. As used herein, removal of one or more ubiquitin molecules is known as “deubiquitination.”

[0168] As used herein, “phosphorylation” refers to the addition of a phosphate (PO4) group to a protein or other organic molecule. As used herein, “dephosphorylation” refers to the removal of a phosphate group from a protein or other organic molecule.

[0169] As used herein, “sumoylation” refers to the process whereby Small Ubiquitin-like Modifier or “SUMO” proteins are covalently attached to other proteins in cells to modify their function. SUMO proteins are similar to ubiquitin, and SUMOylation is directed by an enzymatic cascade analogous to that involved in ubiquitination. As defined herein, “desumoylation” refers to the process whereby SUMO proteins are removed from proteins in cells.

[0170] As used herein, “neddylation” refers to the process by which the ubiquitin-like protein Nedd8 is conjugated to its target proteins. This process is analogous to ubiquitination, although it relies on its own El and E2 enzymes. As used herein, “deneddylation” refers to the process by which the ubiquitin-like protein Nedd8 is unconjugated from its target proteins.

[0171] As used herein, “ADP-ribosylation” refers to the PTM of proteins that involves the addition of one or more ADP and ribose moieties. As used herein, “de-ADP-ribosylation” refers to the removal of one or more ADP and ribose moieties.

[0172] As defined herein, “glycosylation” refers to the enzymatic process that links saccharides to produce glycans, attached to proteins, lipids, or other organic molecules. For the methods described herein, glycosylation includes N-linked glycosylation, O-linked glycosylation (O — N- acetylgalactosamine (O-GalNAc), O-fucose, O-glucose, O — N-acetylglucosamine (O-GlcNAc), O — N-acetylglucosamine, O-mannose, Collagen Glycosylation, Hydroxyproline Glycosylation, Glycosylation of Glycogenin, Glycosylation of Ceramide, Proteoglycans), phospho-Serine Glycosylation and C-mannosylation. As defined herein, “deglycosylation” refers to the enzymatic process that removes saccharides attached to proteins, lipids, or other organic molecules.

[0173] As used herein, “acetylation” (or in IUPAC nomenclature “ethanoylation”) refers to the reaction that introduces an acetyl functional group into a chemical compound, and includes N-alpha- terminal acetylation and lysine acetylation. As used herein, “deacetylation” (or in IUPAC nomenclature “de-ethanoylation”) refers to the reaction that removes an acetyl functional group from a chemical compound.

[0174] As defined herein, “S-nitrosylation” or “nitrosylation” refer to the addition of a nitroso group to a sulfur atom of an amino acid residue of a protein. As defined herein, “de-S-nitrosylation” or “de-nitrosylation” refer to the removal of a nitroso group from a sulfur atom of an amino acid residue of a protein.4934-8116-8174.8Atty. Dkt. No. 043214-000103WO PT

[0175] As used herein, “citrullination” or “deimination” are the terms used for the post- translational modification of the amino acid arginine in a protein into the amino acid citrulline. As used herein, “decitrullination” or “de-deimination” are the terms used for the removal of the amino acid citrulline from a protein.

[0176] As used herein, “methylation” is the term used to denote the addition of a methyl group to a substrate or the substitution of an atom or group by a methyl group. Methylation is a form of alkylation with specifically a methyl group. Protein methylation typically takes place on arginine or lysine amino acid residues in the protein sequence. Arginine can be methylated once (monomethylated arginine) or twice, with either both methyl groups on one terminal nitrogen (asymmetric dimethylated arginine) or one on both nitrogens (symmetric dimethylated arginine) by peptidylarginine methyltransferases (PRMTs). Lysine can be methylated once, twice or three times by lysine methyltransferases. As used herein, “demethylation” refers to the removal of a methyl group from a protein.

[0177] As used herein, “hydroxylation” refers to the chemical process that introduces one or more hydroxyl groups ( — OH) into a compound (or radical) thereby oxidizing it. The principal residue to be hydroxylated in proteins is proline. The hydroxylation occurs at the Cy atom, forming hydroxyproline (Hyp). In some cases, proline may be hydroxylated instead on its C atom. Lysine may also be hydroxylated on its C8 atom, forming hydroxylysine (Hyl). As used herein, “dehydroxylation” refers to the chemical process that removes one or more hydroxyl groups ( — OH) from a protein.

[0178] As used herein, “ufinylation” refers to the process whereby the ubiquitin-like modifier Ufin-1 is covalently attached to a protein. As used herein, “deufinylation” refers to the process whereby the ubiquitin-like modifier Ufm-1 is removed from a protein.

[0179] As used herein, “fattenylation” refers to the process whereby the ubiquitin-like modifier FAT 10 is covalently attached to a protein. As used herein, “defattenylation” refers to the process whereby the ubiquitin-like modifier FAT 10 is removed from a protein.

[0180] As used herein, the terms “prenylation,” “isoprenylation,” or “lipidation” refers to the addition of hydrophobic molecules to a protein. Protein prenylation involves the transfer of either a famesyl or a geranyl-geranyl moiety to C-terminal cysteine(s) of the target protein. As used herein, the terms “deprenylation,” “desoprenylation,” or “delipidation” refers to the removal of hydrophobic molecules from a protein.

[0181] As used herein, “myristoylation” refers to the PTM process wherein myristoyl group (derived from myristic acid) is covalently attached via an amide bond to the alpha-amino group of an N-terminal amino acid of a polypeptide. It is more common on glycine residues but also occurs on other amino acids. Myristoylation occurs post-translationally, for example when previously internal glycine residues become exposed by caspase cleavage during apoptosis. As used herein, “demyristoylation”4934-8116-8174.8Atty. Dkt. No. 043214-000103WO PT refers to the PTM alteration wherein myristoyl group (derived from myristic acid) is removed from the alpha-amino group of an N-terminal amino acid of a polypeptide.

[0182] As used herein, “S-palmitoylation” refers to the covalent attachment of fatty acids, such as palmitic acid, to cysteine residues of proteins. As used herein, “de-S-palmitoylation” refers to the removal of fatty acids, such as palmitic acid, to cysteine residues from proteins.

[0183] As used herein, “tyrosine sulfation” is a PTM where a sulfate group is added to a tyrosine residue of a protein molecule. As used herein, “tyrosine desulfation” is a PTM alteration where a sulfate group is removed from a tyrosine residue of a protein molecule.

[0184] As used herein, “deamidation” refers to the chemical reaction in which an amide functional group is removed from a protein. The reaction damages the amide-containing side chains of the amino acids asparagine and glutamine.

[0185] As used herein, “formylation” is a type of PTM in which a formyl group is added to the N-terminus of a protein. As used herein, “deformylation” is a type of PTM alteration in which a formyl group is removed from the N-terminus of a protein.

[0186] As used herein, “carboxylation” is a PTM in which a carboxylic acid group is added to glutamate residues in proteins. It occurs primarily in proteins involved in the blood clotting cascade, specifically factors II, VII, IX, and X, protein C, and protein S, and also in some bone proteins. As used herein, “decarboxylation” is a PTM alteration in which a carboxylic acid group is removed from glutamate residues in proteins.

[0187] The flexible histone tails that protrude from the globular histone core are often subject to extensive post-translational modification. This modification modulates the transcriptional activity through both positive and negative regulatory mechanisms. The histone tails are highly positively charged, comprising many lysine and arginine residues. The most common PTMs of histone tails are acetylation and methylation of the lysine residues. For more information on the role of the histone tails and their modification see Lorch et al., Nucleic Acids Res., 51(8): 3671-3678 (2023), the contents of which are incorporated herein in their entirety.

[0188] H3 histone is one of the four core histones that make up nucleosomes and comprises a unique N-terminal alpha helix. H3 histone, also referred to as histone H3, is the most extensively modified of the core histones. One reason for this is that the tail of H3 histone is the longest of the core histones, at a length of about 35 amino acids residues. Several variants of H3 histone exist, though it is highly conserved and these variants vary by only a few amino acids. PTMs of H3 histone and sequences of H3 histone variants are known in the art, e.g., see Hake et al., Journal of Biological Chemistry, 281(1): 559-568 (2006), and Maehara et al., Epigenetics & Chromatin, 35(8): 1-17 (2015), the contents of each of which are incorporated herein in their entireties.

[0189] A sortase acts on a histone via transpeptidation. For example, a sortase can act on the peptide bond between a threonine and glycine residue of a sortase recognition motif present in the4934-8116-8174.8Atty. Dkt. No. 043214-000103WO PT histone. A cysteine residue in the active site of sortase attacks the carbonyl of the peptide bond in the histone, cleaving the glycine and forming a thioester intermediate at the threonine. The thioester intermediate is then susceptible to attack from the N-terminus of another peptide, leading to cleavage from the sortase and ligation of the two peptides.

[0190] One aspect of any of the embodiments is a method of producing a tailless H3 histone, the method comprising contacting an engineered polypeptide described herein with a composition comprising at least one tailed H3 histone; and separating at least one cleaved histone tail peptide from at least one tailless H3 histone.

[0191] As used herein, “contacting" refers to any suitable means for delivering, or exposing, a first element (e.g., polypeptide or agent) to at least one other element described herein, e.g., an engineered polypeptide. Exemplary delivery methods include, but are not limited to, direct delivery to cell culture medium, perfusion, injection, or other delivery method well known to one skilled in the art. In some embodiments, contacting comprises physical human activity, e.g., an injection; an act of dispensing, mixing, and / or decanting; and / or manipulation of a delivery device or machine.

[0192] In some embodiments of any of the aspects, the composition comprising at least one tailed H3 histone is a crude nuclear extract. As used herein, “crude nuclear extract” refers to a mixture of proteins and other cellular molecules primarily derived from a cell nucleus.

[0193] In some embodiments of any of the aspects, separating the at least one cleaved histone tail peptide from the at least one tailless H3 histone comprises trichloroacetic acid precipitation. Trichloroacetic acid precipitation is a technique commonly used to concentrate protein samples and / or remove contaminants from crude protein samples. For more information, see Koontz et al., Methods Enzymol., 541:3-10 (2014), the contents of which are incorporated herein in its entirety.

[0194] In some embodiments of any of the aspects, the composition comprising at least one tailless H3 histone further comprises at least one co-expressed unmodified core histone.

[0195] As used herein, “unmodified” refers to a histone, histone tail, histone octamer, or nucleosome which has not undergone post-translational modification. For instance, an unmodified histone, histone tail, histone octamer, or nucleosome does not have covalent or non-covalent modifications to the backbone or sidechains of its peptides. As used herein, “modified” refers to a histone, histone tail, histone octamer, or nucleosome which has undergone post-translational modification. A modified histone, histone tail, histone octamer, or nucleosome may have covalent or non-covalent modifications to the backbone or sidechains of its peptides. Modifications, e.g., PTMs, are described above herein.

[0196] One aspect of any of the embodiments is a method of producing an intact histone octamer, the method comprising producing core histones comprising tailless H3 histones using a method described herein and assembling the intact histone octamer via octamer assembly.4934-8116-8174.8Atty. Dkt. No. 043214-000103WO PT

[0197] As used herein, an “intact histone octamer” refers to a structure comprising two of each of H3, H4, H2A and H2B core histones. The intact histone octamer is the core protein structure of a nucleosome. As used herein, “octamer assembly” refers to the process by which the eight core histones are assembled. All four core histones share a structural motif known as the histone fold, which comprises three alpha helices connected by two loops. In octamer assembly, the histone folds first interact to form two sets of H3-H4 and H2A-H2B dimers. The H3-H4 dimers then combine to form a tetramer, which then binds with the two H2A-H2B dimers to form the intact histone octamer. For more information on histone octamer assembly, see Alberts et al., Molecular Biology of the Cell, 4thEdition, New York: Garland Science; 2002, the contents of which are incorporated herein by reference in its entirety.

[0198] One aspect of any of the embodiments is a method of producing a modified nucleosome, the method comprising producing an intact histone octamer using a method described herein, and wrapping the intact histone octamer with DNA.

[0199] As used herein, a “nucleosome” refers to an intact histone octamer which is wrapped with DNA. The core histones of the histone octamer consist of predominantly positively charged residues, which enables the assembly of the negatively charged phosphate backbone of the DNA around the histones through electrostatic interaction. In some embodiments of any of the aspects, the DNA is 147 base pairs in length. In some embodiments of any of the aspects, the DNA is 185 base pairs in length. For more information on nucleosome assembly, see Alberts et al., Molecular Biology of the Cell, 4thEdition, New York: Garland Science; 2002, the contents of which are incorporated herein by reference in its entirety.

[0200] One aspect of any of the embodiments is a method for producing a tailless H3 nucleosome, the method comprising producing an intact histone octamer comprising tailless H3 histones using a method described herein, and wrapping the intact histone octamer with DNA to form a tailless H3 nucleosome.

[0201] In some embodiments of any of the aspects, the DNA is 147 bp in length. In some embodiments of any of the aspects, the DNA is 185 bp in length. In some embodiments of any of the aspects, the DNA is 100-200 bp in length. In some embodiments of any of the aspects, the DNA is 147 or 185 bp in length.

[0202] One aspect of any of the embodiments is a method for histone tail isolation, the method comprising contacting at least one isolated protein with an engineered polypeptide described herein and an oligoglycine tandem mass tag (TMT) peptide, under conditions comprising at least one of: a temperature of between 37 and 42 °C; a pH of between 7.0 and 7.5; an ionic strength of between 30 and 50 mM; a histone protein concentration of between 10 and 20 pM; an engineered polypeptide concentration of between 100 and 400 pM; an oligoglycine TMT peptide concentration of between 0.25 and 1 mM; a phenylmethylsulfonyl fluoride (PMSF) concentration of between 0 and 2.5 mM; and a4934-8116-8174.8Atty. Dkt. No. 043214-000103WO PT dithiothreitol (DTT) concentration of between 1 and 10 mM; thereby cleaving at least one histone tail from the at least one histone protein; and isolating the at least one cleaved histone tail.

[0203] As used herein, a “tandem mass tag (TMT) peptide” or “TMT” refers to a chemical label which facilitates multiplexing in mass-spectrometry analysis. In some embodiments of any of the aspects, the TMT comprises an oligoglycine.

[0204] In some embodiments of any of the aspects, the oligoglycine TMT peptide has the, p g g y p p ; and Z is H, C(O)NHi, or C(O)OH. In some embodiments of any of the aspects, a is 0. In some embodiments of any of the aspects, a is 1. In some embodiments of any of the aspects, a is 2. In some embodiments of any of the aspects, b is 1. In some embodiments of any of the aspects, b is 2. In some embodiments of any of the aspects, b is 3. In some embodiments of any of the aspects, b is 4. In some embodiments of any of the aspects, X is CH2NH2. In some embodiments of any of the aspects, X is (CH2)4NH2. In some embodiments of any of the aspects, X is (CH2)3NHC(NH)NH2. In some embodiments of any of the aspects,some embodiments of any of the aspects, Y is. In some embodiments of any of the aspects, Z is H.In some embodiments of any of the aspects, Z is C(O)NH2. In some embodiments of any of the aspects,4934-8116-8174.8Atty. Dkt. No. 043214-000103WO PTZ is C(O)OH. In some embodiments of any of the aspects, the oligoglycine TMT peptide is

[0205] In some embodiments of any of the aspects, the conditions for contacting the at least one isolated protein with the engineered polypeptide comprise at least two of: a temperature of between 37 and 42 °C; a pH of between 7.0 and 7.5; an ionic strength of between 30 and 50 mM; a histone protein concentration of between 10 and 20 pM; an engineered polypeptide concentration of between 100 and 400 pM; an oligoglycine TMT peptide concentration of between 0.25 and 1 mM; a phenylmethylsulfonyl fluoride (PMSF) concentration of between 0 and 2.5 mM; and a dithiothreitol (DTT) concentration of between 1 and 10 mM. In some embodiments of any of the aspects, the conditions for contacting the at least one isolated protein with the engineered polypeptide comprise at least three of: a temperature of between 37 and 42 °C; a pH of between 7.0 and 7.5; an ionic strength of between 30 and 50 mM; a histone protein concentration of between 10 and 20 pM; an engineered polypeptide concentration of between 100 and 400 pM; an oligoglycine TMT peptide concentration of between 0.25 and 1 mM; a phenylmethylsulfonyl fluoride (PMSF) concentration of between 0 and 2.5 mM; and a dithiothreitol (DTT) concentration of between 1 and 10 mM. In some embodiments of any of the aspects, the conditions for contacting the at least one isolated protein with the engineered polypeptide comprise at least four of: a temperature of between 37 and 42 °C; a pH of between 7.0 and 7.5; an ionic strength of between 30 and 50 mM; a histone protein concentration of between 10 and 20 pM; an engineered polypeptide concentration of between 100 and 400 pM; an oligoglycine TMT peptide concentration of between 0.25 and 1 mM; a phenylmethylsulfonyl fluoride (PMSF) concentration of between 0 and 2.5 mM; and a dithiothreitol (DTT) concentration of between 1 and 10 mM. In some embodiments of any of the aspects, the conditions for contacting the at least one isolated protein with the engineered polypeptide comprise at least five of: a temperature of between 37 and 42 °C; a pH of between 7.0 and 7.5; an ionic strength of between 30 and 50 mM; a histone protein concentration of between 10 and 20 pM; an engineered polypeptide concentration of between 100 and4934-8116-8174.8Atty. Dkt. No. 043214-000103WO PT400 pM; an oligoglycine TMT peptide concentration of between 0.25 and 1 mM; a phenylmethylsulfonyl fluoride (PMSF) concentration of between 0 and 2.5 mM; and a dithiothreitol (DTT) concentration of between 1 and 10 m . In some embodiments of any of the aspects, the conditions for contacting the at least one isolated protein with the engineered polypeptide comprise at least six of: a temperature of between 37 and 42 °C; a pH of between 7.0 and 7.5; an ionic strength of between 30 and 50 mM; a histone protein concentration of between 10 and 20 pM; an engineered polypeptide concentration of between 100 and 400 pM; an oligoglycine TMT peptide concentration of between 0.25 and 1 mM; a phenylmethylsulfonyl fluoride (PMSF) concentration of between 0 and 2.5 mM; and a dithiothreitol (DTT) concentration of between 1 and 10 mM. In some embodiments of any of the aspects, the conditions for contacting the at least one isolated protein with the engineered polypeptide comprise at least seven of: a temperature of between 37 and 42 °C; a pH of between 7.0 and 7.5; an ionic strength of between 30 and 50 mM; a histone protein concentration of between 10 and 20 pM; an engineered polypeptide concentration of between 100 and 400 pM; an oligoglycine TMT peptide concentration of between 0.25 and 1 mM; a phenylmethylsulfonyl fluoride (PMSF) concentration of between 0 and 2.5 mM; and a dithiothreitol (DTT) concentration of between 1 and 10 mM.

[0206] In some embodiments of any of the aspects, the conditions for contacting the at least one isolated protein with the engineered polypeptide comprise: a temperature of between 37 and 42 °C; a pH of between 7.0 and 7.5; an ionic strength of between 30 and 50 mM; a histone protein concentration of between 10 and 20 pM; an engineered polypeptide concentration of between 100 and 400 pM; an oligoglycine TMT peptide concentration of between 0.25 and 1 mM; a phenylmethylsulfonyl fluoride (PMSF) concentration of between 0 and 2.5 mM; and a dithiothreitol (DTT) concentration of between 1 and 10 mM.

[0207] In some embodiments of any of the aspects, the temperature can be from about 37 to 42 °C. For example, the temperature can be about 37 °C, about 37.05 °C, about 37.1 °C, about 37.15 °C, about 37.2 °C, about 37.25 °C, about 37.3 °C, about 37.35 °C, about 37.4 °C, about 37.45 °C, about 37.5 °C, about 37.55 °C, about 37.6 °C, about 37.65 °C, about 37.7 °C, about 37.75 °C, about 37.8 °C, about 37.85 °C, about 37.9 °C, about 37.95 °C, about 38 °C, about 38.05 °C, about 38.1 °C, about 38.15 °C, about 38.2 °C, about 38.25 °C, about 38.3 °C, about 38.35 °C, about 38.4 °C, about 38.45 °C, about 38.5 °C, about 38.55 °C, about 38.6 °C, about 38.65 °C, about 38.7 °C, about 38.75 °C, about 38.8 °C, about 38.85 °C, about 38.9 °C, about 38.95 °C, about 39 °C, about 39.05 °C, about 39.1 °C, about 39.15 °C, about 39.2 °C, about 39.25 °C, about 39.3 °C, about 39.35 °C, about 39.4 °C, about 39.45 °C, about 39.5 °C, about 39.55 °C, about 39.6 °C, about 39.65 °C, about 39.7 °C, about 39.75 °C, about 39.8 °C, about 39.85 °C, about 39.9 °C, about 39.95 °C, about 40 °C, about 40.05 °C, about 40.1 °C, about 40.15 °C, about 40.2 °C, about 40.25 °C, about 40.3 °C, about 40.35 °C, about 40.4 °C, about 40.45 °C, about 40.5 °C, about 40.55 °C, about 40.6 °C, about 40.65 °C, about 40.7 °C, about 40.75 °C, about 40.8 °C, about 40.85 °C, about 40.9 °C, about 40.95 °C, about 41 °C, about 41.05 °C, 4934-8116-8174.8Atty. Dkt. No. 043214-000103WO PT about 41.1 °C, about 41.15 °C, about 41.2 °C, about 41.25 °C, about 41.3 °C, about 41.35 °C, about41.4 °C, about 41.45 °C, about 41.5 °C, about 41.55 °C, about 41.6 °C, about 41.65 °C, about 41.7 °C, about 41.75 °C, about 41.8 °C, about 41.85 °C, about 41.9 °C, about 41.95 °C, or about 42 °C.

[0208] In some embodiments of any of the aspects, the pH can be from about 7.0 to about 7.5. For example, the pH can be about 7, about 7.005, about 7.01, about 7.015, about 7.02, about 7.025, about 7.03, about 7.035, about 7.04, about 7.045, about 7.05, about 7.055, about 7.06, about 7.065, about 7.07, about 7.075, about 7.08, about 7.085, about 7.09, about 7.095, about 7.1, about 7.105, about 7.11, about 7.115, about 7.12, about 7.125, about 7.13, about 7.135, about 7.14, about 7.145, about 7.15, about 7.155, about 7.16, about 7.165, about 7.17, about 7.175, about 7.18, about 7.185, about 7.19, about 7.195, about 7.2, about 7.205, about 7.21, about 7.215, about 7.22, about 7.225, about 7.23, about 7.235, about 7.24, about 7.245, about 7.25, about 7.255, about 7.26, about 7.265, about 7.27, about 7.275, about 7.28, about 7.285, about 7.29, about 7.295, about 7.3, about 7.305, about 7.31, about 7.315, about 7.32, about 7.325, about 7.33, about 7.335, about 7.34, about 7.345, about 7.35, about 7.355, about 7.36, about 7.365, about 7.37, about 7.375, about 7.38, about 7.385, about 7.39, about 7.395, about 7.4, about 7.405, about 7.41, about 7.415, about 7.42, about 7.425, about 7.43, about 7.435, about 7.44, about 7.445, about 7.45, about 7.455, about 7.46, about 7.465, about 7.47, about 7.475, about 7.48, about 7.485, about 7.49, about 7.495, or about 7.5.

[0209] In some embodiments of any of the aspects, the ionic strength can be from about 30 to about 50 mM. For example, the ionic strength can be about 30 mM, about 30.1 mM, about 30.2 mM, about 30.3 mM, about 30.4 mM, about 30.5 mM, about 30.6 mM, about 30.7 mM, about 30.8 mM, about 30.9 mM, about 31 mM, about 31.1 mM, about 31.2 mM, about 31.3 mM, about 31.4 mM, about31.5 mM, about 31.6 mM, about 31.7 mM, about 31.8 mM, about 31.9 mM, about 32 mM, about 32.1 mM, about 32.2 mM, about 32.3 mM, about 32.4 mM, about 32.5 mM, about 32.6 mM, about 32.7 mM, about 32.8 mM, about 32.9 mM, about 33 mM, about 33.1 mM, about 33.2 mM, about 33.3 mM, about 33.4 mM, about 33.5 mM, about 33.6 mM, about 33.7 mM, about 33.8 mM, about 33.9 mM, about 34 mM, about 34.1 mM, about 34.2 mM, about 34.3 mM, about 34.4 mM, about 34.5 mM, about 34.6 mM, about 34.7 mM, about 34.8 mM, about 34.9 mM, about 35 mM, about 35.1 mM, about 35.2 mM, about 35.3 mM, about 35.4 mM, about 35.5 mM, about 35.6 mM, about 35.7 mM, about 35.8 mM, about 35.9 mM, about 36 mM, about 36.1 mM, about 36.2 mM, about 36.3 mM, about 36.4 mM, about 36.5 mM, about 36.6 mM, about 36.7 mM, about 36.8 mM, about 36.9 mM, about 37 mM, about 37.1 mM, about 37.2 mM, about 37.3 mM, about 37.4 mM, about 37.5 mM, about 37.6 mM, about 37.7 mM, about 37.8 mM, about 37.9 mM, about 38 mM, about 38.1 mM, about 38.2 mM, about 38.3 mM, about 38.4 mM, about 38.5 mM, about 38.6 mM, about 38.7 mM, about 38.8 mM, about 38.9 mM, about 39 mM, about 39.1 mM, about 39.2 mM, about 39.3 mM, about 39.4 mM, about 39.5 mM, about 39.6 mM, about 39.7 mM, about 39.8 mM, about 39.9 mM, about 40 mM, about 40.1 mM, about 40.2 mM, about 40.3 mM, about 40.4 mM, about 40.5 mM, about 40.6 mM, about 40.7 mM, about 40.8 mM, about 40.9 mM, about 41 mM, about 41.1 mM, about 41.2 mM, about 41.3 mM, about 41.4 mM, about 41.5 mM, about4934-8116-8174.8Atty. Dkt. No. 043214-000103WO PT41.6 mM, about 41.7 mM, about 41.8 mM, about 41.9 mM, about 42 mM, about 42.1 mM, about 42.2 mM, about 42.3 mM, about 42.4 mM, about 42.5 mM, about 42.6 mM, about 42.7 mM, about 42.8 mM, about 42.9 mM, about 43 mM, about 43.1 mM, about 43.2 mM, about 43.3 mM, about 43.4 mM, about 43.5 mM, about 43.6 mM, about 43.7 mM, about 43.8 mM, about 43.9 mM, about 44 mM, about 44.1 mM, about 44.2 mM, about 44.3 mM, about 44.4 mM, about 44.5 mM, about 44.6 mM, about 44.7 mM, about 44.8 mM, about 44.9 mM, about 45 mM, about 45.1 mM, about 45.2 mM, about 45.3 mM, about45.4 mM, about 45.5 mM, about 45.6 mM, about 45.7 mM, about 45.8 mM, about 45.9 mM, about 46 mM, about 46.1 mM, about 46.2 mM, about 46.3 mM, about 46.4 mM, about 46.5 mM, about 46.6 mM, about 46.7 mM, about 46.8 mM, about 46.9 mM, about 47 mM, about 47.1 mM, about 47.2 mM, about 47.3 mM, about 47.4 mM, about 47.5 mM, about 47.6 mM, about 47.7 mM, about 47.8 mM, about 47.9 mM, about 48 mM, about 48.1 mM, about 48.2 mM, about 48.3 mM, about 48.4 mM, about 48.5 mM, about 48.6 mM, about 48.7 mM, about 48.8 mM, about 48.9 mM, about 49 mM, about 49.1 mM, about49.2 mM, about 49.3 mM, about 49.4 mM, about 49.5 mM, about 49.6 mM, about 49.7 mM, about 49.8 mM, about 49.9 mM, or about 50 mM.

