Fusion Protein Based on Double-Stranded DNA Deaminase and Method for Detecting Epigenetic Modification

Abstract

The present invention discloses a fusion protein based on double-stranded DNA deaminase and an epigenetic modification detection method. The fusion protein comprises a molecule having specific affinity for an antibody, a linker peptide, and a double-stranded DNA deaminase. The present invention guides the double-stranded DNA deaminase to a specific antibody through the molecule having specific affinity for the antibody, so as to perform a deamination reaction on the DNA sequence at the antibody-targeted site, and detects the position of the chromatin-related protein or chromatin-related protein modification targeted by the antibody on the double-stranded DNA sequence according to the determined position of the DNA deamination site.

Description

Technical Field

[0001] The present invention relates to a fusion protein based on double-stranded DNA deaminase and a method for detecting epigenetic modifications. Background Art

[0002] Transcription factors, chromatin-related proteins, and histone modifications play key roles in gene regulation and epigenetic research, and they coordinately regulate the on or off state of genes. Transcription factors bind to specific DNA sequences to promote or inhibit the transcription of related genes. At the same time, the openness of chromatin and the histone modification state affect the openness of specific genomic regions, thereby controlling the functional state of the genome. For example, different histone modifications such as acetylation, methylation, and ubiquitination can regulate the compactness of chromatin and the gene transcription level, affecting the level of gene expression. In recent years, with the development of high-throughput technologies, a variety of techniques have emerged to detect the localization of transcription factors, chromatin-related proteins, and histone modifications on the genome.

[0003] Chromatin Immunoprecipitation (ChIP) is a classical method for studying the interaction between proteins and DNA, which can reveal transcription factor binding sites and histone modification characteristics. In 2007, ChIP technology was combined with high-throughput second-generation sequencing to form Chromatin Immunoprecipitation Sequencing (ChIP-Seq), which significantly promoted the development of epigenetic and gene regulation research. ChIP-Seq fixes cells or tissues with formaldehyde, randomly breaks chromatin after lysis, precipitates the target protein-DNA complex with a specific antibody, and then reverse cross-links to extract DNA and perform sequencing. The sequencing data can be used to identify protein binding sites across the genome, providing key information for studying gene regulation. However, the application of ChIP-Seq is limited by high sample requirements, limited signal-to-noise ratio, and a high dependence on antibody quality.

[0004] In recent years, researchers have successively developed CUT&RUN (Cleavage Under Targets and Release Using Nuclease) (10,11) and CUT&Tag (Cleavage Under Targets and Tagmentation) technologies. ​Both methods utilize Protein A, which can bind antibodies. By expressing micrococcal nuclease (CUT&RUN) or Tn5 transposase (CUT&Tag) fused with Protein A and binding them to specific antibodies against the target protein, antibody-guided targeted DNA cleavage or transposition is achieved. CUT&RUN cleaves DNA near the target protein using MNase, while CUT&Tag inserts adapters at both ends of the DNA fragment to label the binding region. These two methods significantly simplify the experimental procedure, improve the signal-to-noise ratio and sensitivity, and greatly reduce the required sample amount, and can even be applied to single-cell analysis. However, since both CUT&RUN and CUT&Tag involve DNA fragmentation, they are only applicable to next-generation sequencing technologies, unable to integrate single-molecule long-read sequencing, and there are also problems with the compatibility of multi-genomics technologies.

[0005] In addition, some new technologies developed in recent years have further expanded the possibilities of research. In 2022, the Directed Methylation and Long-read Sequencing technology (DiMeLo-Seq) fused N6-adenine methyltransferase with Protein A to perform 6mA methylation near the target protein binding site, and combined nanopore sequencing or PacBio HiFi sequencing to detect protein-DNA interactions on DNA molecules. Although the amplification-free strategy of DiMeLo-Seq has the advantages of high precision and long reads, in order to retain methylation information, its DNA samples cannot be amplified, and the requirement for starting materials is high (1,000,000 - 2,000,000 cells / experiment), which limits its application in rare samples.

[0006] Both CUT&Tag, CUT&RUN, and DiMeLo-Seq utilize Protein A to bind to the Fc segment of antibodies, achieving the binding of enzymes and antibodies. However, Protein A has different affinities for antibodies from different species. For example, it has a very strong affinity for rabbit antibodies but a very weak affinity for mouse antibodies. In the continuous development of related technologies, two different technical routes have emerged. One route is nano-CT, which further replaces Protein A in Cut&Tag with a nanobody with stronger specificity to recognize the Fc segment of antibodies from specific species. The other is to fuse Protein A and Protein G for expression to achieve a more universal affinity for antibodies of different species through a combined use method. .

[0007] The discovery of double-stranded DNA deaminases has provided new tools for genomic research. Different from single-stranded DNA deaminases, double-stranded DNA deaminases such as DddA can catalyze the deamination reaction of cytosine in double-stranded DNA, converting cytosine into uracil. In 2020, the research groups of Joseph Mougous and David Liu first reported the fusion of the double-stranded deaminase DddA with TALEN to develop DdCBE, achieving single-base precise editing of the mitochondrial genome and providing an important tool for the study of mitochondrial genetic diseases. . Subsequently, the Joseph Mougous research group established the 3D-seq technology. By fusing and overexpressing DddA with specific transcription factors in uracil-tolerant (ung gene knockout) live bacteria, cytosine near the genomic binding site of the transcription factor is converted into uracil, and then uracil is detected as thymine during PCR amplification and next-generation sequencing to determine the localization of the transcription factor. . However, DddA has high toxicity in live cells and