[0210] In some embodiments of any of the aspects, the histone protein concentration can be from about 10 pM to 20 pM. For example, the histone protein concentration can be about 10 pM, about10.05 pM, about 10.1 pM, about 10.15 pM, about 10.2 pM, about 10.25 pM, about 10.3 pM, about10.35 pM, about 10.4 pM, about 10.45 pM, about 10.5 pM, about 10.55 pM, about 10.6 pM, about10.65 pM, about 10.7 pM, about 10.75 pM, about 10.8 pM, about 10.85 pM, about 10.9 pM, about10.95 pM, about 11 pM, about 11.05 pM, about 11.1 pM, about 11.15 pM, about 11.2 pM, about 11.25 pM, about 11.3 pM, about 11.35 pM, about 11.4 pM, about 11.45 pM, about 11.5 pM, about 11.55 pM, about 11.6 pM, about 11.65 pM, about 11.7 pM, about 11.75 pM, about 11.8 pM, about 11.85 pM, about 11.9 pM, about 11.95 pM, about 12 pM, about 12.05 pM, about 12.1 pM, about 12.15 pM, about12.2 pM, about 12.25 pM, about 12.3 pM, about 12.35 pM, about 12.4 pM, about 12.45 pM, about12.5 pM, about 12.55 pM, about 12.6 pM, about 12.65 pM, about 12.7 pM, about 12.75 pM, about12.8 pM, about 12.85 pM, about 12.9 pM, about 12.95 pM, about 13 pM, about 13.05 pM, about 13.1 pM, about 13.15 pM, about 13.2 pM, about 13.25 pM, about 13.3 pM, about 13.35 pM, about 13.4 pM, about 13.45 pM, about 13.5 pM, about 13.55 pM, about 13.6 pM, about 13.65 pM, about 13.7 pM, about 13.75 pM, about 13.8 pM, about 13.85 pM, about 13.9 pM, about 13.95 pM, about 14 pM, about 14.05 pM, about 14.1 pM, about 14.15 pM, about 14.2 pM, about 14.25 pM, about 14.3 pM, about 14.35 pM, about 14.4 pM, about 14.45 pM, about 14.5 pM, about 14.55 pM, about 14.6 pM, about 14.65 pM, about 14.7 pM, about 14.75 pM, about 14.8 pM, about 14.85 pM, about 14.9 pM, about 14.95 pM, about 15 pM, about 15.05 pM, about 15.1 pM, about 15.15 pM, about 15.2 pM, about 15.25 pM, about 15.3 pM, about 15.35 pM, about 15.4 pM, about 15.45 pM, about 15.5 pM, about15.55 pM, about 15.6 pM, about 15.65 pM, about 15.7 pM, about 15.75 pM, about 15.8 pM, about15.85 pM, about 15.9 pM, about 15.95 pM, about 16 pM, about 16.05 pM, about 16.1 pM, about 16.15 pM, about 16.2 pM, about 16.25 pM, about 16.3 pM, about 16.35 pM, about 16.4 pM, about 16.454934-8116-8174.8Atty. Dkt. No. 043214-000103WO PT pM, about 16.5 pM, about 16.55 pM, about 16.6 pM, about 16.65 pM, about 16.7 pM, about 16.75 pM, about 16.8 pM, about 16.85 pM, about 16.9 pM, about 16.95 pM, about 17 pM, about 17.05 pM, about 17.1 pM, about 17.15 pM, about 17.2 pM, about 17.25 pM, about 17.3 pM, about 17.35 pM, about 17.4 pM, about 17.45 pM, about 17.5 pM, about 17.55 pM, about 17.6 pM, about 17.65 pM, about 17.7 pM, about 17.75 pM, about 17.8 pM, about 17.85 pM, about 17.9 pM, about 17.95 pM, about 18 pM, about 18.05 pM, about 18.1 pM, about 18.15 pM, about 18.2 pM, about 18.25 pM, about18.3 pM, about 18.35 pM, about 18.4 pM, about 18.45 pM, about 18.5 pM, about 18.55 pM, about18.6 pM, about 18.65 pM, about 18.7 pM, about 18.75 pM, about 18.8 pM, about 18.85 pM, about18.9 pM, about 18.95 pM, about 19 pM, about 19.05 pM, about 19.1 pM, about 19.15 pM, about 19.2 pM, about 19.25 pM, about 19.3 pM, about 19.35 pM, about 19.4 pM, about 19.45 pM, about 19.5 pM, about 19.55 pM, about 19.6 pM, about 19.65 pM, about 19.7 pM, about 19.75 pM, about 19.8 pM, about 19.85 pM, about 19.9 pM, about 19.95 pM, or about 20 pM.

[0211] In some embodiments of any of the aspects, the engineered polypeptide concentration can be from pM. For example, the engineered polypeptide concentration can be about 100 pM, about102.5 pM, about 105 pM, about 107.5 pM, about 110 pM, about 112.5 pM, about 115 pM, about 117.5 pM, about 120 pM, about 122.5 pM, about 125 pM, about 127.5 pM, about 130 pM, about 132.5 pM, about 135 pM, about 137.5 pM, about 140 pM, about 142.5 pM, about 145 pM, about 147.5 pM, about 150 pM, about 152.5 pM, about 155 pM, about 157.5 pM, about 160 pM, about 162.5 pM, about 165 pM, about 167.5 pM, about 170 pM, about 172.5 pM, about 175 pM, about 177.5 pM, about 180 pM, about 182.5 pM, about 185 pM, about 187.5 pM, about 190 pM, about 192.5 pM, about 195 pM, about197.5 pM, about 200 pM, about 202.5 pM, about 205 pM, about 207.5 pM, about 210 pM, about 212.5 pM, about 215 pM, about 217.5 pM, about 220 pM, about 222.5 pM, about 225 pM, about 227.5 pM, about 230 pM, about 232.5 pM, about 235 pM, about 237.5 pM, about 240 pM, about 242.5 pM, about 245 pM, about 247.5 pM, about 250 pM, about 252.5 pM, about 255 pM, about 257.5 pM, about 260 pM, about 262.5 pM, about 265 pM, about 267.5 pM, about 270 pM, about 272.5 pM, about 275 pM, about 277.5 pM, about 280 pM, about 282.5 pM, about 285 pM, about 287.5 pM, about 290 pM, about292.5 pM, about 295 pM, about 297.5 pM, about 300 pM, about 302.5 pM, about 305 pM, about 307.5 pM, about 310 pM, about 312.5 pM, about 315 pM, about 317.5 pM, about 320 pM, about 322.5 pM, about 325 pM, about 327.5 pM, about 330 pM, about 332.5 pM, about 335 pM, about 337.5 pM, about 340 pM, about 342.5 pM, about 345 pM, about 347.5 pM, about 350 pM, about 352.5 pM, about 355 pM, about 357.5 pM, about 360 pM, about 362.5 pM, about 365 pM, about 367.5 pM, about 370 pM, about 372.5 pM, about 375 pM, about 377.5 pM, about 380 pM, about 382.5 pM, about 385 pM, about387.5 pM, about 390 pM, about 392.5 pM, about 395 pM, about 397.5 pM, or about 400 pM.

[0212] In some embodiments of any of the aspects, the oligoglycine TMT peptide concentration can be from about 0.25 to about 1 mM. For example, the oligoglycine TMT peptide concentration can be about 0.25 mM, about 0.255 mM, about 0.26 mM, about 0.265 mM, about 0.27 mM, about 0.275 mM, about 0.28 mM, about 0.285 mM, about 0.29 mM, about 0.295 mM, about 0.34934-8116-8174.8Atty. Dkt. No. 043214-000103WO PT mM, about 0.305 mM, about 0.31 mM, about 0.315 mM, about 0.32 mM, about 0.325 mM, about 0.33 mM, about 0.335 mM, about 0.34 mM, about 0.345 mM, about 0.35 mM, about 0.355 mM, about 0.36 mM, about 0.365 mM, about 0.37 mM, about 0.375 mM, about 0.38 mM, about 0.385 mM, about 0.39 mM, about 0.395 mM, about 0.4 mM, about 0.405 mM, about 0.41 mM, about 0.415 mM, about 0.42 mM, about 0.425 mM, about 0.43 mM, about 0.435 mM, about 0.44 mM, about 0.445 mM, about 0.45 mM, about 0.455 mM, about 0.46 mM, about 0.465 mM, about 0.47 mM, about 0.475 mM, about 0.48 mM, about 0.485 mM, about 0.49 mM, about 0.495 mM, about 0.5 mM, about 0.505 mM, about 0.51 mM, about 0.515 mM, about 0.52 mM, about 0.525 mM, about 0.53 mM, about 0.535 mM, about 0.54 mM, about 0.545 mM, about 0.55 mM, about 0.555 mM, about 0.56 mM, about 0.565 mM, about 0.57 mM, about 0.575 mM, about 0.58 mM, about 0.585 mM, about 0.59 mM, about 0.595 mM, about 0.6 mM, about 0.605 mM, about 0.61 mM, about 0.615 mM, about 0.62 mM, about 0.625 mM, about 0.63 mM, about 0.635 mM, about 0.64 mM, about 0.645 mM, about 0.65 mM, about 0.655 mM, about 0.66 mM, about 0.665 mM, about 0.67 mM, about 0.675 mM, about 0.68 mM, about 0.685 mM, about 0.69 mM, about 0.695 mM, about 0.7 mM, about 0.705 mM, about 0.71 mM, about 0.715 mM, about 0.72 mM, about 0.725 mM, about 0.73 mM, about 0.735 mM, about 0.74 mM, about 0.745 mM, about 0.75 mM, about 0.755 mM, about 0.76 mM, about 0.765 mM, about 0.77 mM, about 0.775 mM, about 0.78 mM, about 0.785 mM, about 0.79 mM, about 0.795 mM, about 0.8 mM, about 0.805 mM, about 0.81 mM, about 0.815 mM, about 0.82 mM, about 0.825 mM, about 0.83 mM, about 0.835 mM, about 0.84 mM, about 0.845 mM, about 0.85 mM, about 0.855 mM, about 0.86 mM, about 0.865 mM, about 0.87 mM, about 0.875 mM, about 0.88 mM, about 0.885 mM, about 0.89 mM, about 0.895 mM, about 0.9 mM, about 0.905 mM, about 0.91 mM, about 0.915 mM, about 0.92 mM, about 0.925 mM, about 0.93 mM, about 0.935 mM, about 0.94 mM, about 0.945 mM, about 0.95 mM, about 0.955 mM, about 0.96 mM, about 0.965 mM, about 0.97 mM, about 0.975 mM, about 0.98 mM, about 0.985 mM, about 0.99 mM, about 0.995 mM, about 1 mM.

[0213] In some embodiments of any of the aspects, the PMSF concentration is from about 0 to about 2.5 mM. For example, the PMSF concentration can be about 0 mM, about 0.01 mM, about 0.02 mM, about 0.03 mM, about 0.04 mM, about 0.05 mM, about 0.06 mM, about 0.07 mM, about 0.08 mM, about 0.09 mM, about 0.1 mM, about 0.11 mM, about 0.12 mM, about 0.13 mM, about 0.14 mM, about 0.15 mM, about 0.16 mM, about 0.17 mM, about 0.18 mM, about 0.19 mM, about 0.2 mM, about 0.21 mM, about 0.22 mM, about 0.23 mM, about 0.24 mM, about 0.25 mM, about 0.26 mM, about 0.27 mM, about 0.28 mM, about 0.29 mM, about 0.3 mM, about 0.31 mM, about 0.32 mM, about 0.33 mM, about 0.34 mM, about 0.35 mM, about 0.36 mM, about 0.37 mM, about 0.38 mM, about 0.39 mM, about 0.4 mM, about 0.41 mM, about 0.42 mM, about 0.43 mM, about 0.44 mM, about 0.45 mM, about 0.46 mM, about 0.47 mM, about 0.48 mM, about 0.49 mM, about 0.5 mM, about 0.51 mM, about 0.52 mM, about 0.53 mM, about 0.54 mM, about 0.55 mM, about 0.56 mM, about 0.57 mM, about 0.58 mM, about 0.59 mM, about 0.6 mM, about 0.61 mM, about 0.62 mM, about 0.63 mM, about 0.64 mM, about 0.65 mM, about 0.66 mM, about 0.67 mM, about 0.68 mM, about 0.69 mM, about 0.7 mM, about 0.71 mM, about4934-8116-8174.8Atty. Dkt. No. 043214-000103WO PT0.72 mM, about 0.73 mM, about 0.74 mM, about 0.75 mM, about 0.76 mM, about 0.77 mM, about 0.78 mM, about 0.79 mM, about 0.8 mM, about 0.81 mM, about 0.82 mM, about 0.83 mM, about 0.84 mM, about 0.85 mM, about 0.86 mM, about 0.87 mM, about 0.88 mM, about 0.89 mM, about 0.9 mM, about 0.91 mM, about 0.92 mM, about 0.93 mM, about 0.94 mM, about 0.95 mM, about 0.96 mM, about 0.97 mM, about 0.98 mM, about 0.99 mM, about 1 mM, about 1.01 mM, about 1.02 mM, about 1.03 mM, about 1.04 mM, about 1.05 mM, about 1.06 mM, about 1.07 mM, about 1.08 mM, about 1.09 mM, about 1.1 mM, about 1.11 mM, about 1.12 mM, about 1.13 mM, about 1.14 mM, about 1.15 mM, about 1.16 mM, about 1.17 mM, about 1.18 mM, about 1.19 mM, about 1.2 mM, about 1.21 mM, about 1.22 mM, about 1.23 mM, about 1.24 mM, about 1.25 mM, about 1.26 mM, about 1.27 mM, about 1.28 mM, about 1.29 mM, about 1.3 mM, about 1.31 mM, about 1.32 mM, about 1.33 mM, about 1.34 mM, about1.35 mM, about 1.36 mM, about 1.37 mM, about 1.38 mM, about 1.39 mM, about 1.4 mM, about 1.41 mM, about 1.42 mM, about 1.43 mM, about 1.44 mM, about 1.45 mM, about 1.46 mM, about 1.47 mM, about 1.48 mM, about 1.49 mM, about 1.5 mM, about 1.51 mM, about 1.52 mM, about 1.53 mM, about 1.54 mM, about 1.55 mM, about 1.56 mM, about 1.57 mM, about 1.58 mM, about 1.59 mM, about 1.6 mM, about 1.61 mM, about 1.62 mM, about 1.63 mM, about 1.64 mM, about 1.65 mM, about 1.66 mM, about 1.67 mM, about 1.68 mM, about 1.69 mM, about 1.7 mM, about 1.71 mM, about 1.72 mM, about 1.73 mM, about 1.74 mM, about 1.75 mM, about 1.76 mM, about 1.77 mM, about 1.78 mM, about 1.79 mM, about 1.8 mM, about 1.81 mM, about 1.82 mM, about 1.83 mM, about 1.84 mM, about 1.85 mM, about 1.86 mM, about 1.87 mM, about 1.88 mM, about 1.89 mM, about 1.9 mM, about 1.91 mM, about 1.92 mM, about 1.93 mM, about 1.94 mM, about 1.95 mM, about 1.96 mM, about 1.97 mM, about 1.98 mM, about 1.99 mM, about 2 mM, about 2.01 mM, about 2.02 mM, about 2.03 mM, about 2.04 mM, about 2.05 mM, about 2.06 mM, about 2.07 mM, about 2.08 mM, about 2.09 mM, about 2.1 mM, about 2.11 mM, about 2.12 mM, about 2.13 mM, about 2.14 mM, about 2.15 mM, about 2.16 mM, about 2.17 mM, about 2.18 mM, about 2.19 mM, about 2.2 mM, about 2.21 mM, about 2.22 mM, about 2.23 mM, about 2.24 mM, about 2.25 mM, about 2.26 mM, about 2.27 mM, about 2.28 mM, about 2.29 mM, about 2.3 mM, about 2.31 mM, about 2.32 mM, about 2.33 mM, about 2.34 mM, about 2.35 mM, about2.36 mM, about 2.37 mM, about 2.38 mM, about 2.39 mM, about 2.4 mM, about 2.41 mM, about 2.42 mM, about 2.43 mM, about 2.44 mM, about 2.45 mM, about 2.46 mM, about 2.47 mM, about 2.48 mM, about 2.49 mM, or about 2.5 mM.

[0214] In some embodiments of any of the aspects, the temperature can be from 37 to 42 °C. For example, the temperature can be 37 °C, 37.05 °C, 37.1 °C, 37.15 °C, 37.2 °C, 37.25 °C, 37.3 °C, 37.35 °C, 37.4 °C, 37.45 °C, 37.5 °C, 37.55 °C, 37.6 °C, 37.65 °C, 37.7 °C, 37.75 °C, 37.8 °C, 37.85 °C, 37.9 °C, 37.95 °C, 38 °C, 38.05 °C, 38.1 °C, 38.15 °C, 38.2 °C, 38.25 °C, 38.3 °C, 38.35 °C, 38.4 °C, 38.45 °C, 38.5 °C, 38.55 °C, 38.6 °C, 38.65 °C, 38.7 °C, 38.75 °C, 38.8 °C, 38.85 °C, 38.9 °C, 38.95 °C, 39 °C, 39.05 °C, 39.1 °C, 39.15 °C, 39.2 °C, 39.25 °C, 39.3 °C, 39.35 °C, 39.4 °C, 39.45 °C, 39.5 °C, 39.55 °C, 39.6 °C, 39.65 °C, 39.7 °C, 39.75 °C, 39.8 °C, 39.85 °C, 39.9 °C, 39.95 °C, 40 °C, 40.05 °C, 40.1 °C, 40.15 °C, 40.2 °C, 40.25 °C, 40.3 °C, 40.35 °C, 40.4 °C, 40.45 °C, 40.5 °C, 40.55 °C, 40.6 °C,4934-8116-8174.8Atty. Dkt. No. 043214-000103WO PT40.65 °C, 40.7 °C, 40.75 °C, 40.8 °C, 40.85 °C, 40.9 °C, 40.95 °C, 41 °C, 41.05 °C, 41.1 °C, 41.15 °C, 41.2 °C, 41.25 °C, 41.3 °C, 41.35 °C, 41.4 °C, 41.45 °C, 41.5 °C, 41.55 °C, 41.6 °C, 41.65 °C, 41.7 °C, 41.75 °C, 41.8 °C, 41.85 °C, 41.9 °C, 41.95 °C, or 42 °C.

[0215] In some embodiments of any of the aspects, the pH can be from 7.0 to 7.5. For example, the pH can be 7, 7.005, 7.01, 7.015, 7.02, 7.025, 7.03, 7.035, 7.04, 7.045, 7.05, 7.055, 7.06, 7.065, 7.07, 7.075, 7.08, 7.085, 7.09, 7.095, 7.1, 7.105, 7.11, 7.115, 7.12, 7.125, 7.13, 7.135, 7.14, 7.145, 7.15, 7.155, 7.16, 7.165, 7.17, 7.175, 7.18, 7.185, 7.19, 7.195, 7.2, 7.205, 7.21, 7.215, 7.22, 7.225, 7.23, 7.235, 7.24, 7.245, 7.25, 7.255, 7.26, 7.265, 7.27, 7.275, 7.28, 7.285, 7.29, 7.295, 7.3, 7.305, 7.31, 7.315, 7.32, 7.325, 7.33, 7.335, 7.34, 7.345, 7.35, 7.355, 7.36, 7.365, 7.37, 7.375, 7.38, 7.385, 7.39, 7.395, 7.4, 7.405, 7.41, 7.415, 7.42, 7.425, 7.43, 7.435, 7.44, 7.445, 7.45, 7.455, 7.46, 7.465, 7.47, 7.475, 7.48, 7.485, 7.49, 7.495, or 7.5.

[0216] In some embodiments of any of the aspects, the ionic strength can be from 30 to 50 mM. For example, the ionic strength can be 30 mM, 30.1 mM, 30.2 mM, 30.3 mM, 30.4 mM, 30.5 mM,30.6 mM, 30.7 mM, 30.8 mM, 30.9 mM, 31 mM, 31.1 mM, 31.2 mM, 31.3 mM, 31.4 mM, 31.5 mM,31.6 mM, 31.7 mM, 31.8 mM, 31.9 mM, 32 mM, 32.1 mM, 32.2 mM, 32.3 mM, 32.4 mM, 32.5 mM,32.6 mM, 32.7 mM, 32.8 mM, 32.9 mM, 33 mM, 33.1 mM, 33.2 mM, 33.3 mM, 33.4 mM, 33.5 mM,33.6 mM, 33.7 mM, 33.8 mM, 33.9 mM, 34 mM, 34.1 mM, 34.2 mM, 34.3 mM, 34.4 mM, 34.5 mM,34.6 mM, 34.7 mM, 34.8 mM, 34.9 mM, 35 mM, 35.1 mM, 35.2 mM, 35.3 mM, 35.4 mM, 35.5 mM,35.6 mM, 35.7 mM, 35.8 mM, 35.9 mM, 36 mM, 36.1 mM, 36.2 mM, 36.3 mM, 36.4 mM, 36.5 mM,36.6 mM, 36.7 mM, 36.8 mM, 36.9 mM, 37 mM, 37.1 mM, 37.2 mM, 37.3 mM, 37.4 mM, 37.5 mM,37.6 mM, 37.7 mM, 37.8 mM, 37.9 mM, 38 mM, 38.1 mM, 38.2 mM, 38.3 mM, 38.4 mM, 38.5 mM,38.6 mM, 38.7 mM, 38.8 mM, 38.9 mM, 39 mM, 39.1 mM, 39.2 mM, 39.3 mM, 39.4 mM, 39.5 mM,39.6 mM, 39.7 mM, 39.8 mM, 39.9 mM, 40 mM, 40.1 mM, 40.2 mM, 40.3 mM, 40.4 mM, 40.5 mM,40.6 mM, 40.7 mM, 40.8 mM, 40.9 mM, 41 mM, 41.1 mM, 41.2 mM, 41.3 mM, 41.4 mM, 41.5 mM,41.6 mM, 41.7 mM, 41.8 mM, 41.9 mM, 42 mM, 42.1 mM, 42.2 mM, 42.3 mM, 42.4 mM, 42.5 mM,42.6 mM, 42.7 mM, 42.8 mM, 42.9 mM, 43 mM, 43.1 mM, 43.2 mM, 43.3 mM, 43.4 mM, 43.5 mM,43.6 mM, 43.7 mM, 43.8 mM, 43.9 mM, 44 mM, 44.1 mM, 44.2 mM, 44.3 mM, 44.4 mM, 44.5 mM,44.6 mM, 44.7 mM, 44.8 mM, 44.9 mM, 45 mM, 45.1 mM, 45.2 mM, 45.3 mM, 45.4 mM, 45.5 mM,45.6 mM, 45.7 mM, 45.8 mM, 45.9 mM, 46 mM, 46.1 mM, 46.2 mM, 46.3 mM, 46.4 mM, 46.5 mM,46.6 mM, 46.7 mM, 46.8 mM, 46.9 mM, 47 mM, 47.1 mM, 47.2 mM, 47.3 mM, 47.4 mM, 47.5 mM,47.6 mM, 47.7 mM, 47.8 mM, 47.9 mM, 48 mM, 48.1 mM, 48.2 mM, 48.3 mM, 48.4 mM, 48.5 mM,48.6 mM, 48.7 mM, 48.8 mM, 48.9 mM, 49 mM, 49.1 mM, 49.2 mM, 49.3 mM, 49.4 mM, 49.5 mM,49.6 mM, 49.7 mM, 49.8 mM, 49.9 mM, or 50 mM.

[0217] In some embodiments of any of the aspects, the histone protein concentration can be from 10 pM to 20 pM. For example, the histone protein concentration can be 10 pM, 10.05 pM, 10.1 pM, 10.15 pM, 10.2 pM, 10.25 pM, 10.3 pM, 10.35 pM, 10.4 pM, 10.45 pM, 10.5 pM, 10.55 pM,4934-8116-8174.8Atty. Dkt. No. 043214-000103WO PT10.6 pM, 10.65 pM, 10.7 pM, 10.75 pM, 10.8 pM, 10.85 pM, 10.9 pM, 10.95 pM, 11 pM, 11.05 pM,11.1 pM, 11.15 pM, 11.2 pM, 11.25 pM, 11.3 pM, 11.35 pM, 11.4 pM, 11.45 pM, 11.5 pM, 11.55 pM,11.6 pM, 11.65 pM, 11.7 pM, 11.75 pM, 11.8 pM, 11.85 pM, 11.9 pM, 11.95 pM, 12 pM, 12.05 pM,12.1 pM, 12.15 pM, 12.2 pM, 12.25 pM, 12.3 pM, 12.35 pM, 12.4 pM, 12.45 pM, 12.5 pM, 12.55 pM,12.6 pM, 12.65 pM, 12.7 pM, 12.75 pM, 12.8 pM, 12.85 pM, 12.9 pM, 12.95 pM, 13 pM, 13.05 pM,13.1 pM, 13.15 pM, 13.2 pM, 13.25 pM, 13.3 pM, 13.35 pM, 13.4 pM, 13.45 pM, 13.5 pM, 13.55 pM,13.6 pM, 13.65 pM, 13.7 pM, 13.75 pM, 13.8 pM, 13.85 pM, 13.9 pM, 13.95 pM, 14 pM, 14.05 pM,14.1 pM, 14.15 pM, 14.2 pM, 14.25 pM, 14.3 pM, 14.35 pM, 14.4 pM, 14.45 pM, 14.5 pM, 14.55 pM,14.6 pM, 14.65 pM, 14.7 pM, 14.75 pM, 14.8 pM, 14.85 pM, 14.9 pM, 14.95 pM, 15 pM, 15.05 pM,15.1 pM, 15.15 pM, 15.2 pM, 15.25 pM, 15.3 pM, 15.35 pM, 15.4 pM, 15.45 pM, 15.5 pM, 15.55 pM,15.6 pM, 15.65 pM, 15.7 pM, 15.75 pM, 15.8 pM, 15.85 pM, 15.9 pM, 15.95 pM, 16 pM, 16.05 pM,16.1 pM, 16.15 pM, 16.2 pM, 16.25 pM, 16.3 pM, 16.35 pM, 16.4 pM, 16.45 pM, 16.5 pM, 16.55 pM,16.6 pM, 16.65 pM, 16.7 pM, 16.75 pM, 16.8 pM, 16.85 pM, 16.9 pM, 16.95 pM, 17 pM, 17.05 pM,17.1 pM, 17.15 pM, 17.2 pM, 17.25 pM, 17.3 pM, 17.35 pM, 17.4 pM, 17.45 pM, 17.5 pM, 17.55 pM,17.6 pM, 17.65 pM, 17.7 pM, 17.75 pM, 17.8 pM, 17.85 pM, 17.9 pM, 17.95 pM, 18 pM, 18.05 pM,18.1 pM, 18.15 pM, 18.2 pM, 18.25 pM, 18.3 pM, 18.35 pM, 18.4 pM, 18.45 pM, 18.5 pM, 18.55 pM,18.6 pM, 18.65 pM, 18.7 pM, 18.75 pM, 18.8 pM, 18.85 pM, 18.9 pM, 18.95 pM, 19 pM, 19.05 pM,19.1 pM, 19.15 pM, 19.2 pM, 19.25 pM, 19.3 pM, 19.35 pM, 19.4 pM, 19.45 pM, 19.5 pM, 19.55 pM,19.6 pM, 19.65 pM, 19.7 pM, 19.75 pM, 19.8 pM, 19.85 pM, 19.9 pM, 19.95 pM, or 20 pM.

[0218] In some embodiments of any of the aspects, the engineered polypeptide concentration can be from pM. For example, the engineered polypeptide concentration can be 100 pM, 102.5 pM, 105 pM, 107.5 pM, 110 pM, 112.5 pM, 115 pM, 117.5 pM, 120 pM, 122.5 pM, 125 pM, 127.5 pM,130 pM, 132.5 pM, 135 pM, 137.5 pM, 140 pM, 142.5 pM, 145 pM, 147.5 pM, 150 pM, 152.5 pM,155 pM, 157.5 pM, 160 pM, 162.5 pM, 165 pM, 167.5 pM, 170 pM, 172.5 pM, 175 pM, 177.5 pM,180 pM, 182.5 pM, 185 pM, 187.5 pM, 190 pM, 192.5 pM, 195 pM, 197.5 pM, 200 pM, 202.5 pM,205 pM, 207.5 pM, 210 pM, 212.5 pM, 215 pM, 217.5 pM, 220 pM, 222.5 pM, 225 pM, 227.5 pM,230 pM, 232.5 pM, 235 pM, 237.5 pM, 240 pM, 242.5 pM, 245 pM, 247.5 pM, 250 pM, 252.5 pM,255 pM, 257.5 pM, 260 pM, 262.5 pM, 265 pM, 267.5 pM, 270 pM, 272.5 pM, 275 pM, 277.5 pM,280 pM, 282.5 pM, 285 pM, 287.5 pM, 290 pM, 292.5 pM, 295 pM, 297.5 pM, 300 pM, 302.5 pM,305 pM, 307.5 pM, 310 pM, 312.5 pM, 315 pM, 317.5 pM, 320 pM, 322.5 pM, 325 pM, 327.5 pM,330 pM, 332.5 pM, 335 pM, 337.5 pM, 340 pM, 342.5 pM, 345 pM, 347.5 pM, 350 pM, 352.5 pM,355 pM, 357.5 pM, 360 pM, 362.5 pM, 365 pM, 367.5 pM, 370 pM, 372.5 pM, 375 pM, 377.5 pM,380 pM, 382.5 pM, 385 pM, 387.5 pM, 390 pM, 392.5 pM, 395 pM, 397.5 pM, or 400 pM.

[0219] In some embodiments of any of the aspects, the oligoglycine TMT peptide concentration can be from 0.25 to 1 mM. For example, the oligoglycine TMT peptide concentration can be 0.25 mM, 0.255 mM, 0.26 mM, 0.265 mM, 0.27 mM, 0.275 mM, 0.28 mM, 0.285 mM, 0.29 mM, 0.295 mM, 0.3 mM, 0.305 mM, 0.31 mM, 0.315 mM, 0.32 mM, 0.325 mM, 0.33 mM, 0.335 mM, 4934-8116-8174.8Atty. Dkt. No. 043214-000103WO PT0.34 mM, 0.345 mM, 0.35 mM, 0.355 mM, 0.36 mM, 0.365 mM, 0.37 mM, 0.375 mM, 0.38 mM, 0.385 mM, 0.39 mM, 0.395 mM, 0.4 mM, 0.405 mM, 0.41 mM, 0.415 mM, 0.42 mM, 0.425 mM, 0.43 mM, 0.435 mM, 0.44 mM, 0.445 mM, 0.45 mM, 0.455 mM, 0.46 mM, 0.465 mM, 0.47 mM, 0.475 mM, 0.48 mM, 0.485 mM, 0.49 mM, 0.495 mM, 0.5 mM, 0.505 mM, 0.51 mM, 0.515 mM, 0.52 mM, 0.525 mM, 0.53 mM, 0.535 mM, 0.54 mM, 0.545 mM, 0.55 mM, 0.555 mM, 0.56 mM, 0.565 mM, 0.57 mM, 0.575 mM, 0.58 mM, 0.585 mM, 0.59 mM, 0.595 mM, 0.6 mM, 0.605 mM, 0.61 mM, 0.615 mM, 0.62 mM, 0.625 mM, 0.63 mM, 0.635 mM, 0.64 mM, 0.645 mM, 0.65 mM, 0.655 mM, 0.66 mM, 0.665 mM, 0.67 mM, 0.675 mM, 0.68 mM, 0.685 mM, 0.69 mM, 0.695 mM, 0.7 mM, 0.705 mM, 0.71 mM, 0.715 mM, 0.72 mM, 0.725 mM, 0.73 mM, 0.735 mM, 0.74 mM, 0.745 mM, 0.75 mM, 0.755 mM, 0.76 mM, 0.765 mM, 0.77 mM, 0.775 mM, 0.78 mM, 0.785 mM, 0.79 mM, 0.795 mM, 0.8 mM, 0.805 mM, 0.81 mM, 0.815 mM, 0.82 mM, 0.825 mM, 0.83 mM, 0.835 mM, 0.84 mM, 0.845 mM, 0.85 mM, 0.855 mM, 0.86 mM, 0.865 mM, 0.87 mM, 0.875 mM, 0.88 mM, 0.885 mM, 0.89 mM, 0.895 mM, 0.9 mM, 0.905 mM, 0.91 mM, 0.915 mM, 0.92 mM, 0.925 mM, 0.93 mM, 0.935 mM, 0.94 mM, 0.945 mM, 0.95 mM, 0.955 mM, 0.96 mM, 0.965 mM, 0.97 mM, 0.975 mM, 0.98 mM, 0.985 mM, 0.99 mM, 0.995 mM, 1 mM.

[0220] In some embodiments of any of the aspects, the PMSF concentration is from 0 to2.5 mM. For example, the PMSF concentration can be 0 mM, 0.01 mM, 0.02 mM, 0.03 mM, 0.04 mM, 0.05 mM, 0.06 mM, 0.07 mM, 0.08 mM, 0.09 mM, 0.1 mM, 0.11 mM, 0.12 mM, 0.13 mM, 0.14 mM, 0.15 mM, 0.16 mM, 0.17 mM, 0.18 mM, 0.19 mM, 0.2 mM, 0.21 mM, 0.22 mM, 0.23 mM, 0.24 mM, 0.25 mM, 0.26 mM, 0.27 mM, 0.28 mM, 0.29 mM, 0.3 mM, 0.31 mM, 0.32 mM, 0.33 mM, 0.34 mM, 0.35 mM, 0.36 mM, 0.37 mM, 0.38 mM, 0.39 mM, 0.4 mM, 0.41 mM, 0.42 mM, 0.43 mM, 0.44 mM, 0.45 mM, 0.46 mM, 0.47 mM, 0.48 mM, 0.49 mM, 0.5 mM, 0.51 mM, 0.52 mM, 0.53 mM, 0.54 mM, 0.55 mM, 0.56 mM, 0.57 mM, 0.58 mM, 0.59 mM, 0.6 mM, 0.61 mM, 0.62 mM, 0.63 mM, 0.64 mM, 0.65 mM, 0.66 mM, 0.67 mM, 0.68 mM, 0.69 mM, 0.7 mM, 0.71 mM, 0.72 mM, 0.73 mM, 0.74 mM, 0.75 mM, 0.76 mM, 0.77 mM, 0.78 mM, 0.79 mM, 0.8 mM, 0.81 mM, 0.82 mM, 0.83 mM, 0.84 mM, 0.85 mM, 0.86 mM, 0.87 mM, 0.88 mM, 0.89 mM, 0.9 mM, 0.91 mM, 0.92 mM, 0.93 mM, 0.94 mM, 0.95 mM, 0.96 mM, 0.97 mM, 0.98 mM, 0.99 mM , 1 mM, 1.01 mM, 1.02 mM, 1.03 mM, 1.04 mM, 1.05 mM, 1.06 mM, 1.07 mM, 1.08 mM, 1.09 mM, 1.1 mM, l.ll mM, 1.12 mM, 1.13 mM, 1.14 mM, 1.15 mM, 1.16 mM, 1.17 mM, 1.18 mM, 1.19 mM, 1.2 mM, 1.21 mM, 1.22 mM, 1.23 mM, 1.24 mM, 1.25 mM, 1.26 mM, 1.27 mM, 1.28 mM, 1.29 mM, 1.3 mM, 1.31 mM, 1.32 mM, 1.33 mM, 1.34 mM, 1.35 mM, 1.36 mM, 1.37 mM, 1.38 mM, 1.39 mM, 1.4 mM, 1.41 mM, 1.42 mM, 1.43 mM, 1.44 mM, 1.45 mM, 1.46 mM, 1.47 mM, 1.48 mM, 1.49 mM, 1.5 mM, 1.51 mM, 1.52 mM, 1.53 mM, 1.54 mM, 1.55 mM, 1.56 mM, 1.57 mM, 1.58 mM, 1.59 mM, 1.6mM, 1.61 mM, 1.62 mM, 1.63 mM, 1.64 mM, 1.65 mM, 1.66 mM, 1.67 mM, 1.68 mM, 1.69 mM, 1.7 mM, 1.71 mM, 1.72 mM, 1.73 mM, 1.74 mM, 1.75 mM, 1.76 mM, 1.77 mM, 1.78 mM, 1.79 mM, 1.8 mM, 1.81 mM, 1.82 mM, 1.83 mM, 1.84 mM, 1.85 mM, 1.86 mM, 1.87 mM, 1.88 mM, 1.89 mM, 1.9mM, 1.91 mM, 1.92 mM, 1.93 mM, 1.94 mM, 1.95 mM, 1.96 mM, 1.97 mM, 1.98 mM, 1.99 mM , 2 mM, 2.01 mM, 2.02 mM, 2.03 mM, 2.04 mM,4934-8116-8174.8Atty. Dkt. No. 043214-000103WO PT2.05 mM, 2.06 mM, 2.07 mM, 2.08 mM, 2.09 mM, 2.1 mM, 2.11 mM, 2.12 mM, 2.13 mM, 2.14 mM,2.15 mM, 2.16 mM, 2.17 mM, 2.18 mM, 2.19 mM, 2.2 mM, 2.21 mM, 2.22 mM, 2.23 mM, 2.24 mM,2.25 mM, 2.26 mM, 2.27 mM, 2.28 mM, 2.29 mM, 2.3 mM, 2.31 mM, 2.32 mM, 2.33 mM, 2.34 mM,2.35 mM, 2.36 mM, 2.37 mM, 2.38 mM, 2.39 mM, 2.4 mM, 2.41 mM, 2.42 mM, 2.43 mM, 2.44 mM,2.45 mM, 2.46 mM, 2.47 mM, 2.48 mM, 2.49 mM, or 2.5 mM.

[0221] In some embodiments of any of the aspects, the temperature is 37 °C, the pH is 7.5, the ionic strength is 50 mM, the histone protein concentration is 20 pM, the engineered polypeptide concentration is 200 pM, the oligoglycine TMT peptide concentration is 1 mM, the PMSF concentration is 0 mM, and the DTT concentration is 1 mM.

[0222] In some embodiments of any of the aspects, the histone tails are isolated using trichloroacetic acid. In some embodiments of any of the aspects, the peptide solution comprising the histone tails is subsequently exchanged into a trifluoroacetic acid solution.

[0223] One aspect of any of the embodiments is a method of attaching a tail to a tailless nucleosome, the method comprising producing a tailless H3 nucleosome using a method described herein and contacting the tailless H3 nucleosome with a histone tail and a engineered polypeptide described herein, under conditions comprising at least one of: a temperature of 37 °C; an ionic strength of 185 mM; a tailless nucleosome concentration of 1.65 pM; an engineered polypeptide concentration of 200 pM; and a histone tail peptide concentration of between 16.5 and 99 pM; thereby ligating at least the histone tail to the tailless H3 nucleosome.

[0224] In some embodiments of any of the aspects, the conditions under which the tailless H3 nucleosome is contacted with a histone tail and engineered polypeptide comprise at least two of: a temperature of 37 °C; an ionic strength of 185 mM; a tailless nucleosome concentration of 1.65 pM; an engineered polypeptide concentration of 200 pM; and a histone tail peptide concentration of between16.5 and 99 pM. In some embodiments of any of the aspects, the conditions under which the tailless H3 nucleosome is contacted with a histone tail and engineered polypeptide comprise at least three of: a temperature of 37 °C; an ionic strength of 185 mM; a tailless nucleosome concentration of 1.65 pM; an engineered polypeptide concentration of 200 pM; and a histone tail peptide concentration of between16.5 and 99 pM. In some embodiments of any of the aspects, the conditions under which the tailless H3 nucleosome is contacted with a histone tail and engineered polypeptide comprise at least four of: a temperature of 37 °C; an ionic strength of 185 mM; a tailless nucleosome concentration of 1.65 pM; an engineered polypeptide concentration of 200 pM; and a histone tail peptide concentration of between16.5 and 99 pM.

[0225] In some embodiments of any of the aspects, the conditions under which the tailless H3 nucleosome is contacted with a histone tail and engineered polypeptide comprise: a temperature of 37 °C; an ionic strength of 185 mM; a tailless nucleosome concentration of 1.65 pM; an engineered4934-8116-8174.8Atty. Dkt. No. 043214-000103WO PT polypeptide concentration of 200 pM; and a histone tail peptide concentration of between 16.5 and 99 pM.

[0226] In some embodiments of any of the aspects, the temperature is 37 °C. In some embodiments of any of the aspects, the ionic strength is 185 mM. In some embodiments of any of the aspects, the ionic strength is a combination of NaCl, HEPES and KC1. In some embodiments of any of the aspects, the tailless nucleosome concentration is 1.65 pM. In some embodiments of any of the aspects, the engineered polypeptide concentration is 200 pM. In some embodiments of any of the aspects, the histone tail peptide concentration is between 16.5 and 99 pM.

[0227] In some embodiments of any of the aspects, the histone tail peptide concentration can be from about 16.5 to about 99 pM. For example, the histone tail peptide concentration can be about16.5 pM, about 17 pM, about 17.5 pM, about 18 pM, about 18.5 pM, about 19 pM, about 19.5 pM, about 20 pM, about 20.5 pM, about 21 pM, about 21.5 pM, about 22 pM, about 22.5 pM, about 23 pM, about 23.5 pM, about 24 pM, about 24.5 pM, about 25 pM, about 25.5 pM, about 26 pM, about26.5 pM, about 27 pM, about 27.5 pM, about 28 pM, about 28.5 pM, about 29 pM, about 29.5 pM, about 30 pM, about 30.5 pM, about 31 pM, about 31.5 pM, about 32 pM, about 32.5 pM, about 33 pM, about 33.5 pM, about 34 pM, about 34.5 pM, about 35 pM, about 35.5 pM, about 36 pM, about36.5 pM, about 37 pM, about 37.5 pM, about 38 pM, about 38.5 pM, about 39 pM, about 39.5 pM, about 40 pM, about 40.5 pM, about 41 pM, about 41.5 pM, about 42 pM, about 42.5 pM, about 43 pM, about 43.5 pM, about 44 pM, about 44.5 pM, about 45 pM, about 45.5 pM, about 46 pM, about46.5 pM, about 47 pM, about 47.5 pM, about 48 pM, about 48.5 pM, about 49 pM, about 49.5 pM, about 50 pM, about 50.5 pM, about 51 pM, about 51.5 pM, about 52 pM, about 52.5 pM, about 53 pM, about 53.5 pM, about 54 pM, about 54.5 pM, about 55 pM, about 55.5 pM, about 56 pM, about56.5 pM, about 57 pM, about 57.5 pM, about 58 pM, about 58.5 pM, about 59 pM, about 59.5 pM, about 60 pM, about 60.5 pM, about 61 pM, about 61.5 pM, about 62 pM, about 62.5 pM, about 63 pM, about 63.5 pM, about 64 pM, about 64.5 pM, about 65 pM, about 65.5 pM, about 66 pM, about66.5 pM, about 67 pM, about 67.5 pM, about 68 pM, about 68.5 pM, about 69 pM, about 69.5 pM, about 70 pM, about 70.5 pM, about 71 pM, about 71.5 pM, about 72 pM, about 72.5 pM, about 73 pM, about 73.5 pM, about 74 pM, about 74.5 pM, about 75 pM, about 75.5 pM, about 76 pM, about76.5 pM, about 77 pM, about 77.5 pM, about 78 pM, about 78.5 pM, about 79 pM, about 79.5 pM, about 80 pM, about 80.5 pM, about 81 pM, about 81.5 pM, about 82 pM, about 82.5 pM, about 83 pM, about 83.5 pM, about 84 pM, about 84.5 pM, about 85 pM, about 85.5 pM, about 86 pM, about86.5 pM, about 87 pM, about 87.5 pM, about 88 pM, about 88.5 pM, about 89 pM, about 89.5 pM, about 90 pM, about 90.5 pM, about 91 pM, about 91.5 pM, about 92 pM, about 92.5 pM, about 93 pM, about 93.5 pM, about 94 pM, about 94.5 pM, about 95 pM, about 95.5 pM, about 96 pM, about96.5 pM, about 97 pM, about 97.5 pM, about 98 pM, about 98.5 pM, or about 99 pM.4934-8116-8174.8Atty. Dkt. No. 043214-000103WO PT

[0228] In some embodiments of any of the aspects, the histone tail peptide concentration is about 48 pM, about 48.01 pM, about 48.02 pM, about 48.03 pM, about 48.04 pM, about 48.05 pM, about 48.06 pM, about 48.07 pM, about 48.08 pM, about 48.09 pM, about 48.1 pM, about 48.11 pM, about 48.12 pM, about 48.13 pM, about 48.14 pM, about 48.15 pM, about 48.16 pM, about 48.17 pM, about 48.18 pM, about 48.19 pM, about 48.2 pM, about 48.21 pM, about 48.22 pM, about 48.23 pM, about 48.24 pM, about 48.25 pM, about 48.26 pM, about 48.27 pM, about 48.28 pM, about 48.29 pM, about 48.3 pM, about 48.31 pM, about 48.32 pM, about 48.33 pM, about 48.34 pM, about 48.35 pM, about 48.36 pM, about 48.37 pM, about 48.38 pM, about 48.39 pM, about 48.4 pM, about 48.41 pM, about 48.42 pM, about 48.43 pM, about 48.44 pM, about 48.45 pM, about 48.46 pM, about 48.47 pM, about 48.48 pM, about 48.49 pM, about 48.5 pM, about 48.51 pM, about 48.52 pM, about 48.53 pM, about 48.54 pM, about 48.55 pM, about 48.56 pM, about 48.57 pM, about 48.58 pM, about 48.59 pM, about 48.6 pM, about 48.61 pM, about 48.62 pM, about 48.63 pM, about 48.64 pM, about 48.65 pM, about 48.66 pM, about 48.67 pM, about 48.68 pM, about 48.69 pM, about 48.7 pM, about 48.71 pM, about 48.72 pM, about 48.73 pM, about 48.74 pM, about 48.75 pM, about 48.76 pM, about 48.77 pM, about 48.78 pM, about 48.79 pM, about 48.8 pM, about 48.81 pM, about 48.82 pM, about 48.83 pM, about 48.84 pM, about 48.85 pM, about 48.86 pM, about 48.87 pM, about 48.88 pM, about 48.89 pM, about 48.9 pM, about 48.91 pM, about 48.92 pM, about 48.93 pM, about 48.94 pM, about 48.95 pM, about 48.96 pM, about 48.97 pM, about 48.98 pM, about 48.99 pM, about 49 pM, about 49.01 pM, about 49.02 pM, about 49.03 pM, about 49.04 pM, about 49.05 pM, about 49.06 pM, about 49.07 pM, about 49.08 pM, about 49.09 pM, about 49.1 pM, about 49.11 pM, about 49.12 pM, about 49.13 pM, about 49.14 pM, about 49.15 pM, about 49.16 pM, about 49.17 pM, about 49.18 pM, about 49.19 pM, about 49.2 pM, about 49.21 pM, about 49.22 pM, about 49.23 pM, about 49.24 pM, about 49.25 pM, about 49.26 pM, about 49.27 pM, about 49.28 pM, about 49.29 pM, about 49.3 pM, about 49.31 pM, about 49.32 pM, about 49.33 pM, about 49.34 pM, about 49.35 pM, about 49.36 pM, about 49.37 pM, about 49.38 pM, about 49.39 pM, about 49.4 pM, about 49.41 pM, about 49.42 pM, about 49.43 pM, about 49.44 pM, about 49.45 pM, about 49.46 pM, about 49.47 pM, about 49.48 pM, about 49.49 pM, about 49.5 pM, about 49.51 pM, about 49.52 pM, about 49.53 pM, about 49.54 pM, about 49.55 pM, about 49.56 pM, about 49.57 pM, about 49.58 pM, about 49.59 pM, about 49.6 pM, about 49.61 pM, about 49.62 pM, about 49.63 pM, about 49.64 pM, about 49.65 pM, about 49.66 pM, about 49.67 pM, about 49.68 pM, about 49.69 pM, about 49.7 pM, about 49.71 pM, about 49.72 pM, about 49.73 pM, about 49.74 pM, about 49.75 pM, about 49.76 pM, about 49.77 pM, about 49.78 pM, about 49.79 pM, about 49.8 pM, about 49.81 pM, about 49.82 pM, about 49.83 pM, about 49.84 pM, about 49.85 pM, about 49.86 pM, about 49.87 pM, about 49.88 pM, about 49.89 pM, about 49.9 pM, about 49.91 pM, about 49.92 pM, about 49.93 pM, about 49.94 pM, about 49.95 pM, about 49.96 pM, about 49.97 pM, about 49.98 pM, about 49.99 pM, about 50 pM, about 50.01 pM, about 50.02 pM, about 50.03 pM, about 50.04 pM, about 50.05 pM, about 50.06 pM, about 50.07 pM, about 50.08 pM, about 50.09 pM, about 50.1 pM, about 50.11 pM, about 50.12 pM, about 50.13 pM, about 50.14 pM, about 50.15 pM,4934-8116-8174.8Atty. Dkt. No. 043214-000103WO PT about 50.16 pM, about 50.17 pM, about 50.18 pM, about 50.19 pM, about 50.2 pM, about 50.21 pM, about 50.22 pM, about 50.23 pM, about 50.24 pM, about 50.25 pM, about 50.26 pM, about 50.27 pM, about 50.28 pM, about 50.29 pM, about 50.3 pM, about 50.31 pM, about 50.32 pM, about 50.33 pM, about 50.34 pM, about 50.35 pM, about 50.36 pM, about 50.37 pM, about 50.38 pM, about 50.39 pM, about 50.4 pM, about 50.41 pM, about 50.42 pM, about 50.43 pM, about 50.44 pM, about 50.45 pM, about 50.46 pM, about 50.47 pM, about 50.48 pM, about 50.49 pM, about 50.5 pM, about 50.51 pM, about 50.52 pM, about 50.53 pM, about 50.54 pM, about 50.55 pM, about 50.56 pM, about 50.57 pM, about 50.58 pM, about 50.59 pM, about 50.6 pM, about 50.61 pM, about 50.62 pM, about 50.63 pM, about 50.64 pM, about 50.65 pM, about 50.66 pM, about 50.67 pM, about 50.68 pM, about 50.69 pM, about 50.7 pM, about 50.71 pM, about 50.72 pM, about 50.73 pM, about 50.74 pM, about 50.75 pM, about 50.76 pM, about 50.77 pM, about 50.78 pM, about 50.79 pM, about 50.8 pM, about 50.81 pM, about 50.82 pM, about 50.83 pM, about 50.84 pM, about 50.85 pM, about 50.86 pM, about 50.87 pM, about 50.88 pM, about 50.89 pM, about 50.9 pM, about 50.91 pM, about 50.92 pM, about 50.93 pM, about 50.94 pM, about 50.95 pM, about 50.96 pM, about 50.97 pM, about 50.98 pM, about 50.99 pM, or about 51 pM.

[0229] In some embodiments of any of the aspects, the histone tail peptide concentration can be from 16.5 to 99 pM. For example, the histone tail peptide concentration can be 16.5 pM, 17 pM,17.5 pM, 18 pM, 18.5 pM, 19 pM, 19.5 pM, 20 pM, 20.5 pM, 21 pM, 21.5 pM, 22 pM, 22.5 pM, 23 pM, 23.5 pM, 24 pM, 24.5 pM, 25 pM, 25.5 pM, 26 pM, 26.5 pM, 27 pM, 27.5 pM, 28 pM,28.5 pM, 29 pM, 29.5 pM, 30 pM, 30.5 pM, 31 pM, 31.5 pM, 32 pM, 32.5 pM, 33 pM, 33.5 pM, 34 pM, 34.5 pM, 35 pM, 35.5 pM, 36 pM, 36.5 pM, 37 pM, 37.5 pM, 38 pM, 38.5 pM, 39 pM,39.5 pM, 40 pM, 40.5 pM, 41 pM, 41.5 pM, 42 pM, 42.5 pM, 43 pM, 43.5 pM, 44 pM, 44.5 pM, 45 pM, 45.5 pM, 46 pM, 46.5 pM, 47 pM, 47.5 pM, 48 pM, 48.5 pM, 49 pM, 49.5 pM, 50 pM,50.5 pM, 51 pM, 51.5 pM, 52 pM, 52.5 pM, 53 pM, 53.5 pM, 54 pM, 54.5 pM, 55 pM, 55.5 pM, 56 pM, 56.5 pM, 57 pM, 57.5 pM, 58 pM, 58.5 pM, 59 pM, 59.5 pM, 60 pM, 60.5 pM, 61 pM,61.5 pM, 62 pM, 62.5 pM, 63 pM, 63.5 pM, 64 pM, 64.5 pM, 65 pM, 65.5 pM, 66 pM, 66.5 pM, 67 pM, 67.5 pM, 68 pM, 68.5 pM, 69 pM, 69.5 pM, 70 pM, 70.5 pM, 71 pM, 71.5 pM, 72 pM,72.5 pM, 73 pM, 73.5 pM, 74 pM, 74.5 pM, 75 pM, 75.5 pM, 76 pM, 76.5 pM, 77 pM, 77.5 pM, 78 pM, 78.5 pM, 79 pM, 79.5 pM, 80 pM, 80.5 pM, 81 pM, 81.5 pM, 82 pM, 82.5 pM, 83 pM,83.5 pM, 84 pM, 84.5 pM, 85 pM, 85.5 pM, 86 pM, 86.5 pM, 87 pM, 87.5 pM, 88 pM, 88.5 pM, 89 pM, 89.5 pM, 90 pM, 90.5 pM, 91 pM, 91.5 pM, 92 pM, 92.5 pM, 93 pM, 93.5 pM, 94 pM,94.5 pM, 95 pM, 95.5 pM, 96 pM, 96.5 pM, 97 pM, 97.5 pM, 98 pM, 98.5 pM, or 99 pM.

[0230] In some embodiments of any of the aspects, the histone tail peptide concentration is 48 pM, 48.01 pM, 48.02 pM, 48.03 pM, 48.04 pM, 48.05 pM, 48.06 pM, 48.07 pM, 48.08 pM,48.09 pM, 48.1 pM, 48.11 pM, 48.12 pM, 48.13 pM, 48.14 pM, 48.15 pM, 48.16 pM, 48.17 pM,48.18 pM, 48.19 pM, 48.2 pM, 48.21 pM, 48.22 pM, 48.23 pM, 48.24 pM, 48.25 pM, 48.26 pM,48.27 pM, 48.28 pM, 48.29 pM, 48.3 pM, 48.31 pM, 48.32 pM, 48.33 pM, 48.34 pM, 48.35 pM,4934-8116-8174.8Atty. Dkt. No. 043214-000103WO PT48.36 pM, 48.37 pM, 48.38 pM, 48.39 pM, 48.4 pM, 48.41 pM, 48.42 pM, 48.43 pM, 48.44 pM,48.45 pM, 48.46 pM, 48.47 pM, 48.48 pM, 48.49 pM, 48.5 pM, 48.51 pM, 48.52 pM, 48.53 pM,48.54 pM, 48.55 pM, 48.56 pM, 48.57 pM, 48.58 pM, 48.59 pM, 48.6 pM, 48.61 pM, 48.62 pM,48.63 pM, 48.64 pM, 48.65 pM, 48.66 pM, 48.67 pM, 48.68 pM, 48.69 pM, 48.7 pM, 48.71 pM,48.72 pM, 48.73 pM, 48.74 pM, 48.75 pM, 48.76 pM, 48.77 pM, 48.78 pM, 48.79 pM, 48.8 pM,48.81 pM, 48.82 pM, 48.83 pM, 48.84 pM, 48.85 pM, 48.86 pM, 48.87 pM, 48.88 pM, 48.89 pM, 48.9 pM, 48.91 pM, 48.92 pM, 48.93 pM, 48.94 pM, 48.95 pM, 48.96 pM, 48.97 pM, 48.98 pM, 48.99 pM, 49 pM, 49.01 pM, 49.02 pM, 49.03 pM, 49.04 pM, 49.05 pM, 49.06 pM, 49.07 pM,49.08 pM, 49.09 pM, 49.1 pM, 49.11 pM, 49.12 pM, 49.13 pM, 49.14 pM, 49.15 pM, 49.16 pM,49.17 pM, 49.18 pM, 49.19 pM, 49.2 pM, 49.21 pM, 49.22 pM, 49.23 pM, 49.24 pM, 49.25 pM,49.26 pM, 49.27 pM, 49.28 pM, 49.29 pM, 49.3 pM, 49.31 pM, 49.32 pM, 49.33 pM, 49.34 pM,49.35 pM, 49.36 pM, 49.37 pM, 49.38 pM, 49.39 pM, 49.4 pM, 49.41 pM, 49.42 pM, 49.43 pM,49.44 pM, 49.45 pM, 49.46 pM, 49.47 pM, 49.48 pM, 49.49 pM, 49.5 pM, 49.51 pM, 49.52 pM,49.53 pM, 49.54 pM, 49.55 pM, 49.56 pM, 49.57 pM, 49.58 pM, 49.59 pM, 49.6 pM, 49.61 pM,49.62 pM, 49.63 pM, 49.64 pM, 49.65 pM, 49.66 pM, 49.67 pM, 49.68 pM, 49.69 pM, 49.7 pM,49.71 pM, 49.72 pM, 49.73 pM, 49.74 pM, 49.75 pM, 49.76 pM, 49.77 pM, 49.78 pM, 49.79 pM,49.8 pM, 49.81 pM, 49.82 pM, 49.83 pM, 49.84 pM, 49.85 pM, 49.86 pM, 49.87 pM, 49.88 pM,49.89 pM, 49.9 pM, 49.91 pM, 49.92 pM, 49.93 pM, 49.94 pM, 49.95 pM, 49.96 pM, 49.97 pM,49.98 pM, 49.99 pM, 50 pM, 50.01 pM, 50.02 pM, 50.03 pM, 50.04 pM, 50.05 gM, 50.06 gM,50.07 gM, 50.08 gM, 50.09 gM, 50.1 gM, 50.11 gM, 50.12 gM, 50.13 gM, 50.14 gM, 50.15 gM,50.16 gM, 50.17 gM, 50.18 gM, 50.19 gM, 50.2 gM, 50.21 gM, 50.22 gM, 50.23 gM, 50.24 gM,50.25 gM, 50.26 gM, 50.27 gM, 50.28 gM, 50.29 gM, 50.3 gM, 50.31 gM, 50.32 gM, 50.33 gM,50.34 jiM, 50.35 jiM, 50.36 jiM, 50.37 jiM, 50.38 jiM, 50.39 jiM, 50.4 jiM, 50.41 jiM, 50.42 jiM,50.43 gM, 50.44 gM, 50.45 gM, 50.46 gM, 50.47 gM, 50.48 gM, 50.49 gM, 50.5 gM, 50.51 gM,50.52 gM, 50.53 gM, 50.54 gM, 50.55 gM, 50.56 gM, 50.57 gM, 50.58 gM, 50.59 gM, 50.6 gM,50.61 jiM, 50.62 jiM, 50.63 jiM, 50.64 jiM, 50.65 jiM, 50.66 jiM, 50.67 jiM, 50.68 jiM, 50.69 jiM,50.7 gM, 50.71 gM, 50.72 gM, 50.73 gM, 50.74 gM, 50.75 gM, 50.76 gM, 50.77 gM, 50.78 gM,50.79 gM, 50.8 gM, 50.81 gM, 50.82 gM, 50.83 gM, 50.84 gM, 50.85 gM, 50.86 gM, 50.87 gM,50.88 jiM, 50.89 jiM, 50.9 jiM, 50.91 jiM, 50.92 jiM, 50.93 jiM, 50.94 jiM, 50.95 jiM, 50.96 jiM,50.97 jiM, 50.98 jiM, 50.99 jiM, or 51 gM.

[0231] In some embodiments of any of the aspects, the temperature is 37 °C, the ionic strength is 185 mM, the tailless nucleosome concentration is 1.65 pM, the engineered polypeptide concentration is 200 pM, and the histone tail peptide concentration is 49.5 pM.

[0232] One aspect of any of the embodiments is a method for late-stage tail modification of a nucleosome, or a method of chemo-enzymatic H3 histone tagging, the method comprising producing a modified nucleosome using a method described herein, and modifying at least one residue of at least one histone tail. In some embodiments of any of the aspects, the modification is an acylation, an4934-8116-8174.8Atty. Dkt. No. 043214-000103WO PT alkylation, a ubiquitination, a citrullination, or a phosphorylation. In some embodiments of any of the aspects, the modification is an acylation. In some embodiments of any of the aspects, the modification is an alkylation. In some embodiments of any of the aspects, the modification is a ubiquitination. In some embodiments of any of the aspects, the modification is a citrullination. In some embodiments of any of the aspects, the modification is a phosphorylation.

[0233] As used herein, “late-stage modification” refers to the process of chemically modifying a peptide after its initial synthesis and purification. Late-stage modification can include PTMs or functionalization with analytical tags and can be useful in the preparation of a library of peptide analogs for screening purposes.

[0234] In some embodiments of any of the aspects, the modification is selected from the group consisting of: K4mel, K4me2, K4me3, K9ac, K14ac, K18ac, K23ac, K27ac, Kacyl, K9propionyl, K9butyryl, K9crotonyl, K9octanoyl, K91actoyl, K9succinyl, K9-a-hydroxyisobutyryl, K9- - hydroxybutyryl, K9-N-methylthiourea, K9norleucine, K9myristoyl, S28phosphoryl, S3 Iphosphoryl, K4me / K9ac, R8Cit / K9ac, K4me2 / K14ac, K9me2 / K14ac, K9me3 / K14ac, K9me2 / K18ac, K18ac / K27me2, K14ac / K18ac, K18UB / K23UB, K9me3 / K18Ub / K23Ub, K9ac / K14ac / K18ac / K27ac, and K9ac / K14ac / K18ac / K23ac / K27ac.

[0235] In some embodiments of any of the aspects, the modification is K4mel. In some embodiments of any of the aspects, the modification is K4me2. In some embodiments of any of the aspects, the modification is K4me3. In some embodiments of any of the aspects, the modification is K9ac. In some embodiments of any of the aspects, the modification is K14ac. In some embodiments of any of the aspects, the modification is K18ac. In some embodiments of any of the aspects, the modification is K23ac. In some embodiments of any of the aspects, the modification is K27ac. In some embodiments of any of the aspects, the modification is Kacyl. In some embodiments of any of the aspects, the modification is K9propionyl. In some embodiments of any of the aspects, the modification is K9butyryl. In some embodiments of any of the aspects, the modification is K9crotonyl. In some embodiments of any of the aspects, the modification is K9octanoyl. In some embodiments of any of the aspects, the modification is K9-a-hydroxyisobutyryl. In some embodiments of any of the aspects, the modification is K9-P-hydroxybutyryl. In some embodiments of any of the aspects, the modification is K9-N-methylthiourea. In some embodiments of any of the aspects, the modification is K9norleucine. In some embodiments of any of the aspects, the modification is K9myristoyl. In some embodiments of any of the aspects, the modification is S28phosphoryl. In some embodiments of any of the aspects, the modification is S3 Iphosphoryl. In some embodiments of any of the aspects, the modification is K4me3 / K9ac. In some embodiments of any of the aspects, the modification is R8Cit / K9ac. In some embodiments of any of the aspects, the modification is K4me2 / K14ac. In some embodiments of any of the aspects, the modification is K9me2 / K14ac. In some embodiments of any of the aspects, the modification is K9me3 / K14ac. In some embodiments of any of the aspects, the modification is4934-8116-8174.8Atty. Dkt. No. 043214-000103WO PTK9me2 / K18ac. In some embodiments of any of the aspects, the modification is K9me2 / K18ac. In some embodiments of any of the aspects, the modification is K18ac / K27me2. In some embodiments of any of the aspects, the modification is K14ac / K18ac. In some embodiments of any of the aspects, the modification is K18Ub / K23Ub. In some embodiments of any of the aspects, the modification is K9me3 / K18Ub / K23Ub. In some embodiments of any of the aspects, the modification is K9ac / K14ac / K18ac / K27ac. In some embodiments of any of the aspects, the modification is K9ac / K14ac / Kl 8ac / K23ac / K27ac.

[0236] One aspect of any of the embodiments is a method for late-stage tail modification of nucleosomes, or a method of chemo-enzymatic H3 histone tagging, the method comprising, producing a modified nucleosome using a method described herein, and modifying at least one residue of each of a first histone tail and at least one residues of a second histone tail, wherein the modification of at least one residue of the first histone is the same modification as the modification of at least one residue of the second histone tail. In some embodiments of any of the aspects, wherein the modification is K18ac. In some embodiments of any of the aspects, the modification is K23ac. In some embodiments of any of the aspects, the modification is K27ac. In some embodiments of any of the aspects, the modification is K18Ub / K23Ub. In some embodiments of any of the aspects, the modification is K9me3 / Kl 8UB / K23Ub.

[0237] In some embodiments of any of the aspects, the first and second tails are each on one copy of H3 histone in the same nucleosome. In some embodiments of any of the aspects, the first tail is on H3 histone and the second tail is on another core histone in the same nucleosome. In some embodiments of any of the aspects, the second tail is on H3 histone and the first tail is on another core histone in the same nucleosome. In some embodiments of any of the aspects, the first and second tails are on H3 histone in two different nucleosomes. In some embodiments of any of the aspects, the first tail is on H3 histone and the second tail is on another core histone in a different nucleosome. In some embodiments of any of the aspects, the second tail is on H3 histone and the first tail is on another core histone in a different nucleosome.

[0238] One aspect of any of the embodiments is a method for late-stage tail modification of nucleosomes, the method comprising, producing a modified nucleosome using a method described herein, and modifying at least one residue of at least one of a first histone tail or at least one residue of a second histone tail.

[0239] In some embodiments of any of the aspects, at least one residue of a first histone tail is modified, and at least one residue of a second histone tail is unmodified. In some embodiments of any of the aspects, at least one residue of a first histone tail is unmodified, and at least one residue of a second histone tail is modified. In some embodiments of any of the aspects, at least one residue of a first histone tail is modified, and at least one residue of a second histone tail is modified. In some embodiments of any of the aspects, at least one residue of the first histone tail is a different type of4934-8116-8174.8Atty. Dkt. No. 043214-000103WO PT modification from the type of modification of at least one residue of the second histone tail. In some embodiments of any of the aspects, the first histone tail is unmodified and the second histone tail modification is K18ac. In some embodiments of any of the aspects, the first histone tail is unmodified and the second histone tail modification is K23ac. In some embodiments of any of the aspects, the first histone tail is unmodified and the second histone tail modification is K27ac. In some embodiments of any of the aspects, wherein the first histone tail is unmodified and the second histone tail modification is K4me2. In some embodiments of any of the aspects, the first histone tail is unmodified and the second histone tail modification isa K4me2 / K14ac. In some embodiments of any of the aspects, the first histone tail modification is K18ac and the second histone tail modification is K9ac / K14ac / K23ac / K27ac. In some embodiments of any of the aspects, the first histone tail modification is K23ac and the second histone tail modification is K9ac / K14ac / K18ac / K27ac. In some embodiments of any of the aspects, the first histone tail modification is K27ac and the second histone tail modification is K9ac / K14ac / K18ac / K23ac. In some embodiments of any of the aspects, the first histone tail modification is K18Ub / K23Ub and the second histone tail modification is K9me3. In some embodiments of any of the aspects, the first histone tail modification is K18Ub / K23Ub and the second histone tail is unmodified. In some embodiments of any of the aspects, the first histone tail modification is K9me3 / K18Ub / K23Ub and the second histone tail is unmodified.

[0240] One aspect of any of the embodiments is a method of histone tagging, the method comprising, producing a tailless H3 histone according to a method described herein, and contacting the tailless H3 histone with a histone tail comprising a tag and the engineered polypeptide described herein to provide a tagged H3 histone.

[0241] Histone tagging is a method of ligating an analytical label to a histone protein for use in quantitative or qualitative analysis. For instance, such a tag can be a TMT peptide, which provides a unique readout from mass-spectrometry. In this way, mass-spectrometric readout can be correlated back to specific histone proteins based on the analytical signal of the tag.

[0242] One aspect of any of the embodiments is a method of chemo-enzymatic tagged mass spectrometry, the method comprising, producing a chemo-enzymatic tagged H3 histone using a method described herein, and performing mass spectrometry on the chemo-enzymatic tagged H3 histone.

[0243] Chemo-enzymatic tagging is a labeling or targeting technique which leverages the ability of enzymes to attach chemical groups to target molecules. The modification resulting from the enzyme can then be specifically chemically reacted with a complementary tag, such as a fluorescent or affinity label. Such tagging can be a useful tool in conjunction with mass-spectrometric and other analytical techniques, allowing for specific identification of target molecules via the readout from the tag. Chemo-enzymatic tagging is known in the art, e.g.„ see Rashidian et al., Bioconjug Chem., 24(8): 1277-1294 (2013), the contents of which are incorporated herein in its entirety.4934-8116-8174.8Atty. Dkt. No. 043214-000103WO PT

[0244] One aspect of any of the embodiments is a method of middle-down proteomics of histone tails, the method comprising, producing at least one isolated cleaved histone tail according to a method described herein, and performing mass spectrometry on the at least one isolated cleaved histone tail.

[0245] Middle-down proteomics is a mass-spectrometry based analytical technique to analyze peptides generally between 3 and 10 kDa in size. Bridging the gap between bottom-up and top-down proteomics, middle-down proteomics offers enhanced capability for the detection of multiple PTMs on a single peptide. Middle-down proteomics is known in the art, e.g., see Cristobal et al., Analytical Chemistry, 89(6): 3318-3325 (2017), the contents of which are incorporated herein in its entirety.

[0246] In one aspect of any of the embodiments, described herein is a nucleic acid encoding a polypeptide as described herein. In one aspect of any of the embodiments, described herein is a vector comprising nucleic acid encoding a polypeptide as described herein.

[0247] As used herein, the term “nucleic acid” or “nucleic acid sequence” refers to any molecule, preferably a polymeric molecule, incorporating units of ribonucleic acid, deoxyribonucleic acid or an analog thereof. The nucleic acid can be either single-stranded or double-stranded. A singlestranded nucleic acid can be one nucleic acid strand of a denatured double-stranded DNA. Alternatively, it can be a single-stranded nucleic acid not derived from any double-stranded DNA. In one aspect, the nucleic acid can be DNA. In another aspect, the nucleic acid can be RNA. Suitable nucleic acid molecules are DNA, including genomic DNA or cDNA. Other suitable nucleic acid molecules are RNA, including mRNA.

[0248] In some embodiments, a nucleic acid encoding a polypeptide as described herein (e.g. a engineered sortase polypeptide) is comprised by a vector. In some of the aspects described herein, a nucleic acid sequence encoding a given polypeptide as described herein, or any module thereof, is operably linked to a vector. The term “vector”, as used herein, refers to a nucleic acid construct designed for delivery to a host cell or for transfer between different host cells. As used herein, a vector can be viral or non-viral. The term “vector” encompasses any genetic element that is capable of replication when associated with the proper control elements and that can transfer gene sequences to cells. A vector can include, but is not limited to, a cloning vector, an expression vector, a plasmid, phage, transposon, cosmid, chromosome, virus, virion, etc.

[0249] As used herein, the term “expression vector” refers to a vector that directs expression of an RNA or polypeptide from sequences linked to transcriptional regulatory sequences on the vector. The sequences expressed will often, but not necessarily, be heterologous to the cell. An expression vector may comprise additional elements, for example, the expression vector may have two replication systems, thus allowing it to be maintained in two organisms, for example in human cells for expression and in a prokaryotic host for cloning and amplification. The term “expression” refers to the cellular processes involved in producing RNA and proteins and as appropriate, secreting proteins, including4934-8116-8174.8Atty. Dkt. No. 043214-000103WO PT where applicable, but not limited to, for example, transcription, transcript processing, translation and protein folding, modification and processing. “Expression products” include RNA transcribed from a gene, and polypeptides obtained by translation of mRNA transcribed from a gene. The term “gene” means the nucleic acid sequence, which is transcribed (DNA) to RNA in vitro or in vivo when operably linked to appropriate regulatory sequences. The gene may or may not include regions preceding and following the coding region, e.g. 5' untranslated (5TJTR) or “leader” sequences and 3' UTR or “trailer” sequences, as well as intervening sequences (introns) between individual coding segments (exons).

[0250] As used herein, the term “viral vector” refers to a nucleic acid vector construct that includes at least one element of viral origin and has the capacity to be packaged into a viral vector particle. The viral vector can contain the nucleic acid encoding a polypeptide as described herein in place of non-essential viral genes. The vector and / or particle may be utilized for the purpose of transferring any nucleic acids into cells either in vitro or in vivo. Numerous forms of viral vectors are known in the art.

[0251] By “recombinant vector” is meant a vector that includes a heterologous nucleic acid sequence, or “transgene” that is capable of expression in vivo. It should be understood that the vectors described herein can, in some embodiments, be combined with other suitable compositions and therapies. In some embodiments, the vector is episomal. The use of a suitable episomal vector provides a means of maintaining the nucleotide of interest in the subject in high copy number extra chromosomal DNA thereby eliminating potential effects of chromosomal integration.

[0252] In one aspect of any of the embodiments described herein, there is provided a cell comprising an engineered polypeptide or vector as described herein. In some embodiments of any of the aspects, the cell is a eukaryotic cell, optionally a mammalian cell, optionally a human cell. In one aspect of any of the embodiments, the cell is able to express any of the engineered polypeptides provided herein.

[0253] “Express” or “expression” as used herein refers to the translation from a nucleic acid molecule to give a protein or polypeptide or a portion thereof. In some embodiments, “expression” may refer to the production of protein to elicit the intended effect.

[0254] In another aspect provided herein is a kit. The kit can comprise any of the engineered polypeptides or compositions comprising same provided herein and packaging and materials. Accordingly, in some embodiments the kit comprises an engineered polypeptide as provided herein.

[0255] Generally, the kit comprises an effective amount of the composition, e.g., the engineered polypeptide. As will be appreciated by one of skill in the art, the components of the kit can be supplied in a lyophilized form or a concentrated form that can be diluted or suspended in liquid prior to use. The kit components described herein can be supplied in aliquots or in unit doses.4934-8116-8174.8Atty. Dkt. No. 043214-000103WO PT

[0256] In some embodiments, the compositions described herein can be provided singularly or in any combination as a kit. Such a kit includes the components described herein and packaging materials thereof.

[0257] In addition, a kit optionally comprises informational material. The informational material can be descriptive, instructional, marketing or other material that relates to the methods described herein. The informational material of the kits is not limited in its form. In some embodiments, the informational material can include information about production of the components, concentration, date of expiration, batch or production site information, and so forth. In some embodiments, the informational material relates to methods for using or administering the components of the kit.

[0258] The packaging material can comprise a label or package insert with relevant information.

[0259] In some embodiments, the components in a kit, e.g. the engineered polypeptide(s), can be provided in a watertight or gas tight container which in some embodiments is substantially free of other components of the kit. For example, the compositions described herein can be supplied in more than one container, e.g., it can be supplied in a container having sufficient amount of the compositions for a predetermined number of applications, i.e., doses or administrations, e.g., 1, 2, 3 or greater. One or more components as described herein can be provided in any form, e.g., liquid, dried or lyophilized form. Liquids or components for suspension or solution of the reagents can be provided in sterile form and should not contain microorganisms or other contaminants. When the components described herein are provided in a liquid solution, the liquid solution preferably is an aqueous solution.

[0260] The kit will typically be provided with its various elements included in one package, e.g., a fiber-based, e.g., a cardboard, or polymeric, e.g., a Styrofoam box. The enclosure can be configured so as to maintain a temperature differential between the interior and the exterior, e.g., it can provide insulating properties to keep the reagents at a preselected temperature for a preselected time.Definitions

[0261] As used herein, the terms “protein" and “polypeptide" are used interchangeably to designate a series of amino acid residues, connected to each other by peptide bonds between the alpha-amino and carboxy groups of adjacent residues. The terms "protein", and "polypeptide" refer to a polymer of amino acids, including modified amino acids (e.g., phosphorylated, glycated, glycosylated, etc.) and amino acid analogs, regardless of its size or function. "Protein" and “polypeptide” are often used in reference to relatively large polypeptides, whereas the term "peptide" is often used in reference to small polypeptides, but usage of these terms in the art overlaps. The terms "protein" and "polypeptide" are used interchangeably herein when referring to a gene product and fragments thereof. Thus, exemplary polypeptides or proteins include gene products, naturally occurring proteins, homologs, orthologs, paralogs, fragments and other equivalents, variants, fragments, and analogs of the foregoing.4934-8116-8174.8Atty. Dkt. No. 043214-000103WO PT

[0262] In any of the embodiments described herein, the engineered polypeptide may comprise one or more of the following sequences,4934-8116-8174.8Atty. Dkt. No. 043214-000103WO PT4934-8116-8174.8Atty. Dkt. No. 043214-000103WO PTwherein X is any amino acids and Xnu is one or two amino acids.

[0263] In the various embodiments described herein, it is further contemplated that variants (naturally occurring or otherwise), alleles, homologs, conservatively modified variants, and / or conservative substitution variants of any of the particular polypeptides described are encompassed. As to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters a single amino acid or a small percentage of amino acids in the encoded sequence is a “conservatively modified variant" where the alteration results in the substitution of an amino acid with a chemically similar amino acid and retains the desired activity of the polypeptide. Such conservatively modified variants are in4934-8116-8174.8Atty. Dkt. No. 043214-000103WO PT addition to and do not exclude polymorphic variants, interspecies homologs, and alleles consistent with the disclosure.

[0264] A given amino acid can be replaced by a residue having similar physiochemical characteristics, e.g., substituting one aliphatic residue for another (such as He, Vai, Leu, or Ala for one another), or substitution of one polar residue for another (such as between Lys and Arg; Glu and Asp; or Gin and Asn). Other such conservative substitutions, e.g., substitutions of entire regions having similar hydrophobicity characteristics, are well known. Polypeptides comprising conservative amino acid substitutions can be tested to confirm that a desired activity.

[0265] Amino acids can be grouped according to similarities in the properties of their side chains (in A. L. Lehninger, in Biochemistry, second ed., pp. 73-75, Worth Publishers, New York (1975)): (1) non-polar: Ala (A), Vai (V), Leu (L), He (I), Pro (P), Phe (F), Trp (W), Met (M); (2) uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gin (Q); (3) acidic: Asp (D), Glu (E); (4) basic: Lys (K), Arg (R), His (H). Alternatively, naturally occurring residues can be divided into groups based on common side-chain properties: (1) hydrophobic: Norleucine, Met, Ala, Vai, Leu, He; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; (6) aromatic: Trp, Tyr, Phe. Nonconservative substitutions will entail exchanging a member of one of these classes for another class. Particular conservative substitutions include, for example; Ala into Gly or into Ser; Arg into Lys; Asn into Gin or into His; Asp into Glu; Cys into Ser; Gin into Asn; Glu into Asp; Gly into Ala or into Pro; His into Asn or into Gin; He into Leu or into Vai; Leu into He or into Vai; Lys into Arg, into Gin or into Glu; Met into Leu, into Tyr or into He; Phe into Met, into Leu or into Tyr; Ser into Thr; Thr into Ser; Trp into Tyr; Tyr into Trp; and / or Phe into Vai, into He or into Leu.

[0266] In some embodiments, the polypeptide described herein (or a nucleic acid encoding such a polypeptide) can be a functional fragment of one of the amino acid sequences described herein. As used herein, a “functional fragment” is a fragment or segment of a peptide which retains at least 50% of the wildtype reference polypeptide’s activity according to the assays described herein. A functional fragment can comprise conservative substitutions of the sequences disclosed herein.

[0267] In some embodiments, the polypeptide described herein can be a variant of a sequence described herein. In some embodiments, the variant is a conservatively modified variant. Conservative substitution variants can be obtained by mutations of native nucleotide sequences, for example. A “variant," as referred to herein, is a polypeptide substantially homologous to a native or reference polypeptide, but which has an amino acid sequence different from that of the native or reference polypeptide because of one or a plurality of deletions, insertions or substitutions. Variant polypeptide-encoding DNA sequences encompass sequences that comprise one or more additions, deletions, or substitutions of nucleotides when compared to a native or reference DNA sequence, but that encode a variant protein or fragment thereof that retains activity. A wide variety of PCR-based4934-8116-8174.8Atty. Dkt. No. 043214-000103WO PT site-specific mutagenesis approaches are known in the art and can be applied by the ordinarily skilled artisan.

[0268] A variant amino acid or DNA sequence can be at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more, identical to a native or reference sequence, e.g., to the entirety of the reference sequence. The degree of homology (percent identity) between a native and a mutant sequence can be determined, for example, by comparing the two sequences using freely available computer programs commonly employed for this purpose on the world wide web (e.g. BLASTp or BLASTn with default settings).

[0269] In some embodiments of any of the aspects, a variant can be a polypeptide having at least 90%, at least 95%, at least 98% or greater sequence homology to one of the reference sequences provided herein (e.g., to the entirety of the reference sequence) and retaining the wild-type activity of that reference sequence, e.g., sortase activity. In some embodiments of any of the aspects, a variant can be a polypeptide having at least 90%, at least 95%, at least 98% or greater sequence homology to one of the naturally-occurring reference sequences provided herein (e.g., to the entirety of the reference sequence) and retaining the wild-type activity of that reference sequence, e.g., incretin activity. In some embodiments of any of the aspects, a variant can be a naturally-occurring polypeptide having at least 90%, at least 95%, at least 98% or greater sequence homology to one of the reference sequences provided herein (e.g., to the entirety of the reference sequence) and retaining the wild-type activity of that reference sequence, e.g., sortase activity.

[0270] Alterations of the native amino acid sequence can be accomplished by any of a number of techniques known to one of skill in the art. Mutations can be introduced, for example, at particular loci by synthesizing oligonucleotides containing a mutant sequence, flanked by restriction sites enabling ligation to fragments of the native sequence. Following ligation, the resulting reconstructed sequence encodes an analog having the desired amino acid insertion, substitution, or deletion. Alternatively, oligonucleotide-directed site-specific mutagenesis procedures can be employed to provide an altered nucleotide sequence having particular codons altered according to the substitution, deletion, or insertion required. Techniques for making such alterations are very well established and include, for example, those disclosed by Walder et al. (Gene 42: 133, 1986); Bauer et al. (Gene 37:73, 1985); Craik (BioTechniques, January 1985, 12-19); Smith et al. (Genetic Engineering: Principles and Methods, Plenum Press, 1981); and U.S. Pat. Nos. 4,518,584 and 4,737,462, which are herein incorporated by reference in their entireties. Any cysteine residue not involved in maintaining the proper conformation of the polypeptide also can be substituted, generally with serine, to improve the oxidative stability of the molecule and prevent aberrant crosslinking. Conversely, cysteine bond(s) can be added to the polypeptide to improve its stability or facilitate oligomerization.4934-8116-8174.8Atty. Dkt. No. 043214-000103WO PT

[0271] In some embodiments of any of the aspects, a polypeptide, nucleic acid, or cell as described herein can be engineered. As used herein, “engineered" refers to the aspect of having been manipulated by the hand of man. For example, a polypeptide is considered to be “engineered" when at least one aspect of the polypeptide, e.g., its sequence, has been manipulated by the hand of man to differ from the aspect as it exists in nature. As is common practice and is understood by those in the art, progeny of an engineered cell are typically still referred to as “engineered" even though the actual manipulation was performed on a prior entity.

[0272] In some embodiments of any of the aspects, an engineered polypeptide does not comprise a naturally occurring sortase domain. In some embodiments of any of the aspects, an engineered polypeptide does not comprise a naturally occurring sortase domain sequence. In some embodiments of any of the aspects, the sortase domain’s sequence is not naturally occurring. In some embodiments of any of the aspects, the sortase domain is not naturally occurring.

[0273] In some embodiments of any of the aspects, an engineered polypeptide does not comprise a sortase domain naturally occurring in Staphylococcus aureus. In some embodiments of any of the aspects, an engineered polypeptide does not comprise a sortase domain sequence naturally occurring in Staphylococcus aureus. In some embodiments of any of the aspects, the sortase domain’s sequence is not naturally occurring in Staphylococcus aureus. In some embodiments of any of the aspects, the sortase domain is not naturally occurring in Staphylococcus aureus.

[0274] “Operably linked” refers to an arrangement of elements wherein the components so described are configured so as to perform their usual function. Thus, control elements operably linked to a coding sequence are capable of effecting the expression of the coding sequence. The control elements need not be contiguous with the coding sequence, so long as they function to direct the expression thereof. Thus, for example, intervening untranslated yet transcribed sequences can be present between a promoter sequence and the coding sequence, and the promoter sequence can still be considered "operably linked" to the coding sequence.

[0275] As used herein, “contacting" refers to any suitable means for delivering, or exposing, a first element (e.g., polypeptide or agent) to at least one other element described herein, e.g., an engineered polypeptide. Exemplary delivery methods include, but are not limited to, direct delivery to cell culture medium, perfusion, injection, or other delivery method well known to one skilled in the art. In some embodiments, contacting comprises physical human activity, e.g., an injection; an act of dispensing, mixing, and / or decanting; and / or manipulation of a delivery device or machine.

[0276] The term “statistically significant" or “significantly" refers to statistical significance and generally means a two standard deviation (2SD) or greater difference.

[0277] Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood as4934-8116-8174.8Atty. Dkt. No. 043214-000103WO PT modified in all instances by the term “about.” The term “about” when used in connection with percentages can mean ±1%.

[0278] As used herein, the term “comprising” means that other elements can also be present in addition to the defined elements presented. The use of “comprising” indicates inclusion rather than limitation.

[0279] The term "consisting of' refers to compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment.

[0280] As used herein the term "consisting essentially of' refers to those elements required for a given embodiment. The term permits the presence of additional elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment of the invention.

[0281] The singular terms "a," "an," and "the" include plural referents unless context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below. The abbreviation, "e.g." is derived from the Latin exempli gratia, and is used herein to indicate a non-limiting example. Thus, the abbreviation "e.g." is synonymous with the term "for example".

[0282] It should be understood that this invention is not limited to the particular methodology, protocols, and reagents, etc., described herein and as such can vary. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention, which is defined solely by the claims.

[0283] All patents and other publications; including literature references, issued patents, published patent applications, and co-pending patent applications; cited throughout this application are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications that might be used in connection with the technology described herein. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicants and does not constitute any admission as to the correctness of the dates or contents of these documents.

[0284] Specific elements of any of the foregoing embodiments can be combined or substituted for elements in other embodiments. Furthermore, while advantages associated with certain embodiments of the disclosure have been described in the context of these embodiments, other embodiments may also exhibit such advantages, and not all embodiments need necessarily exhibit such advantages to fall within the scope of the disclosure.4934-8116-8174.8Atty. Dkt. No. 043214-000103WO PT

[0285] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in, or deleted from, a group for reasons of convenience and / or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fillfilling the written description of all Markush groups used in the appended claims.

[0286] Unless otherwise defined herein, scientific and technical terms used in connection with the present application shall have the meanings that are commonly understood by those of ordinary skill in the art to which this disclosure belongs. It should be understood that this invention is not limited to the particular methodology, protocols, and reagents, etc., described herein and as such can vary. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention, which is defined solely by the claims. Definitions of common terms in cell biology, immunology, and molecular biology can be found in The Merck Manual of Diagnosis and Therapy, 20th Edition, published by Merck Sharp & Dohme Corp., 2018 (ISBN 0911910190, 978-0911910421); Robert S. Porter et al. (eds.), The Encyclopedia of Molecular Cell Biology and Molecular Medicine, published by Blackwell Science Ltd., 1999-2012 (ISBN 9783527600908); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8); Immunology by Werner Luttmann, published by Elsevier, 2006; Janeway's Immunobiology, Kenneth Murphy, Allan Mowat, Casey Weaver (eds.), W. W. Norton & Company, 2016 (ISBN 0815345054, 978-0815345053); Lewin's Genes XI, published by Jones & Bartlett Publishers, 2014 (ISBN- 1449659055); Michael Richard Green and Joseph Sambrook, Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (2012) (ISBN 1936113414); Davis et al., Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (2012) (ISBN 044460149X); Laboratory Methods in Enzymology: DNA, Jon Lorsch (ed.) Elsevier, 2013 (ISBN 0124199542); Current Protocols in Molecular Biology (CPMB), Frederick M. Ausubel (ed.), John Wiley and Sons, 2014 (ISBN 047150338X, 9780471503385), Current Protocols in Protein Science (CPPS), John E. Coligan (ed.), John Wiley and Sons, Inc., 2005; and Current Protocols in Immunology (CPI) (John E. Coligan, ADA M Kruisbeek, David H Margulies, Ethan M Shevach, Warren Strobe, (eds.) John Wiley and Sons, Inc., 2003 (ISBN 0471142735, 9780471142737), the contents of which are all incorporated by reference herein in their entireties.

[0287] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. All documents, or portions of documents, cited in this application, including, but not limited to, patents, patent applications, articles, books, and treatises, are hereby expressly incorporated by reference in their entirety for any purpose.4934-8116-8174.8Atty. Dkt. No. 043214-000103WO PT

[0288] The description of embodiments of the disclosure is not intended to be exhaustive or to limit the disclosure to the precise form disclosed. While specific embodiments of, and examples for, the disclosure are described herein for illustrative purposes, various equivalent modifications are possible within the scope of the disclosure, as those skilled in the relevant art will recognize. For example, while method steps or functions are presented in a given order, alternative embodiments may perform functions in a different order, or functions may be performed substantially concurrently. The teachings of the disclosure provided herein can be applied to other procedures or methods as appropriate. The various embodiments described herein can be combined to provide further embodiments. Aspects of the disclosure can be modified, if necessary, to employ the compositions, functions and concepts of the above references and application to provide yet further embodiments of the disclosure. Moreover, due to biological functional equivalency considerations, some changes can be made in protein structure without affecting the biological or chemical action in kind or amount. These and other changes can be made to the disclosure in light of the detailed description. All such modifications are intended to be included within the scope of the appended claims.

[0289] In some embodiments, the present technology may be defined in any of the following numbered paragraphs:Paragraph 1: An engineered polypeptide comprising: a sortase domain comprising the sequence of SEQ ID NO: 14.Paragraph 2: The engineered polypeptide of paragraph 1, wherein Xi of SEQ ID NO: 14 is P, S, or R. Paragraph 3: The engineered polypeptide of paragraph 1, wherein Xi of SEQ ID NO: 14 is S or R. Paragraph 4: The engineered polypeptide of any one of the preceding paragraphs, wherein X2 of SEQ ID NO: 14 is E, A, D, Q, orN.Paragraph 5: The engineered polypeptide of any one of the preceding paragraphs, wherein X2 of SEQ ID NO: 14 is A, D, Q, or N.Paragraph 6: The engineered polypeptide of any one of the preceding paragraphs, wherein X3 of SEQ ID NO: 14 is E, A, D, Q, orN.Paragraph 7: The engineered polypeptide of any one of the preceding paragraphs, wherein X3 of SEQ ID NO: 14 is A, D, Q, or N.Paragraph 8: The engineered polypeptide of any one of the preceding paragraphs, wherein X4 of SEQ ID NO: 14 is E or Q.Paragraph 9: The engineered polypeptide of any one of the preceding paragraphs, wherein X4 of SEQ ID NO: 14 is Q.Paragraph 10: The engineered polypeptide of any one of the preceding paragraphs, wherein X5 of SEQ ID NO: 14 is D, A, E, Q, or N.Paragraph 11: The engineered polypeptide of any one of the preceding paragraphs, wherein X5 of SEQ ID NO: 14 is A, E, Q, or N.4934-8116-8174.8Atty. Dkt. No. 043214-000103WO PTParagraph 12: The engineered polypeptide of any one of the preceding paragraphs, wherein X6 of SEQ ID NO: 14 is S or V.Paragraph 13: The engineered polypeptide of any one of the preceding paragraphs, wherein X6 of SEQ ID NO: 14 is V.Paragraph 14: The engineered polypeptide of any one of the preceding paragraphs, wherein X7 of SEQ ID NO: 14 is M or K.Paragraph 15: The engineered polypeptide of any one of the preceding paragraphs, wherein X7 of SEQ ID NO: 14 is M.Paragraph 16: The engineered polypeptide of any one of the preceding paragraphs, wherein X8 of SEQ ID NO: 14 is D or N.Paragraph 17: The engineered polypeptide of any one of the preceding paragraphs, wherein X8 of SEQ ID NO: 14 is N.Paragraph 18: The engineered polypeptide of any one of the preceding paragraphs, wherein X9 of SEQ ID NO: 14 is Q or T.Paragraph 19: The engineered polypeptide of any one of the preceding paragraphs, wherein X9 of SEQ ID NO: 14 is Q.Paragraph 20: The engineered polypeptide of any one of the preceding paragraphs, wherein X10 of SEQ ID NO: 14 is D or A.Paragraph 21: The engineered polypeptide of any one of the preceding paragraphs, wherein X10 of SEQ ID NO: 14 is A.Paragraph 22: The engineered polypeptide of any one of the preceding paragraphs, wherein XI 112 of SEQ ID NO: 14 is VG, VE or V.Paragraph 23: The engineered polypeptide of any one of the preceding paragraphs, wherein XI 112 of SEQ ID NO: 14 is V.Paragraph 24: The engineered polypeptide of any one of the preceding paragraphs, wherein X13 of SEQ ID NO: 14 is M or V.Paragraph 25: The engineered polypeptide of any one of the preceding claims, wherein XI 3 of SEQ ID NO: 14 is M.Paragraph 26: The engineered polypeptide of any one of the preceding paragraphs, wherein X14 of SEQ ID NO: 14 is H or L.Paragraph 27: The engineered polypeptide of any one of the preceding paragraphs, wherein X14 of SEQ ID NO: 14 is H.Paragraph 28: The engineered polypeptide of any one of the preceding paragraphs, wherein XI 5 of SEQ ID NO: 14 is L or D.Paragraph 29: The engineered polypeptide of any one of the preceding paragraphs, wherein XI 5 of SEQ ID NO: 14 is L.4934-8116-8174.8Atty. Dkt. No. 043214-000103WO PTParagraph 30: The engineered polypeptide of any one of the preceding paragraphs, wherein X16 of SEQ ID NO: 14 is A or E.Paragraph 31: The engineered polypeptide of any one of the preceding paragraphs, wherein XI 6 of SEQ ID NO: 14 is A.Paragraph 32: The engineered polypeptide of any one of the preceding paragraphs, wherein X17 of SEQ ID NO: 14 is Q, E, or R.Paragraph 33: The engineered polypeptide of any one of the preceding paragraphs, wherein X17 of SEQ ID NO: 14 is E or R.Paragraph 34: The engineered polypeptide of any one of the preceding paragraphs, wherein X17 of SEQ ID NO: 14 is E.Paragraph 35: The engineered polypeptide of any one of the preceding paragraphs, wherein XI 8 of SEQ ID NO: 14 is K or E.Paragraph 36: The engineered polypeptide of any one of the preceding paragraphs, wherein XI 8 of SEQ ID NO: 14 is E.Paragraph 37: The engineered polypeptide of any one of the preceding paragraphs, wherein X19 of SEQ ID NO: 14 is K or T.Paragraph 38: The engineered polypeptide of any one of the preceding paragraphs, wherein XI 9 of SEQ ID NO: 14 is T.Paragraph 39: The engineered polypeptide of any one of the preceding paragraphs, wherein X8 of SEQ ID NO: 14 is N, X18 of SEQ ID NO: 14 is E, and X19 of SEQ ID NO: 14 is T.Paragraph 40: The engineered polypeptide of any one of the preceding paragraphs, further comprising at least one of the following: XI of SEQ ID NO: 14 is S or R; X2 of SEQ ID NO: 14 is A, D, Q, or N; X3 of SEQ ID NO: 14 is A, D, Q, or N; X5 of SEQ ID NO: 14 is A, E, Q, or N, X17 of SEQ ID NO: 14 is E or R.Paragraph 41 : The engineered polypeptide of any one of the preceding paragraphs, wherein: XI of SEQ ID NO: 14 is S or R; X2 of SEQ ID NO: 14 is A, D, Q, or N; X3 of SEQ ID NO: 14 is A, D, Q, or N; X5 of SEQ ID NO: 14 is A, E, Q, or N, X17 of SEQ ID NO: 14 is E or R.Paragraph 42: The engineered polypeptide of paragraph 1, wherein XI of SEQ ID NO: 14 is P, X2 is E, X3 is E, X4 is E, X5 is D, X6 is S, X7 is M, X8 is D, X9 is Q, X10 is D, XI 112 is VG, X13 is M, X14 is H, X15 is L, X16 is A, X17 is E, X18 is K, and X19 is K (SEQ ID NO: 3).Paragraph 43: The engineered polypeptide of paragraph 1, wherein XI of SEQ ID NO: 14 is S, X2 is E, X3 is E, X4 is E, X5 is D, X6 is S, X7 is M, X8 is N, X9 is T, X10 is A, XI 112 is V, X13 is M, X14 is H, X15 is L, X16 is A, X17 is E, X18 is K, and X19 is K (SEQ ID NO: 4).Paragraph 44: The engineered polypeptide of paragraph 1, wherein XI of SEQ ID NO: 14 is P, X2 is E, X3 is E, X4 is E, X5 is D, X6 is S, X7 is M, X8 is N, X9 is Q, X10 is D, XI 112 is V, X13 is M, X14 is H, X15 is L, X16 is A, X17 is E, X18 is E, and X19 is T (SEQ ID NO: 5).4934-8116-8174.8Atty. Dkt. No. 043214-000103WO PTParagraph 45: The engineered polypeptide of paragraph 1, wherein XI of SEQ ID NO: 14 is S, X2 is E, X3 is E, X4 is E, X5 is D, X6 is S, X7 is M, X8 is N, X9 is Q, X10 is D, XI 112 is V, X13 is M, X14 is H, X15 is L, X16 is A, X17 is E, X18 is K, and X19 is T (SEQ ID NO: 6).Paragraph 46: The engineered polypeptide of paragraph 1, wherein XI of SEQ ID NO: 14 is S, X2 is E, X3 is E, X4 is E, X5 is D, X6 is S, X7 is M, X8 is N, X9 is Q, X10 is D, XI 112 is V, X13 is M, X14 is H, X15 is L, X16 is A, X17 is E, X18 is E, and X19 is T (SEQ ID NO: 7).Paragraph 47: The engineered polypeptide of paragraph 1, wherein XI of SEQ ID NO: 14 is S, X2 is E, X3 is E, X4 is E, X5 is D, X6 is E, X7 is S, X8 is N, X9 is T, X10 is A, XI 112 is V, X13 is M, X14 is H, X15 is L, X16 is A, X17 is E, X18 is K, and X19 is T (SEQ ID NO: 8).Paragraph 48: The engineered polypeptide of paragraph 1, wherein XI of SEQ ID NO: 14 is S, X2 is E, X3 is E, X4 is E, X5 is D, X6 is S, X7 is M, X8 is N, X9 is Q, X10 is A, XI 112 is V , X13 is M, X14 is H, X15 is L, X16 is A, X17 is E, X18 is E, and X19 is T (SEQ ID NO: 9).Paragraph 49: The engineered polypeptide of paragraph 1, wherein XI of SEQ ID NO: 14 is R, X2 is E, X3 is E, X4 is E, X5 is D, X6 is S, X7 is M, X8 is N, X9 is Q, X10 is ,D XI 112 is V, X13 is M, X14 is H, X15 is L, X16 is A, X17 is E, X18 is E, and X19 is T (SEQ ID NO: 10).Paragraph 50: The engineered polypeptide of paragraph 1, wherein XI of SEQ ID NO: 14 is R, X2 is E, X3 is E, X4 is E, X5 is D, X6 is S, X7 is M, X8 is N, X9 is Q, X10 is A, XI 112 is V, X13 is M, X14 is H, X15 is L, X16 is A, X17 is E, X18 is K, and X19 is T (SEQ ID NO: 11).Paragraph 51: The engineered polypeptide of paragraph 1, wherein XI of SEQ ID NO: 14 is S, X2 is E, X3 is E, X4 is Q, X5 is D, X6 is V, X7 is M, X8 is N, X9 is Q, X10 is D, XI 112 is V, X13 is M, X14 is H, X15 is L, X16 is A, X17 is E, X18 is E, and X19 is T (SEQ ID NO: 12).Paragraph 52: The engineered polypeptide of paragraph 1, wherein the sortase domain does not comprise SEQ ID NO: 3, SEQ ID NO: 24, SEQ ID NO: 19, SEQ ID NO: 23, SEQ ID NO: 20, or SEQ ID NO 25.Paragraph 53 : The engineered polypeptide of any of paragraphs 42-51 , further comprising SEQ ID NO: 36.Paragraph 54: The engineered polypeptide of any of paragraphs 42-51 , further comprising SEQ ID NO: 36 at the C-terminus.Paragraph 55: The engineered polypeptide of any one of the preceding paragraphs, further comprising at least one split intein sequence.Paragraph 56: The engineered polypeptide of any one of the preceding paragraphs, further comprising a pair of split intein sequences.Paragraph 57: The engineered polypeptide of paragraph 56, wherein the pair of split intein sequences flank the sortase domain.Paragraph 58: The engineered polypeptide of paragraphs 56 or 57, further comprising linker domains between each of the pair of split intein sequences and the sortase domain.4934-8116-8174.8Atty. Dkt. No. 043214-000103WO PTParagraph 59: The engineered polypeptide of paragraphs 56-58, further comprising, from N-terminus to C-terminus, an N-terminus split intein comprising the sequence of SEQ ID NO: 15, an N-terminus linker comprising the sequence of SEQ ID NO: 16, the sortase domain, a C-terminus linker comprising the sequence of SEQ ID NO: 17, and a C-terminus split intein comprising the sequence of SEQ ID NO: 18.Paragraph 60: The engineered polypeptide of any one of the preceding paragraphs, wherein the polypeptide is circularized.Paragraph 61 : A method of producing a circularized engineered polypeptide comprising a sortase domain, the method comprising: expressing the engineered polypeptide of paragraphs 55-59, thereby excising the intein sequences and producing the circularized engineered polypeptide comprising the sortase domain.Paragraph 62: The method of paragraph 61, wherein the circularized engineered polypeptide is produced through peptide bond formation between the N-terminus of the N-terminus linker and the C- terminus of the C-terminus linker.Paragraph 63: A method of producing a tailless H3 histone, the method comprising: contacting the engineered polypeptide of any of paragraphs 1 - 62 with a composition comprising at least one tailed H3 histone; and separating at least one cleaved histone tail peptide from at least one tailless H3 histone.Paragraph 64: The method of paragraph 63, wherein the composition comprising at least one tailed H3 histone is a crude nuclear extract.Paragraph 65: The method of any one of paragraphs 63 and 64, wherein separating the at least one cleaved histone tail peptide from the at least one tailless H3 histone comprises trichloroacetic acid precipitation.Paragraph 66: The method of paragraph 63, wherein the composition comprising at least one tailless H3 histone further comprises at least one co-expressed unmodified core histone.Paragraph 67: A method for producing an intact histone octamer, the method comprising: producing core histones comprising tailless H3 histones using the method of any one of paragraphs 63-66, and assembling the intact histone octamer via octamer assembly.Paragraph 68: A method of producing a modified nucleosome, the method comprising: producing an intact histone octamer using the method of paragraph 67, and wrapping the intact histone octamer with DNA.Paragraph 69: A method for producing a tailless H3 nucleosome, the method comprising: producing an intact histone octamer comprising tailless H3 histones using the method of paragraph 67, and wrapping the intact histone octamer with DNA to form a H3 tailless nucleosome.Paragraph 70: The method of paragraphs 68 or 69, wherein the DNA is 147 bp in length. Paragraph 71 : The method of paragraphs 68 or 69, wherein the DNA is 185 bp in length.4934-8116-8174.8Atty. Dkt. No. 043214-000103WO PTParagraph 72: A method for histone tail isolation, the method comprising: contacting at least one isolated histone protein with: an engineered polypeptide of any one of paragraphs 1-60 and an oligoglycine tandem mass tag (TMT) peptide, under conditions comprising at least one of: a temperature of between 37 and 42 °C; a pH of between 7.0 and 7.5; an ionic strength of between 30 and 50 mM; a histone protein concentration of between 10 and 20 pM; an engineered polypeptide concentration of between 100 and 400 pM; an oligoglycine TMT peptide concentration of between 0.25 and 1 mM; a phenylmethylsulfonyl fluoride (PMSF) concentration of between 0 and 2.5 mM; and a dithiothreitol (DTT) concentration of between 1 and 10 mM; thereby cleaving at least one histone tail from the at least one histone protein; and isolating the at least one cleaved histone tail. Paragraph 73: The method of paragraph 72, wherein the temperature is between 37 and 42 °C. Paragraph 74: The method of paragraphs 72 or 73, wherein the pH is between 7.0 and 7.5.Paragraph 75: The method of any of paragraphs 72-74, wherein the ionic strength is between 30 and 50 mM.Paragraph 76: The method of any of paragraphs 72-75, wherein the histone protein concentration is between 10-20 pM.Paragraph 77: The method of any of paragraphs 72-76, wherein the engineered polypeptide concentration is between 100 and 400 pM.Paragraph 78: The method of any of paragraphs 72-77, wherein the oligoglycine TMT peptide concentration is between 0.25 and 1 mM.Paragraph 79: The method of any of paragraphs 72-78, wherein the PMSF concentration is less than2.5 mM.Paragraph 80: The method of paragraph 72, wherein the temperature is 37 °C, the pH is 7.5, the ionic strength is 50 mM, the histone protein concentration is 20 pM, the engineered polypeptide concentration is 200 pM, the oligoglycine TMT peptide concentration is 1 mM, the PMSF concentration is 0 mM, and the DTT concentration is 1 mM.Paragraph 81: The method of any of paragraphs 72-80, wherein the oligoglycine TMT peptide has the structure, wherein: a is 0, 1, or 2; b is 1, 2, 3, or 4; X is4934-8116-8174.8Atty. Dkt. No. 043214-000103WO PT, denotes the attachment point to the oligoglycine TMT peptide; and Z is H, C(O)NH2, or C(O)OH.Paragraph 82: The method of any of paragraphs 72-80, wherein the oligoglycine TMT peptide isParagraph 83: The method of any of paragraphs 72-82, wherein the histone tails are isolated using trichloroacetic acid.Paragraph 84: The method of any of paragraphs 72-83, wherein the peptide solution comprising the histone tails is subsequently exchanged into a trifluoroacetic acid solution.Paragraph 85: A method of attaching a tail to a tailless nucleosome, the method comprising: producing a tailless H3 nucleosome using the method paragraph 69; and contacting the tailless H3 nucleosome with a histone tail and the engineered polypeptide of any of paragraphs 1-60; under conditions comprising at least one of: a temperature of 37 °C; an ionic strength of 185 mM; a tailless nucleosome concentration of 1.65 pM; an engineered polypeptide concentration of 200 pM; and a histone tail peptide concentration of between 16.5 and 99 pM; thereby ligating at least the histone tail to the tailless H3 nucleosome.Paragraph 86: The method of paragraph 85, wherein the temperature is 37 °C.Paragraph 87: The method of paragraphs 85 or 86, wherein the ionic strength is 185 mM.Paragraph 88: The method of any of paragraphs 85-87, wherein the ionic strength is a combination of NaCl, HEPES, and KC1.Paragraph 89: The method of any of paragraphs 85-88, wherein the tailless nucleosome concentration is 1.65 pM.Paragraph 90: The method of any of paragraphs 85-89, wherein the engineered polypeptide concentration is 200 pM.Paragraph 91 : The method of any of paragraphs 85-90, wherein the histone tail peptide concentration is between 16.5 and 99 pM.4934-8116-8174.8Atty. Dkt. No. 043214-000103WO PTParagraph 92:The method of paragraph 85, wherein the temperature is 37 °C, the ionic strength is 185 mM, the tailless nucleosome concentration is 1.65 pM, the engineered polypeptide concentration is 200 pM, and the histone tail peptide concentration is 49.5 pM.Paragraph 93: A method for late-stage tail modification of a nucleosome, or a method of chemo- enzymatic H3 histone tagging, the method comprising: producing a modified nucleosome using the method of any one of paragraphs 68-71, and modifying at least one residue of at least one histone tail. Paragraph 94: The method of paragraphs 93, wherein the modification is an acylation, an alkylation, a ubiquitination, a citrullination, or a phosphorylation.Paragraph 95: The method of paragraphs 93 or 94, wherein the modification is selected from the group consisting of: K4mel, K4me2, K4me3, K9ac, K14ac, K18ac, K23ac, K27ac, Kacyl, K9propionyl, K9butyryl, K9crotonyl, K9octanoyl, K91actoyl, K9succinyl, K9-a-hydroxyisobutyryl, K9-P-hydroxybutyryl, K9-N-methylthiourea, K9norleucine, K9myristoyl, S28phosphoryl, S31phosphoryl, K4me / K9ac, R8Cit / K9ac, K4me2 / K14ac, K9me2 / K14ac, K9me3 / K14ac, K9me2 / K18ac, K18ac / K27me2, K14ac / K18ac, K18UB / K23UB, K9me3 / K18Ub / K23Ub, K9ac / K14ac / K18ac / K27ac, and K9ac / K14ac / K18ac / K23ac / K27ac.Paragraph 96: The method of paragraphs 93 or 94, wherein the modification is K4mel . Paragraph 97: The method of paragraphs 93 or 94, wherein the modification is K4me2. Paragraph 98: The method of paragraphs 93 or 94, wherein the modification is K4me3. Paragraph 99: The method of paragraphs 93 or 94, wherein the modification is K9ac. Paragraph 100: The method of paragraphs 93 or 94, wherein the modification is K14ac. Paragraph 101: The method of paragraphs 93 or 94, wherein the modification is K18ac. Paragraph 102: The method of paragraphs 93 or 94, wherein the modification is K23ac. Paragraph 103: The method of paragraphs 93 or 94, wherein the modification is K27ac. Paragraph 104: The method of paragraphs 93 or 94, wherein the modification is Kacyl.Paragraph 105: The method of paragraphs 93 or 94, wherein the modification is K9propionyl. Paragraph 106: The method of paragraphs 93 or 94, wherein the modification is K9butyryl. Paragraph 107: The method of paragraphs 93 or 94, wherein the modification is K9crotonyl. Paragraph 108: The method of paragraphs 93 or 94, wherein the modification is K9octanoyl. Paragraph 109: The method of paragraphs 93 or 94, wherein the modification is K9-a- hydroxyisobutyryl.Paragraph 110: The method of paragraphs 93 or 94, wherein the modification is K9-P-hydroxybutyryl. Paragraph 111: The method of paragraphs 93 or 94, wherein the modification is K9-N-methylthiourea. Paragraph 112: The method of paragraphs 93 or 94, wherein the modification is K9norleucine. Paragraph 113: The method of paragraphs 93 or 94, wherein the modification is K9myristoyl. Paragraph 114: The method of paragraphs 93 or 94, wherein the modification is S28phosphoryl. Paragraph 115: The method of paragraphs 93 or 94, wherein the modification is S3 Iphosphoryl. Paragraph 116: The method of paragraphs 93 or 94, wherein the modification is K4me3 / K9ac.4934-8116-8174.8Atty. Dkt. No. 043214-000103WO PTParagraph 117: The method of paragraphs 93 or 94, wherein the modification is R8Cit / K9ac. Paragraph 118: The method of paragraphs 93 or 94, wherein the modification is K4me2 / K14ac. Paragraph 119: The method of paragraphs 93 or 94, wherein the modification is K9me2 / K14ac. Paragraph 120: The method of paragraphs 93 or 94, wherein the modification is K9me3 / K14ac. Paragraph 121: The method of paragraphs 93 or 94, wherein the modification is K9me2 / K18ac. Paragraph 122: The method of paragraphs 93 or 94, wherein the modification is K18ac / K27me2. Paragraph 123: The method of paragraphs 93 or 94, wherein the modification is K14ac / K18ac. Paragraph 124: The method of paragraphs 93 or 94, wherein the modification is K18Ub / K23Ub. Paragraph 125: The method of paragraphs 93 or 94, wherein the modification is K9me3 / K18Ub / K23Ub.Paragraph 126: The method of paragraphs 93 or 94, wherein the modification is K9ac / K14ac / Kl 8ac / K27ac.Paragraph 127: The method of paragraphs 93 or 94, wherein the modification is K9ac / K14ac / Kl 8ac / K23ac / K27ac.Paragraph 128: A method for late-stage tail modification of nucleosomes, or a method of chemo- enzymatic H3 histone tagging, the method comprising: producing a modified nucleosome using the method of any one of paragraphs 68-71, and modifying at least one residue of each of a first histone tail and at least one residues of a second histone tail, wherein the modification of at least one residue of the first histone is the same modification as the modification of at least one residue of the second histone tail.Paragraph 129: The method of paragraph 128, wherein the modification is K18ac.Paragraph 130: The method of paragraph 128, wherein the modification is K23ac.Paragraph 131: The method of paragraph 128, wherein the modification is K27ac.Paragraph 132: The method of paragraph 128, wherein the modification is K18Ub / K23Ub.Paragraph 133: The method of paragraph 128, wherein the modification is K9me3 / K18UB / K23Ub.Paragraph 134: A method for late-stage tail modification of nucleosomes, the method comprising: producing a modified nucleosome using the method of any one of paragraphs 68-71, and modifying at least one residue of at least one of a first histone tail or at least one residue of a second histone tail. Paragraph 135: The method of paragraph 134, wherein at least one residue of a first histone tail is modified, and at least one residue of a second histone tail is unmodified.Paragraph 136: The method of paragraph 134, wherein at least one residue of a first histone tail is unmodified, and at least one residue of a second histone tail is modified.Paragraph 137: The method of paragraph 134, wherein at least one residue of a first histone tail is modified, and at least one residue of a second histone tail is modified.Paragraph 138: The method of paragraph 134, wherein the modification of at least one residue of the first histone tail is a different type of modification from the type of modification of at least one residue of the second histone tail.4934-8116-8174.8Atty. Dkt. No. 043214-000103WO PTParagraph 139: The method of paragraphs 134 or 136, wherein the first histone tail is unmodified and the second histone tail modification is K18ac.Paragraph 140: The method of paragraphs 134 or 136, wherein the first histone tail is unmodified and the second histone tail modification is K23ac.Paragraph 141: The method of paragraphs 134 or 136, wherein the first histone tail is unmodified and the second histone tail modification is K27ac.Paragraph 142: The method of paragraphs 134 or 136, wherein the first histone tail is unmodified and the second histone tail modification is K4me2.Paragraph 143: The method of paragraphs 134 or 136, wherein the first histone tail is unmodified and the second histone tail modification isa K4me2 / K14ac.Paragraph 144: The method of paragraphs 134 or 137, wherein the first histone tail modification is K18ac and the second histone tail modification is K9ac / K14ac / K23ac / K27ac.Paragraph 145: The method of paragraphs 134 or 137, wherein the first histone tail modification is K23ac and the second histone tail modification is K9ac / K14ac / K18ac / K27ac.Paragraph 146: The method of paragraphs 134 or 137, wherein the first histone tail modification is K27ac and the second histone tail modification is K9ac / K14ac / K18ac / K23ac.Paragraph 147: The method of paragraphs 134 or 137, wherein the first histone tail modification is K18Ub / K23Ub and the second histone tail modification is K9me3.Paragraph 148: The method of paragraphs 134 or 135, wherein the first histone tail modification is K18Ub / K23Ub and the second histone tail is unmodified.Paragraph 149: The method of paragraphs 134 or 135, wherein the first histone tail modification is K9me3 / K18Ub / K23Ub and the second histone tail is unmodified.Paragraph 150: A method of histone tagging, the method comprising: producing the tailless H3 histone according to the method of any one of paragraphs 63-66, and contacting the tailless H3 histone with a histone tail comprising a tag and the engineered polypeptide of any of claims 1-60 to provide a tagged H3 histone.Paragraph 151 : A method of chemo-enzymatic tagged mass spectrometry, the method comprising: producing a chemo-enzymatic tagged H3 histone using the method of paragraph 150, and performing mass spectrometry on the chemo-enzymatic tagged H3 histone.Paragraph 152: A method of middle-down proteomics of histone tails, the method comprising: producing at least one isolated cleaved histone tail according to the method of any one of paragraphs 72-83, and performing mass spectrometry on the at least one isolated cleaved histone tail.

[0290] The technology described herein is further illustrated by the following examples which in no way should be construed as being further limiting.EXAMPLES4934-8116-8174.8Atty. Dkt. No. 043214-000103WO PT

[0291] Example 1: A circular engineered sortase for interrogating H3 histone in chromatin

[0292] Reversible modification of the H3 histone N-terminal tail is critical in regulating chromatin structure, gene expression, and cell states, while its dysregulation contributes to disease pathogenesis. Understanding the crosstalk between H3 tail modifications in nucleosomes constitutes a central challenge in epigenetics. Herein an engineered sortase transpeptidase, cWll, that displays highly favorable properties for introducing scarless H3 tails onto nucleosomes is discussed. This approach significantly accelerates the production of both symmetrically and asymmetrically modified nucleosomes. As demonstrated herein, asymmetrically modified nucleosomes produced in this way in are useful in dissecting the impact of multiple modifications on eraser enzyme processing and molecular recognition by a reader protein. Moreover, discussed herein is how cWll sortase is very effective at cutting and tagging H3 histone tails from endogenous histones, facilitating multiplex “cut-and-paste” middle down proteomics with tandem mass tags. This cut-and-paste proteomics approach permits the quantitative analysis of H3 histone modification crosstalk after treatment with different histone deacetylase inhibitors. These chemoenzymatic tail isolation and modification strategies made possible with cWll sortase will broadly power epigenetics discovery and therapeutic development.

[0293] Understanding the patterns and functional interactions of histone tail posttranslational modifications (PTMs) has emerged as a central challenge in epigenetics.1The building blocks of cellular chromatin are nucleosomes, which are comprised of a histone octamer (four pairs of histones H2A, H2B, H3, and H4) wrapped by 147 bp DNA. Conformational changes in chromatin contribute to the regulation of cell growth, differentiation, and gene expression.2’3Such chromatin structural changes are influenced by reversible histone modifications which are inscribed by “writer” enzymes, removed by “eraser” enzymes, and functionally interpreted by “reader” domain proteins. H3 histone N-terminal modifications are subjects of particular attention due to their central importance in gene regulation. H3 histone N- terminal tail modifications include well-established Lys acetylation, methylation, and ubiquitination and lesser studied acylation modifications like propionylation, butyrylation, crotonylation, and succinylation.4’5These and other PTMs are found in various combinations on H3 histone tails. This complex pattern of modifications affects chromatin structure and the writers, erasers, and readers that act on histones, but detailed molecular insights into how histone PTM crosstalk modulates these processes are generally lacking.6’7

[0294] Addressed herein are two significant challenges in the field: limitations on the ready availability of designer nucleosomes and middle-down mass spectrometric analysis of H3 histone modifications. Although progress has been made over the past 15 years in the ability to prepare nucleosomes containing site-specific modifications on the H3 tail, this is still an onerous multistep task. This workflow requires individual expression and purification of all four histone proteins, including a truncated form of the modified histone, semisynthesis of the modified histone, octamer refolding, DNA4934-8116-8174.8Atty. Dkt. No. 043214-000103WO PT isolation and finally nucleosome reconstitution.8’9Starting from scratch this process takes about a month even in labs with experience. Moreover, substantial additional labor is required to produce nucleosomes containing distinct H3 histone tails, “asymmetric nucleosomes,” that can be employed to distinguish between biochemical effects mediated by modifications of H3 that occur on the same tails versus different tails.10-13

[0295] Middle-down mass spectrometric analysis of H3 histone can provide precise information about the interplay between modifications within individual H3 histone tails by evaluating intact H3 protein tails isolated from cellular histones.14Current middle-down methods require purification of cellular H3 histone prior to treatment with the protease GluC.15-17The resultant aal-51 H3 peptide tails typically require complex and specialized chromatography for separation and characterization by tandem mass spectrometry, which is particularly challenging on such large peptide segments. Furthermore, this, like other middle-down proteomics approaches, lacks the quantitative strength of current tandem mass tag (TMT) isotopic labels, which are widely used in bottom-up mass spectrometry.18

[0296] Herein these limitations in H3 histone analysis are addressed through the application of a novel engineered sortase transpeptidase, cWH.

[0297] F40 sortase was developed as a chemoenzymatic tool to catalyze H3 tail attachment to tailless H3 histone tail recombinant protein which contains the sequence APXTG (SEQ ID NO: 51) (aa29-33) rather than the natural sortase recognition epitope LPXTG (SEQ ID NO: 57).19’20F40 sortase shows relatively slow rates of H3 tail attachment with standard amide bond sequence and relatively low yields of semisynthetic H3 histones.

[0298] The sortases described herein, e.g., sortase cWll, have been found to be a more effective catalyst of H3 histone transpeptidation reactions, facilitating H3 tail ligation to prefabricated tailless nucleosomes. Moreover, cWll sortase can be employed to create asymmetric nucleosomes bearing distinct patterns of modifications on the H3 tails. The availability of such asymmetric nucleosomes has allowed new insights into molecular recognition of nucleosomes by eraser and reader proteins.7,21In addition, efficient isolation of H3 peptide tails from crude extracts of endogenous histones with cWll permits concurrent labeling with tandem mass tags. This ‘cut-and-paste’ approach enhances the middledown proteomics analysis of H3 histone tails.Results

[0299] Development of cWll sortase. Mutant sortases containing amino acid replacements were designed. These mutations were introduced individually, then in combination, and mutant sortases were screened for transpeptidation-based cleavage of H3 histone in the presence of excess oligoglycine peptide. First pass screening employed purified H3 histone with no modifications (FIGs. 7A-7C). D165A, the single most activating mutation identified in eSrtA, falls within the helix mutagenized in F40, and generally hinders the cleavage reaction. Combined mutation of D160, K190 and K196 synergistically enhanced activity without disrupting the selectivity imparted by F40 mutations (T164 and V168-Q172).4934-8116-8174.8Atty. Dkt. No. 043214-000103WO PTThe best performing enzyme was tested against synthetic histone substrates with single modifications, and no significant bias was observed in the cleavage reaction (FIGs. 8A-8F).

[0300] With a sequence-selective sortase, sample handling steps in the typical middle-down proteomic workflow can be reduced (FIG. 1A). Conducting the sortase reaction in a crude nuclear extract eliminates histone purification as a prerequisite for proteolytic isolation of histone tail (FIGs. IB, 9A-9B). Sortase performs optimally at neutral pH and low salt concentrations, however, this compromises the solubility of endogenous histones. Sortase exhibits minimal tolerance for detergents, co-solvents and chaotropes, which we sought to mitigate (FIGs. 10A-10E).22Individual thermostabilizing mutations were identified using the FireProt web server and screened in combination (FIGs. 10A-10E).23Sortase backbone cyclization was previously reported to enhance stability, and was also incorporated, leading to enzyme cW 11.22,23Sortase cWll exhibits superior activity in nuclear acid extracts at physiological temperatures. The cleaved histone tail peptides are readily separated from sortase and other components of the nuclear acid extract reaction by trichloroacetic acid precipitation (FIGs. 1C, 11). Following supernatant buffer exchange and cleanup with Cl 8 stage tips, the peptides are suitable for LC-MS / MS analysis.

[0301] ‘Cut-and-paste’ middle-down proteomics of H3 histone modifications. In the sortase transpeptidation an N-terminal Gly is required for reaction with the enzyme-thioester intermediate, while C-terminal diversity is tolerated. By solid-phase peptide synthesis a GGGKX (SEQ ID NO: 58) peptide was prepared with -aminoalanine at the C-terminal position. After orthogonal deprotection of the side chain -amino group the peptide resin was split into 6 portions, each of which was reacted with a unique TMT 6-plex NHS-ester reagent (FIG. ID). This peptide tag 6-plex system allowed concurrent analysis of histone tails from two cell treatment conditions, each in biological triplicate.

[0302] To explore cWll sortase-mediated mass spectrometry analysis of H3 tails in a pharmacological application, this approach was utilized to evaluate the changes in the patterns of tail modifications in response to two different histone deacetylase (HDAC) inhibitors: MS275 and Corin.24MS275 is a class I selective HDAC inhibitor, targeting HDACs 1-3 whereas Corin is a bivalent inhibitor that contains HDAC as well as an LSD1 demethylase targeting warhead. In this way, Corin appears to show selectivity toward CoREST-containing HDAC complexes. Treatment of HEK293T cells with these HDAC inhibitors or DMSO was performed for 6 hours, after which nuclear acid extracts were isolated. Samples from these experiments were divided for separate bottom-up and middle-down proteomic analyses. H3 histone tails isolated for middle down were tandem mass tagged by cWl 1 sortase, with each replicate assigned a unique tandem mass tag. Quantification of individual H3 PTMs by GluC-based middle-down and bottom-up proteomics typically results in a Pearson correlation between 0.3 and 0.6.25 In this case, middle-down analysis of the sortase-derived H3 peptides resulted in a stronger correlation with bottom-up analysis of the same acid extracted histone samples (Pearson correlations of ~0.8) (Table 5).4934-8116-8174.8Atty. Dkt. No. 043214-000103WO PT

[0303] By LC-MS / MS ~150 uniquely modified peptides (proteoforms) were identified in a combined DMSO vehicle I Corin sample, and ~180 in a combined DMSO vehicle I MS275 sample (FIG. IE, Table 1, Table 2). Nearly half of the proteoforms identified had sufficient TMT ion signals for quantitation (>2 per sample, FIG. IF, Table 3, Table 4). This quantifiable fraction accounted for ~45-50% of all signal intensity attributable to H3 tail peptides. Relative to the DMSO vehicle, both HDAC inhibitors significantly decreased H3 tail proteoforms with one or two modifications, while increasing those with three or four modifications (FIGS. 1G, 1H). The most significant increase was seen in H3K9acK14acK23acK27me2, however, a greater increase was seen with MS275 than with Corin (155% v. 110% increase). It was hypothesized that H3K4mel / 2 proteoforms would increase following Corin treatment due to LSD1 inhibition by Corin, however H3K4 modifications were detected infrequently in this analysis, consistent with prior middle-down analyses.26 H3K4melK14acK23acK27me2 was the sole H3K4me proteoform to exhibit a significant change, an increase following MS275 treatment. This increase is in line with recent work pointing toward a general stimulatory effect of H3 acetylation on the MLL family K4 methyltransferases.214934-8116-8174.8Table 1. DMSO Corin proteoforms.4934-8116-8174.84934-8116-8174.84934-8116-8174.84934-8116-8174.84934-8116-8174.84934-8116-8174.84934-8116-8174.84934-8116-8174.84934-8116-8174.84934-8116-8174.84934-8116-8174.84934-8116-8174.84934-8116-8174.8Table 2. DMSO MS275 proteofomis.4934-8116-8174.84934-8116-8174.84934-8116-8174.84934-8116-8174.84934-8116-8174.84934-8116-8174.84934-8116-8174.84934-8116-8174.84934-8116-8174.84934-8116-8174.84934-8116-8174.84934-8116-8174.84934-8116-8174.84934-8116-8174.84934-8116-8174.84934-8116-8174.8Table 3. DMSO Corin quantifiable.4934-8116-8174.84934-8116-8174.84934-8116-8174.84934-8116-8174.84934-8116-8174.84934-8116-8174.84934-8116-8174.8Table 4. DMSO MS275 quantifiable.4934-8116-8174.84934-8116-8174.84934-8116-8174.84934-8116-8174.84934-8116-8174.84934-8116-8174.84934-8116-8174.84934-8116-8174.84934-8116-8174.84934-8116-8174.84934-8116-8174.8Atty. Dkt. No. 043214-000103WO PT

[0304] Streamlined production of modified nucleosomes. Histone octamers are typically prepared by the individual purification of each core histone protein followed by in vitro octamer assembly. Coexpression of the four core Xenopus histones in E. coli has been used to directly furnish octamer.27In the course of this work, co-expression of the core histones was attempted with tailless H3 histone (aa33-135) and it was found that deletion of the H3 tail facilitated the isolation of intact histone octamer about 5-fold over comparable full-length H3 octamer (5 ± 2.5 mg / L culture) (FIGs 12A- 12C). This octamer could be readily wrapped by DNA (147 or 185 bp) to produce H3 tailless nucleosomes. Late-stage tail attachment to tailless nucleosomes by cWll sortase was pursued. This would permit rapid designer nucleosome production from a common late-stage precursor and minimize the consumption of synthetic H3 tail peptide. Enhanced sortase variants eSrtA and 5M have been used to ligate H3 histone tail to nucleosomes and endogenous chromatin respectively but require an A29L mutation that complicates recognition of the heavily modified H3K27 site.28,29F40 sortase is ineffective in this setting.

[0305] The activity of cWll sortase in ligating H3 tails to tailless nucleosomes (aa33-135) was investigated, and western blot revealed reasonable efficiency (~90% ligation of H3) (FIG. 13A-13B). Analytical anion exchange chromatography was used to precisely delineate the distribution of products, resolving nucleosomes with zero, one or two H3 tails ligated (FIG. 14). This revealed a similar ligation efficiency with respect to H3 histone (79 ± 13%), with optimized conditions converting all tailless nucleosomes to products with either one or two H3 tails ligated. Nucleosomes with two ligated H3 tails typically accounted for 58 ± 6% of products (25 ± 3% isolated yield) (FIG. 2). At standard nanomole scale, 100 pg of nucleosome is obtained for every 100 pg of peptide, which is ~ 100-fold less peptide than required to make the same amount of nucleosome by sortase semisynthesis of histone protein.30

[0306] Late-stage H3 functionalization by cWll ligation was evaluated for compatibility with modifications of lysine (acylations, alkylations, ubiquitination), arginine (citrullination) and serine / threonine (phosphorylation) (FIGs. 2, 15A-15M, 16A-16D). Ligation of alkylated peptides resulted in yields similar to those of an unmodified peptide (LC: 53 ± 11%; isolated: 26 ± 9%), however, modifications decreasing the peptide positive charge were observed to decrease yield. For single acylations this could be overcome by substituting the Thr32 Gly33 amide linkage for a depsipeptide ester linkage.19The first step in sortase transpeptidation, cleavage of the T-G amide bond, liberates a glycine dipeptide that competes with H3 in the transpeptidation reaction, however, the alcohol terminated byproduct of ester cleavage is incompatible with the reverse reaction. Irreversible cleavage of the synthetic peptide C-terminus proved invaluable in the synthesis of multiply acetylated nucleosomes, where the charge masking effect of the acetylations was offset by addition of multiple cationic residues after G34. Introduction of this cleavable C-terminal cationic auxiliary enabled ligation of peptides with five concurrent acetylations spanning all the major acetylation sites (K9, K14, K18, K23 and K27) (FIG. 2, FIGs. 23A-23X). Though broadly compatible with histone PTMs, there are limitations to the cWll ligation. Introduction of a negative charge near the sorting motif (FIGs. 24A-24E, 25A-25C) hinders4934-8116-8174.8Atty. Dkt. No. 043214-000103WO PT ligation progress, resulting in isolable yields of 5-10%. Phosphorylation of S28 was found to have no effect on H3 tail cleavage during cWll development (FIGs. 8A-8F). Thus, we hypothesize that the proximity of Gly33 to the DNA backbone (-10-12A, PDBID: A1OI) leads to repulsion between the DNA backbone and proximal histone tail peptide phosphorylation.28’31Anion exchange chromatography resolves nucleosome ligation products from free DNA, and also separates nucleosomes with zero, one or two copies of H3(33-135). Isolation of nucleosomes with one copy of full length H3 and one copy of H3(33-135), followed by a second round of sortase ligation allows production of nucleosomes with asymmetric modifications of the H3 tail (FIGs. 3, 17A-17E). Asymmetric, or heterotypic, nucleosomes are sought after tools for unraveling crosstalk between modifications of the histone tails, but current strategies for their production are cumbersome.11 The cW 11 ligation simplifies their production, requiring only minor changes in peptide stoichiometry to favor production of the intermediate single tail nucleosome and subsequent asymmetric nucleosome.

[0307] Confirmation of nucleosome biochemical integrity. Site-specific nucleosome deacetylation rates for multiple enzymes and complexes at most of the major acetylation sites on H3 histone are known.30’32’33In theory, this new approach to preparing nucleosomes by late-stage tail addition could lead to unintended consequences in biochemical analysis. For example, residual peptide could be present, which might compete with nucleosome as an enzyme substrate and alter nucleosome recognition by masking DNA, either of which was predicted to affect deacetylase rate. As a quality control for the cWl 1 ligation, parallel deacetylation rate studies were conducted using nucleosome substrates prepared by this new approach and using previously established methods. Using those HDACs most extensively characterized no significant difference in deacetylation rates was found (FIGs. 26A-26E, 27A-27C).34,35Finally, cryo-EM was used to validate nucleosome structure and integrity, and resulted in a structure consistent with numerous other 147 bp nucleosome structures (FIGs. 18A-18C).

[0308] Revealing histone deacylase activity with acylated nucleosomes. Reports of histone lysine succinylation, propionylation and butyrylation, among others have steadily accumulated in recent years, while characterization of their erasers has lagged. Broad functional group compatibility in the sortase ligation initiated characterization of histone deacylase activity toward these variant acylations. Drawing upon previously reported histone acylation selectivity and proposed modes of nucleosome-recognition, we selected the nuclear or nucleocytoplasmic HDACs mitosis-associated HD AC complex (MiDAC) and LSDl / HDAC / CoREST (LHC) complex as representatives of class I HD AC complexes, and Sirtuins 1, 2, and 6 as representatives of class III HDACs with which to explore the class- or complex-specific modes of deacylation.30,34’36’37

[0309] Across all sites on the H3 tail K9 is generally the most rapidly deacetylated, and was therefore selected to probe reactivity trends. Removal of acetyl, propionyl, butyryl and octanoyl modifications were compared (FIGs. 4A, 28A-28D, 29A-29C, 30A-30C, 31A-31D) while myristoylation proved intractable in the sortase nucleosome ligation (FIG. 3). Sirtuins 2 and 6 were superior long chain deacylases4934-8116-8174.8Atty. Dkt. No. 043214-000103WO PT processing K9octanoyl substrates 18-fold faster than K9ac and ~5-fold faster than K9ac respectively. Sirtuin 1 exhibited no activity toward any acylation, consistent with prior observations. Long chain deacylation by the class I HDACs is not much evidenced, so MiDAC was selected for comparison as its baseline deacetylation of K9 is the fastest measured by an order of magnitude. Deacylation by MiDAC slowed with chain length, decreasing ~180-fold (K9butyryl) and ~16-fold (K9octanoyl) relative to K9ac. That octanoylation was processed at all is chemically interesting, and particularly that MiDAC favors it ~10-fold over butyrylation (FIG. 4A, Table 6).

[0310] For each deacylase enzyme with detectable activity, butyrylated nucleosomes were consistently the slow substrate, perhaps accounting for its observed metabolic accumulation in specific tissue types.38However, sirtuin catalysis is reported to be enhanced by long chain acylations that pack within the active site. Exploring the conformational contribution to substrate selectivity for deacylases, four carbon acylations with links to metabolic state were tested, including crotonylation, hydroxyisobutyrylation and succinylation (FIGs. 4B, 32A-32C, 33A-33C, 34A-34C, 35A-35C, 36A- 36D, 37A-37D). Of these decrotonylation exhibited a rate similar to debutyrylation for Sirt6, and faster than debutyrylation for MiDAC and LHC (Table 7). Removal of the branched and charged modifications was unmeasurable for all but Sirt6, which was exceptionally slow. Succinylation is the preferred substrate of Sirt5, which desuccinylated nucleosomes at the fastest rate measured among all deacylases and acylations surveyed here (V / [E] = 5.7 min1) (FIGs. 4C, 38A-38B, Table 8).

[0311] Symmetric and asymmetric nucleosome tools for testing consequences of histone hyperacetylation. Among the synthetic nucleosomes prepared were those with five acetylations of H3 at K9, K14, KI 8, K23, and K27, which are the predominant sites of H3 acetylation detected in the sortase middle-down data, and reported by others. The sharp rise in multiply acetylated peptides following HD AC inhibition, and prior reports of histone acetyltransferase ‘acetyl spray’ activity, prompted investigation of these hyperacetylated substrates.39Extensive acetylation increases histone tail dynamic motion and alters the local electrostatic environment encountered by regulators like HDACs.4<M2Whether this influences the rate of site-specific deacylation was tested using a K9ac / K14ac / K18ac / K23ac / K27ac penta-acetylated substrate, and site-specific rates were compared to previously reported rates for each of K9ac, K14ac, K18ac, K23ac, and K27ac.32,33The rate of deacetylation of penta-acetylated nucleosomes by different enzymes was monitored by western blot with site-selective anti-acetyl-Lys antibodies, the site-specificity of which was validated (FIG. 39) with designer nucleosomes (FIGs. 5A-5C, 40A-40E, 41A-41C, 42A- 42C, 43A-43C). Penta-acetylation influenced removal for a select set of acetyl-Lys locations with distinct impacts among the different enzymes. Deacetylation of H3K27 by both Sirt2 and Sirt6 slowed ~2-fold, while deacetylation by MiDAC increased nearly 4-fold. Sirt2 exhibited a striking change in site-selectivity from K27 to KI 8, due to ~2-fold deacetylation rate increases at KI 8 and K23. To try to better understand the mechanistic basis for these effects, we used the sequential ligations enabled by cW 11 sortase to prepare asymmetrically modified nucleosome substrates.4934-8116-8174.8Atty. Dkt. No. 043214-000103WO PT

[0312] For each site exhibiting a rate changed by penta-acetylation, an asymmetrically modified nucleosome was prepared to isolate that site from the other four acetylations. For example, all three enzymes surveyed exhibited altered rates of deacetylation at K27, and the nature of those rate changes could be assayed using nucleosomes with one copy of H3K27ac and one copy of H3K9ac / K14ac / K18ac / K23ac. Compared to the symmetrically acetylated substrates assayed previously, this substrate has half the effective concentration of K27ac, one copy per nucleosome (FIGs. 19, 20A- 20F). To control for this, rate comparisons were made using asymmetric nucleosomes with one unmodified copy of H3 and one copy of H3K27ac (FIGs. 20A-20F). Deacetylation of asymmetric K9ac / K14ac / K18ac / K23ac and K27ac nucleosomes by Sirt2 and Sirt6 was ~2-fold slower than the matched control (FIGs. 5D, 5E, 44A-44E, 45A-45B, Table 9, Table 10). This is consistent with competition between acetylation sites driving the rate decrease, rather than a local acetylation slowing the rate through altered enzyme recognition. With the same substrates MiDAC showed ~3-fold faster deacetylation of the more acetylated asymmetric substrate than the singly acetylated asymmetric substrate (FIGs. 5F, 46A-46D, Table 11). This in trans stimulation (FIGs. 5G-5H) suggests a processive mechanism at the level of the nucleosome rather than a single histone tail and suggests that histone tail mobility alone does not explain accelerated deacetylation of K27 by MiDAC in a hyperacetylated context. Notably, MiDAC exists as a predominantly tetrameric complex with four catalytic HDAC1 / 2 modules and the dimeric MiDAC subunit DNTTIP1 is a nucleosome acidic patch binder.36,43We speculate that multivalent binding through these domains could drive crosstalk between H3 tails.

[0313] Deacetylation of KI 8 and K23 by Sirt2 was accelerated in the penta-acetylated context, prompting assays in which each site was isolated individually (FIG. 5D). In each case, whether the opposite H3 tail had zero or four acetylations, the deacetylation rate was insignificantly different. Here, accelerated deacetylation depends on in cis modification of one H3 tail with multiple acetylations.

[0314] Symmetric and asymmetric nucleosomes as tools for unraveling methylation-acetylation crosstalk. We have previously reported that demethylation of H3K4mel / 2 by LC or LHC slows >5-fold in the presence of H3K14ac specifically.44Recent crystallographic evidence points at a candidate in cis mechanism but cannot rule out in trans inhibition.35Final resolution of the inhibitory effect was attainable using a series of asymmetrically modified nucleosome substrates in which K4me2 and K14ac modifications could be sequentially isolated. Demethylation rates for nucleosomes with H3K4me2 and one of either unmodified H3 or H3K14ac were insignificantly different (FIGs. 51, 47A-47C). To control for absolute PTM concentration these were both compared to asymmetric nucleosomes with H3K4me2 / K14ac and unmodified H3, which again exhibited >5-fold reduction in demethylase activity by LC.

[0315] DNMT1 recognition of symmetric versus asymmetric nucleosomes. Multi- monoubiquitination of H3 histone has been reported to direct binding by DNMT1, as has H3K9me3. The contribution of each modification has been partially evaluated using peptide, protein, and recently4934-8116-8174.8Atty. Dkt. No. 043214-000103WO PT nucleosome substrates.45^7The replication foci targeting sequence domain (RFTS) of DNMT1 has an atypical H3K9me3 binding site, but whether this exclusively reads methylation in cis was untestable in prior investigations. To address this, peptides, and subsequently asymmetric nucleosomes were prepared with unmodified H3 and either K18Ub / K23Ub or K9me3 / Kl 8Ub / K23Ub, as well as H3K9me3 and KI 8Ub / K23Ub (FIGs. 48A-48F, 49A-49C). An electrophoretic mobility shift assay (EMSA) was used to assess differences in binding of each substrate with a fluorescent RFTS domain fusion construct. The combination of K9me3 / Kl 8Ub / K23Ub nucleosome and RFTS domain resulted in a clearly resolved band shift at single digit nanomolar concentrations (FIG. 6, left). The other two nucleosomes produced diffuse and smeared shifts of the RFTS band, and consistently showed a weak signal for the complex band (FIG. 6, center, right). This was observed with either component (RFTS or nucleosome) used as titrant, and appeared constant over time (FIGs. 50A-50D, 51A-51D). Based on this EMSA behavior we deduce that the in cis K9me3 / K18Ub / K23Ub modifications confer substantially enhanced stability of the RFTS- nucleosome interaction, which are not maintained when K9me3 is present in trans.Discussion

[0316] Described herein is a powerful transpeptidase, cWll sortase, that can efficiently ligate and cleave N-terminal H3 tails from H3 histone. This has facilitated three important methods in chromatin analysis. First, cWll sortase can be used to simply and rapidly produce site-specifically modified nucleosomes by permitting late-stage attachment of H3 tails to H3 tailless nucleosomes. An unexpected dividend that accelerates this approach is that the H3 tailless histone octamer can be efficiently generated by co-expressing tailless H3 with the other three core histones. This not only reduces the time needed to generate designer nucleosomes from about one month to one week, this approach dramatically reduces synthetic tail peptide amounts needed for ligation. It is shown herein that this cWl 1 sortase-driven strategy can easily incorporate a wide range of single and multiple PTMs (acetyl, acyl, methyl) into nucleosomes. This is especially useful for ubiquitin-like modifications, which present a formidable synthetic challenge, particularly when introduced along with other PTMs.48-50Streamlining such syntheses facilitates analysis of these nucleosomes in enzymatic and binding experiments.47,51These studies have uncovered interesting new selectivities of deacylases that interconnect metabolism and chromatin structure. Of particular note, both class I and class IV HDACs remove acylations with linear chains up to eight carbons, however, branched or polar acylations are exclusively processed by specific sirtuins.

[0317] Second, cWl 1 sortase provides an attractive and reliable route to asymmetric nucleosomes in which the two different H3 tails contain distinct patterns of site-specific PTMs. Prior work on the construction of asymmetric nucleosomes with PTMs on histones H2A, H2B, and H3 have been reported but are technically demanding and not widely used.11,12The present simple stepwise addition of H3 tails with a single chromatographic isolation of the intermediate single tail form represents a relatively convenient alternative. Using this approach, the crosstalk between H3K4me2 and H3K14ac by LSD1 demethylase, multi-acetylated H3 tails by various histone deacetylases, and Lys ubiquitination and methylation by the RFTS reader domain of DNMT1 was examined in asymmetric nucleosomes. These4934-8116-8174.8Atty. Dkt. No. 043214-000103WO PT experiments have revealed cases where specific patterns can impact eraser or reader interactions specifically in cis or trans depending on the site of modification and eraser / reader involved.7,17While the precise mechanisms for these PTM crosstalk influences remain to be elucidated, these multifaceted regulatory features provide interesting and novel insights into histone mark crosstalk and highlight the necessity of studying asymmetric nucleosomes to understand molecular recognition of individual chromatin interactors.

[0318] Third, cWll sortase has been adopted to perform a “cut-and-paste” method to isolate and analyze purified intact H3 histone tails from a human cell line. Though the position of the sortase recognition motif precludes quantification of H3K36 modifications, this procedure imparts quantitative power to LC-MS / MS analysis of these tails through multiplex tandem mass tagging. With this approach, it was possible to discern some overlapping but also distinct H3 tail PTM patterns after treatment with two types of HD AC inhibitors. It is contemplated herein that this method can be generally useful for analyzing other epigenetic agents and to analyze specific cell states. The use of the chemoenzymatic labeling step with isotopic barcodes avoids the more complicated electrophilic chemical tandem mass tagging which is avoided on large peptides because of complex reactivities. The use of engineered sortase tagging may be broadly useful in middle-down proteomics.Methods

[0319] Protein expression and purification. Plasmids for the relevant proteins and nucleic acids were transformed into E. coli, or co-transformed in the case of octamer, then selected with the relevant antibiotics, and grown from cell stocks. Sortase cWll (from LOBSTR Rosetta E. coli) was grown at 37 °C from an overnight starter culture of Luria-Bertani (LB) medium (Sigma) with ampicillin (100 mg / L), of which 10 mL was used to inoculate each 1 L of the same medium used for overexpression. Cells were grown to an OD600 of 0.6-0.8, then induced with 0.5 mM IPTG for 3 hours at 37 °C. Cells were harvested by centrifugation (4 k x g, 30 min, 4°C), and the cell pellet was resuspended in 5 volumes of chilled (4 °C) lysis buffer (10 mM Tris, pH 7.5 at 25 C, 0.1 % Tween-20). Resuspended pellets were mixed on a dounce homogenizer to uniformity and lysed by three passages through a microfluidizer. The supernatant was cleared by centrifugation (12 k x g, 30 min, 4 °C) and the supernatant was passed over 5 mL Ni NTA agarose twice by gravity. Resin was washed with 10 column volumes (CV) lysis buffer, followed by 10 CV wash buffer (10 mM Tris, pH 7.5 at 25 °C, 500 mM NaCl), then 5 CV imidazole wash buffer (10 mM Tris, pH 7.5 at 25 °C, 0.1 % Tween-20, 10 mM imidazole), and eluted with 5 CV elution buffer (10 mM Tris, pH 7.5 at 25 °C, 400 mM imidazole). The elution was dialyzed three times (Spectra / Por 12-14 kDa MWCO membrane, Spectrum Labs, 50 mM Tris, pH 7.5 at 25 °C, 150 mM NaCl, 5 mM CaC12) at 4 °C, then concentrated (Amicon, 10 kDa MWCO, 4 °C) to 2-6 mM with frequent sample mixing to prevent aggregation.

[0320] Histone octamer (from LOBSTR Rosetta E. coli) was grown from an overnight starter culture (30 °C) of LB medium (Sigma) with ampicillin (100 mg / L), kanamycin (50 mg / L), streptomycin (1004934-8116-8174.8Atty. Dkt. No. 043214-000103WO PT mg / L) and chloramphenicol (34 mg / L), which was not allowed to reach saturation. Growth for overexpression was initiated by adding 10 mL of starter culture to inoculate each 1 L of the same medium. Octamer growth is typically slow, taking up to 12 hours to reach the target induction OD600 of 0.8. Expression was induced by adding IPTG to 0.5 mM, and cultures were grown overnight (12-16 h) at 25 °C. Cells were harvested by centrifugation (4 k x g, 30 min, 4 °C), and the cell pellet was resuspended in 5 volumes of chilled (4 °C) lysis buffer (20 mM Tris, pH 7.5 at 25 °C, 2 M NaCl, 0.5 mM TCEP). Resuspended pellets were dounced to uniformity and lysed by three passages through a microfluidizer. Supernatant was cleared by centrifugation (12 k x g, 30 min, 4 °C) and the supernatant was batch bound to 10 mL Ni NTA agarose with gentle agitation at 4 °C. Resin was pelleted by centrifugation (500 x g, 5 min, 4 °C), and the supernatant was removed. Resin was washed with 5 CV lysis buffer, followed by 5 CV wash buffer (20 mM Tris, pH 7.5 at 25 °C, 2 M NaCl, 0.5 mM TCEP, 20 mM imidazole), then eluted with 5 CV elution buffer (20 mM Tris, pH 7.5 at 25 C, 2 M NaCl, 0.5 mM TCEP, 200 mM imidazole). The elution was concentrated (Amicon, 10 kDa MWCO, 4 °C) to 5-10 mg / mL by 280 nm absorbance (nanodrop), and incubated overnight with 0.01 mass equivalents of TEV protease at 4 °C. Octamer was further purified on an FPLC using a superdex 200 column (20 mM Tris, pH 7.5 at 25 °C, 2 M NaCl, 0.5 mM TCEP), then purified fractions were pooled and concentrated to 5-10 mg / mL. Glycerol and NaCl (5 M) were added to final concentrations of 15% and 2 M respectively, the samples mixed thoroughly, and aliquoted before flash freezing for storage at -80 °C.

[0321] Widom 601 DNAs (147 and 185 bp) were produced from multi-copy plasmids (16 x 147, 12 x 185) isolated from transformed DH5a E. coli grown for 36-48 h in CircleGrow media supplemented with antibiotic (147 bp: Ampicillin; 185 bp: Kanamycin). Cells were harvested by centrifugation (4 k x g, 30 min, 4 °C), and the cell pellet was resuspended in 5 volumes of lysis buffer (10 mM Tris, pH 8 at 25 °C, 10 mM EDTA). Lysis was initiated with lysozyme, after which 0.8 volumes of alkaline lysis buffer (2% SDS, 0.2 M NaOH) was added and the sample was mixed by inverting. One volume of neutralization buffer (1.5 M potassium acetate / acetic acid (pH 4.9)) was added and the sample was mixed by inverting, then insoluble material was removed by centrifugation. Clarified supernatant was filtered, then crude product was precipitated with isopropanol (0.6 volumes). Crude product was resuspended in 10 mM Tris (pH 8), 10 mM EDTA, and one volume of 5 M LiCl was added to precipitate RNA and proteins. The solution was clarified by centrifugation and the supernatant was combined with isopropanol (0.6 volumes) to precipitate crude product. The crude product was again resuspended in 10 mM Tris (pH 8), 1 mM EDTA and treated with RNase A, before precipitating again with isopropanol (3 volumes). The pelleted plasmid was resuspended in 10 mM Tris (pH 8), 1 mM EDTA, and digested with EcoRV in CutSmart buffer (New England Biolabs) at 37 °C. Insert DNA was purified by diluting the reaction 10-fold with dilution buffer (10 mM Tris, pH 8 at 25 °C, 1 mM EDTA) and passing twice over Sepharose fast-flow Q resin. The resin was washed with 20 volumes of wash buffer (10 mM Tris, pH 8 at 25 °C, 300 mM NaCl, 1 mM EDTA) and eluted with 5 volumes of elution buffer (10 mM Tris, pH 8 at 25 °C, 700 mM NaCl, 1 mM EDTA) then concentrated and stored at -20 °C.4934-8116-8174.8Atty. Dkt. No. 043214-000103WO PT

[0322] Mammalian cell growth, nuclear acid extraction and histone tail isolation. HEK293T cells (ATCC) were grown in DMEM supplemented with 5% FBS and 1% penn / strep (37 °C, 5% CO2), and regularly tested by PCR for mycoplasma contamination. All experiments were conducted within 10 passages of stock thawing. For treatment with DMSO vehicle, MS275 (LC Laboratories, Cat# E-3866, Lot: ENT- 102) or Corin (Med Chem Express, Cat# HY-111048 / CS0034060, Lot: 41808), a single 15 cm plate of HEK293T cells was grown to near confluence and split. Cells were washed with Dulbecco’s phosphate buffered saline (DPBS, D8537, Sigma-Aldrich), liberated with TryplE, and quenched with DMEM, then cell count and viability (99%) were determined by Trypan blue staining (Countess III). Plates (10 cm) were seeded in triplicate (4.4e6 cells / plate) and recovered for two days in DMEM to ~50% confluence. Following media change, stocks of drug were prepared in sterile filtered DMSO, then diluted in DMEM and added to plates to a final concentration of 10 pM MS275, or 2 pM Corin, and 1% DMSO. Cells were incubated for 6 hours in the presence of vehicle or drug, after which the media was aspirated, cells were washed with DPBS, liberated with TryplE, and quenched with DMEM. Cells were pelleted and the supernatant removed by vacuum, then washed once with cold (4 °C) DPBS, pelleted again, and the supernatant was again removed by vacuum before flash freezing the pellets.

[0323] Pellets were thawed on ice and gently resuspended in 10 pellet volumes of nuclear isolation buffer (15 mM Tris, pH 7.5 at 25 °C, 60 mM KC1, 15 mM NaCl, 5 mM MgC12, 1 mM CaC12, 250 mM sucrose, 5 mM sodium butyrate, ...

Claims

Atty. Dkt. No. 043214-000103WO PTCLAIMSWe claim:

1. An engineered polypeptide comprising: a sortase domain comprising the sequence of SEQ ID NO: 14.

2. The engineered polypeptide of claim 1, wherein Xi of SEQ ID NO: 14 is P, S, or R.

3. The engineered polypeptide of claim 1, wherein Xi of SEQ ID NO: 14 is S or R.

4. The engineered polypeptide of any one of the preceding claims, wherein X2 of SEQ ID NO: 14 is E, A, D, Q, or N.

5. The engineered polypeptide of any one of the preceding claims, wherein X2 of SEQ ID NO: 14 is A, D, Q, or N.

6. The engineered polypeptide of any one of the preceding claims, wherein X3 of SEQ ID NO: 14 is E, A, D, Q, or N.

7. The engineered polypeptide of any one of the preceding claims, wherein X3 of SEQ ID NO: 14 is A, D, Q, or N.

8. The engineered polypeptide of any one of the preceding claims, wherein X4 of SEQ ID NO: 14 is E or Q.

9. The engineered polypeptide of any one of the preceding claims, wherein X4 of SEQ ID NO: 14 is Q.

10. The engineered polypeptide of any one of the preceding claims, wherein X5 of SEQ ID NO: 14 is D, A, E, Q, or N.

11. The engineered polypeptide of any one of the preceding claims, wherein X5 of SEQ ID NO: 14 is A, E, Q, or N.

12. The engineered polypeptide of any one of the preceding claims, wherein X& of SEQ ID NO: 14 is S or V.

13. The engineered polypeptide of any one of the preceding claims, wherein X& of SEQ ID NO: 14 is V.

14. The engineered polypeptide of any one of the preceding claims, wherein X7 of SEQ ID NO: 14 is M or K.

15. The engineered polypeptide of any one of the preceding claims, wherein X7 of SEQ ID NO: 14 is M.

16. The engineered polypeptide of any one of the preceding claims, wherein Xg of SEQ ID NO: 14 is D or N.

17. The engineered polypeptide of any one of the preceding claims, wherein Xg of SEQ ID NO: 14 is N.4934-8116-8174.8Atty. Dkt. No. 043214-000103WO PT18. The engineered polypeptide of any one of the preceding claims, wherein X9 of SEQ ID NO:14 is Q or T.

19. The engineered polypeptide of any one of the preceding claims, wherein X9 of SEQ ID NO: 14 is Q.

20. The engineered polypeptide of any one of the preceding claims, wherein X10 of SEQ ID NO: 14 is D or A.

21. The engineered polypeptide of any one of the preceding claims, wherein Xu of SEQ ID NO: 14 is A.

22. The engineered polypeptide of any one of the preceding claims, wherein Xnu of SEQ ID NO: 14 is VG, VE or V.

23. The engineered polypeptide of any one of the preceding claims, wherein Xnu of SEQ ID NO: 14 is V.

24. The engineered polypeptide of any one of the preceding claims, wherein X13 of SEQ ID NO: 14 is M or V.

25. The engineered polypeptide of any one of the preceding claims, wherein X13 of SEQ ID NO: 14 is M.

26. The engineered polypeptide of any one of the preceding claims, wherein Xu of SEQ ID NO: 14 is H or L.

27. The engineered polypeptide of any one of the preceding claims, wherein Xu of SEQ ID NO: 14 is H.

28. The engineered polypeptide of any one of the preceding claims, wherein X15 of SEQ ID NO: 14 is L or D.

29. The engineered polypeptide of any one of the preceding claims, wherein X15 of SEQ ID NO: 14 is L.

30. The engineered polypeptide of any one of the preceding claims, wherein Xu of SEQ ID NO: 14 is A or E.

31. The engineered polypeptide of any one of the preceding claims, wherein Xu of SEQ ID NO: 14 is A.

32. The engineered polypeptide of any one of the preceding claims, wherein X17 of SEQ ID NO:14 is Q, E, or R.

33. The engineered polypeptide of any one of the preceding claims, wherein X17 of SEQ ID NO: 14 is E or R.

34. The engineered polypeptide of any one of the preceding claims, wherein X17 of SEQ ID NO: 14 is E.

35. The engineered polypeptide of any one of the preceding claims, wherein Xu of SEQ ID NO: 14 is K or E.4934-8116-8174.8Atty. Dkt. No. 043214-000103WO PT36. The engineered polypeptide of any one of the preceding claims, wherein Xis of SEQ ID NO:14 is E.

37. The engineered polypeptide of any one of the preceding claims, wherein X19 of SEQ ID NO: 14 is K or T.

38. The engineered polypeptide of any one of the preceding claims, wherein X19 of SEQ ID NO: 14 is T.

39. The engineered polypeptide of any one of the preceding claims, wherein X8of SEQ ID NO: 14 is N, Xu of SEQ ID NO: 14 is E, and X19 of SEQ ID NO: 14 is T.

40. The engineered polypeptide of any one of the preceding claims, further comprising at least one of the following:Xi of SEQ ID NO: 14 is S or R;X2of SEQ ID NO: 14 is A, D, Q, or N;X3of SEQ ID NO: 14 is A, D, Q, or N;X5of SEQ ID NO: 14 is A, E, Q, or NX17 of SEQ ID NO: 14 is E or R.

41. The engineered polypeptide of any one of the preceding claims, wherein:Xi of SEQ ID NO: 14 is S or R;X2of SEQ ID NO: 14 is A, D, Q, or N;X3of SEQ ID NO: 14 is A, D, Q, or N;X5of SEQ ID NO: 14 is A, E, Q, or NX17 of SEQ ID NO: 14 is E or R.

42. The engineered polypeptide of claim 1, wherein Xi of SEQ ID NO: 14 is P, X2 is E, X3is E, X4 is E, X5 is D, Xeis S, X7 is M, X8is D, X9 is Q, X10 is D, X1112 is VG, Xi3is M, X14 is H, X15 is L, Xi6 is A, X17 is E, Xi8is K, and X19 is K (SEQ ID NO: 3).

43. The engineered polypeptide of claim 1, wherein Xi of SEQ ID NO: 14 is S, X2 is E, X3is E, X4 is E, X5 is D, Xe is S, X7 is M, X8is N, X9 is T, X10 is A, X1112 is V, Xi3is M, Xu is H, X15 is L, Xu is A, X17 is E, Xi8is K, and X19 is K (SEQ ID NO: 4).

44. The engineered polypeptide of claim 1, wherein Xi of SEQ ID NO: 14 is P, X2 is E, X3is E, X4 is E, X5 is D, Xeis S, X7 is M, X8is N, X9 is Q, X10 is D, X1112 is V, Xi3is M, X is H, X15 is L, Xu is A, X17 is E, Xi8is E, and X19 is T (SEQ ID NO: 5).

45. The engineered polypeptide of claim 1, wherein Xi of SEQ ID NO: 14 is S, X2 is E, X3is E, X4 is E, X5 is D, Xeis S, X7 is M, X8is N, X9 is Q, X10 is D, X1112 is V, Xi3is M, Xu is H, X15 is L, Xu is A, X17 is E, Xi8is K, and X19 is T (SEQ ID NO: 6).

46. The engineered polypeptide of claim 1, wherein Xi of SEQ ID NO: 14 is S, X2 is E, X3is E, X4 is E, X5 is D, Xeis S, X7 is M, X8is N, X9 is Q, X10 is D, X1112 is V, Xi3is M, Xu is H, X15 is L, Xu is A, X17 is E, Xi8is E, and X19 is T (SEQ ID NO: 7).4934-8116-8174.8Atty. Dkt. No. 043214-000103WO PT47. The engineered polypeptide of claim 1, wherein Xi of SEQ ID NO: 14 is S, X2 is E, X3 is E, X4 is E, X5 is D, Xeis E, X7 is S, Xg is N, X9 is T, X10 is A, X1112 is V, X13 is M, Xu is H, X15 is L, Xu is A, X17 is E, Xis is K, and X19 is T (SEQ ID NO: 8).

48. The engineered polypeptide of claim 1, wherein Xi of SEQ ID NO: 14 is S, X2 is E, X3 is E, X4 is E, X5 is D, Xeis S, X7 is M, Xg is N, X9 is Q, X10 is A, X1112 is V , X13 is M, Xu is H, X15 is L, Xu is A, X17 is E, Xig is E, and X19 is T (SEQ ID NO: 9).

49. The engineered polypeptide of claim 1, wherein Xi of SEQ ID NO: 14 is R, X2 is E, X3 is E, X4 is E, X5 is D, Xeis S, X7 is M, Xg is N, X9 is Q, X10 is ,D X1112 is V, X13 is M, Xu is H, X15 is L, Xu is A, X17 is E, Xig is E, and X19 is T (SEQ ID NO: 10).

50. The engineered polypeptide of claim 1, wherein Xi of SEQ ID NO: 14 is R, X2 is E, X3 is E, X4 is E, X5 is D, Xeis S, X7 is M, Xg is N, X9 is Q, X10 is A, X1112 is V, X13 is M, Xu is H, X15 is L, Xu is A, X17 is E, Xig is K, and X19 is T (SEQ ID NO: 11).

51. The engineered polypeptide of claim 1 , wherein Xi of SEQ ID NO: 14 is S, X2 is E, X3 is E, X4 is Q, X5 is D, Xeis V, X7 is M, Xg is N, X9 is Q, X10 is D, X1112 is V, X13 is M, Xu is H, X15 is L, Xu is A, X17 is E, Xig is E, and X19 is T (SEQ ID NO: 12).

52. The engineered polypeptide of claim 1, wherein the sortase domain does not comprise SEQ ID NO: 3, SEQ ID NO: 24, SEQ ID NO: 19, SEQ ID NO: 23, SEQ ID NO: 20, or SEQ ID NO 25.

53. The engineered polypeptide of any of claims 42-51 , further comprising SEQ ID NO: 36.

54. The engineered polypeptide of any of claims 42-51, further comprising SEQ ID NO: 36 at the C-terminus.

55. The engineered polypeptide of any one of the preceding claims, further comprising at least one split intein sequence.

56. The engineered polypeptide of any one of the preceding claims, further comprising a pair of split intein sequences.

57. The engineered polypeptide of claim 56, wherein the pair of split intein sequences flank the sortase domain.

58. The engineered polypeptide of claims 56 or 57, further comprising linker domains between each of the pair of split intein sequences and the sortase domain.

59. The engineered polypeptide of claims 56-58, further comprising, from N-terminus to C- terminus, an N-terminus split intein comprising the sequence of SEQ ID NO: 15, an N- terminus linker comprising the sequence of SEQ ID NO: 16, the sortase domain, a C-terminus linker comprising the sequence of SEQ ID NO: 17, and a C-terminus split intein comprising the sequence of SEQ ID NO: 18.

60. The engineered polypeptide of any one of the preceding claims, wherein the polypeptide is circularized.4934-8116-8174.8Atty. Dkt. No. 043214-000103WO PT61. A method of producing a circularized engineered polypeptide comprising a sortase domain, the method comprising: expressing the engineered polypeptide of claims 55-59, thereby excising the intein sequences and producing the circularized engineered polypeptide comprising the sortase domain.

62. The method of claim 61, wherein the circularized engineered polypeptide is produced through peptide bond formation between the N-terminus of the N-terminus linker and the C-terminus of the C-terminus linker.

63. A method of producing a tailless H3 histone, the method comprising: a) contacting the engineered polypeptide of any of claims 1 - 62 with a composition comprising at least one tailed H3 histone; and b) separating at least one cleaved histone tail peptide from at least one tailless H3 histone.

64. The method of claim 63, wherein the composition comprising at least one tailed H3 histone is a crude nuclear extract.

65. The method of any one of claims 63 and 64, wherein separating the at least one cleaved histone tail peptide from the at least one tailless H3 histone comprises trichloroacetic acid precipitation.

66. The method of claim 63, wherein the composition comprising at least one tailless H3 histone further comprises at least one co-expressed unmodified core histone.

67. A method for producing an intact histone octamer, the method comprising: a) producing core histones comprising tailless H3 histones using the method of any one of claims 63-66, and b) assembling the intact histone octamer via octamer assembly.

68. A method of producing a modified nucleosome, the method comprising: a) producing an intact histone octamer using the method of claim 67, and b) wrapping the intact histone octamer with DNA.

69. A method for producing a tailless H3 nucleosome, the method comprising: a) producing an intact histone octamer comprising tailless H3 histones using the method of claim 67, and b) wrapping the intact histone octamer with DNA to form a H3 tailless nucleosome.

70. The method of claims 68 or 69, wherein the DNA is 147 bp in length.

71. The method of claims 68 or 69, wherein the DNA is 185 bp in length.

72. A method for histone tail isolation, the method comprising: a) contacting at least one isolated histone protein with: i) an engineered polypeptide of any one of claims 1-60 and ii) an oligoglycine tandem mass tag (TMT) peptide,4934-8116-8174.8Atty. Dkt. No. 043214-000103WO PT under conditions comprising at least one of: i) a temperature of between 37 and 42 °C; ii) a pH ofbetween 7.0 and 7.5; iii) an ionic strength of between 30 and 50 mM; iv) a histone protein concentration of between 10 and 20 pM; v) an engineered polypeptide concentration of between 100 and 400 pM; vi) an oligoglycine TMT peptide concentration of between 0.25 and 1 mM; vii) a phenylmethylsulfonyl fluoride (PMSF) concentration of between 0 and 2.5 m ; and viii) a dithiothreitol (DTT) concentration of between 1 and 10 mM; thereby cleaving at least one histone tail from the at least one histone protein; and b) isolating the at least one cleaved histone tail.

73. The method of claim 72, wherein the temperature is between 37 and 42 °C.

74. The method of claim 72 or 73, wherein the pH is between 7.0 and 7.5.

75. The method of any of claims 72-74, wherein the ionic strength is between 30 and 50 mM.

76. The method of any of claims 72-75, wherein the histone protein concentration is between 10- 20 pM.

77. The method of any of claims 72-76, wherein the engineered polypeptide concentration is between 100 and 400 pM.

78. The method of any of claims 72-77, wherein the oligoglycine TMT peptide concentration is between 0.25 and 1 mM.

79. The method of any of claims 72-78, wherein the PMSF concentration is less than 2.5 mM.

80. The method of claim 72, wherein the temperature is 37 °C, the pH is 7.5, the ionic strength is50 mM, the histone protein concentration is 20 pM, the engineered polypeptide concentration is 200 pM, the oligoglycine TMT peptide concentration is 1 mM, the PMSF concentration is 0 mM, and the DTT concentration is 1 mM.

81. The method of any of claims 72-80, wherein the oligoglycine TMT peptide has the structurewherein: a is 0, 1, or 2; b is 1, 2, 3, or 4;4934-8116-8174.8Atty. Dkt. No. 043214-000103WO PTwherein •»jJJdenotes the attachment point to the oligoglycine TMT peptide; andZ is H, C(O)NH2, or C(O)OH.

82. The method of any of claims 72-80, wherein the oligoglycine TMT peptide is83. The method of any of claims 72-82, wherein the histone tails are isolated using trichloroacetic acid.

84. The method of any of claims 72-83, wherein the peptide solution comprising the histone tails is subsequently exchanged into a trifluoroacetic acid solution.

85. A method of attaching a tail to a tailless nucleosome, the method comprising: a) producing a tailless H3 nucleosome using the method claim 69; and b) contacting the tailless H3 nucleosome with a histone tail and the engineered polypeptide of any of claims 1-60; under conditions comprising at least one of:a temperature of 37 °C; ii) an ionic strength of 185 mM; iii) a tailless nucleosome concentration of 1.65 pM; iv) an engineered polypeptide concentration of 200 pM; and v) a histone tail peptide concentration of between 16.5 and 99 pM; thereby ligating at least the histone tail to the tailless H3 nucleosome.

86. The method of claim 85, wherein the temperature is 37 °C.4934-8116-8174.8Atty. Dkt. No. 043214-000103WO PT87. The method of claim 85 or 86, wherein the ionic strength is 185 mM.

88. The method of any of claims 85-87, wherein the ionic strength is a combination of NaCl, HEPES, and KC1.

89. The method of any of claims 85-88, wherein the tailless nucleosome concentration is 1.65 pM.

90. The method of any of claims 85-89, wherein the engineered polypeptide concentration is 200 pM.

91. The method of any of claims 85-90, wherein the histone tail peptide concentration is between 16.5 and 99 pM.

92. The method of claim 85, wherein the temperature is 37 °C, the ionic strength is 185 mM, the tailless nucleosome concentration is 1.65 pM, the engineered polypeptide concentration is 200 pM, and the histone tail peptide concentration is 49.5 pM.

93. A method for late-stage tail modification of a nucleosome, or a method of chemo-enzymatic H3 histone tagging, the method comprising: a) producing a modified nucleosome using the method of any one of claims 68-71 , and b) modifying at least one residue of at least one histone tail.

94. The method of claim 93, wherein the modification is an acylation, an alkylation, a ubiquitination, a citrullination, or a phosphorylation.

95. The method of claim 93 or 94, wherein the modification is selected from the group consisting of:K4mel, K4me2, K4me3, K9ac, K14ac, K18ac, K23ac, K27ac, Kacyl, K9propionyl, K9butyryl, K9crotonyl, K9octanoyl, K91actoyl, K9succinyl, K9-a-hydroxyisobutyryl, K9-P-hydroxybutyryl, K9-N-methylthiourea, K9norleucine, K9myristoyl, S28phosphoryl, S3 Iphosphoryl, K4me / K9ac, R8Cit / K9ac, K4me2 / K14ac, K9me2 / K14ac, K9me3 / K14ac, K9me2 / K18ac, K18ac / K27me2, K14ac / K18ac, K18UB / K23UB, K9me3 / K18Ub / K23Ub, K9ac / K14ac / K18ac / K27ac, and K9ac / K14ac / K18ac / K23ac / K27ac.

96. The method of claims 93 or 94, wherein the modification is K4mel.

97. The method of claims 93 or 94, wherein the modification is K4me2.

98. The method of claims 93 or 94, wherein the modification is K4me3.

99. The method of claims 93 or 94, wherein the modification is K9ac.

100. The method of claims 93 or 94, wherein the modification is K14ac.

101. The method of claims 93 or 94, wherein the modification is KI 8ac.

102. The method of claims 93 or 94, wherein the modification is K23ac.

103. The method of claims 93 or 94, wherein the modification is K27ac.

104. The method of claims 93 or 94, wherein the modification is Kacyl.4934-8116-8174.8Atty. Dkt. No. 043214-000103WO PT105. The method of claims 93 or 94, wherein the modification is K9propionyl.

106. The method of claims 93 or 94, wherein the modification is K9butyryl.

107. The method of claims 93 or 94, wherein the modification is K9crotonyl.

108. The method of claims 93 or 94, wherein the modification is K9octanoyl.

109. The method of claims 93 or 94, wherein the modification is K9-a-hydroxyisobutyryl.

110. The method of claims 93 or 94, wherein the modification is K9-P-hydroxybutyryl.

111. The method of claims 93 or 94, wherein the modification is K9-N-methylthiourea.

112. The method of claims 93 or 94, wherein the modification is K9norleucine.

113. The method of claims 93 or 94, wherein the modification is K9myristoyl.

114. The method of claims 93 or 94, wherein the modification is S28phosphoryl.

115. The method of claims 93 or 94, wherein the modification is S3 Iphosphoryl.

116. The method of claims 93 or 94, wherein the modification is K4me3 / K9ac.

117. The method of claims 93 or 94, wherein the modification is R8Cit / K9ac.

118. The method of claims 93 or 94, wherein the modification is K4me2 / K14ac.

119. The method of claims 93 or 94, wherein the modification is K9me2 / K14ac.

120. The method of claims 93 or 94, wherein the modification is K9me3 / K14ac.

121. The method of claims 93 or 94, wherein the modification is K9me2 / K18ac.

122. The method of claims 93 or 94, wherein the modification is K18ac / K27me2.

123. The method of claims 93 or 94, wherein the modification is K14ac / K18ac.

124. The method of claims 93 or 94, wherein the modification is KI 8Ub / K23Ub.

125. The method of claims 93 or 94, wherein the modification is K9me3 / K18Ub / K23Ub.

126. The method of claims 93 or 94, wherein the modification isK9ac / K14ac / K18ac / K27ac.

127. The method of claims 93 or 94, wherein the modification is K9ac / K14ac / K18ac / K23ac / K27ac.

128. A method for late-stage tail modification of nucleosomes, or a method of chemo- enzymatic H3 histone tagging, the method comprising: a) producing a modified nucleosome using the method of any one of claims 68-71 , and b) modifying at least one residue of each of a first histone tail and at least one residues of a second histone tail, wherein the modification of at least one residue of the first histone is the same modification as the modification of at least one residue of the second histone tail.

129. The method of claim 128, wherein the modification is KI 8ac.

130. The method of claim 128, wherein the modification is K23ac.

131. The method of claim 128, wherein the modification is K27ac.

132. The method of claim 128, wherein the modification is K18Ub / K23Ub.

133. The method of claim 128, wherein the modification is K9me3 / K18UB / K23Ub.4934-8116-8174.8Atty. Dkt. No. 043214-000103WO PT134. A method for late-stage tail modification of nucleosomes, the method comprising: a) producing a modified nucleosome using the method of any one of claims 68-71 , and b) modifying at least one residue of at least one of a first histone tail or at least one residue of a second histone tail.

135. The method of claim 134, wherein at least one residue of a first histone tail is modified, and at least one residue of a second histone tail is unmodified.

136. The method of claim 134, wherein at least one residue of a first histone tail is unmodified, and at least one residue of a second histone tail is modified.

137. The method of claim 134, wherein at least one residue of a first histone tail is modified, and at least one residue of a second histone tail is modified.

138. The method of claim 134, wherein the modification of at least one residue of the first histone tail is a different type of modification from the type of modification of at least one residue of the second histone tail.

139. The method of claims 134 or 136, wherein the first histone tail is unmodified and the second histone tail modification is K18ac.

140. The method of claims 134 or 136, wherein the first histone tail is unmodified and the second histone tail modification is K23ac.

141. The method of claims 134 or 136, wherein the first histone tail is unmodified and the second histone tail modification is K27ac.

142. The method of claims 134 or 136, wherein the first histone tail is unmodified and the second histone tail modification is K4me2.

143. The method of claims 134 or 136, wherein the first histone tail is unmodified and the second histone tail modification isa K4me2 / K14ac.

144. The method of claims 134 or 137, wherein the first histone tail modification is K18ac and the second histone tail modification is K9ac / K14ac / K23ac / K27ac.

145. The method of claims 134 or 137, wherein the first histone tail modification is K23ac and the second histone tail modification is K9ac / K14ac / K18ac / K27ac.

146. The method of claims 134 or 137, wherein the first histone tail modification is K27ac and the second histone tail modification is K9ac / K14ac / K18ac / K23ac.

147. The method of claims 134 or 137, wherein the first histone tail modification is K18Ub / K23Ub and the second histone tail modification is K9me3.

148. The method of claims 134 or 135, wherein the first histone tail modification is K18Ub / K23Ub and the second histone tail is unmodified.

149. The method of claims 134 or 135, wherein the first histone tail modification is K9me3 / K18Ub / K23Ub and the second histone tail is unmodified.

150. A method of histone tagging, the method comprising:4934-8116-8174.8Atty. Dkt. No. 043214-000103WO PT a) producing the tailless H3 histone according to the method of any one of claims 63-66, and b) contacting the tailless H3 histone with a histone tail comprising a tag and the engineered polypeptide of any of claims 1-60 to provide a tagged H3 histone.

151. A method of chemo-enzymatic tagged mass spectrometry, the method comprising: a) producing a chemo-enzymatic tagged H3 histone using the method of claim 150, and b) performing mass spectrometry on the chemo-enzymatic tagged H3 histone.

152. A method of middle-down proteomics of histone tails, the method comprising: a) producing at least one isolated cleaved histone tail according to the method of any one of claims 72-83, and b) performing mass spectrometry on the at least one isolated cleaved histone tail.4934-8116-8174.8

Images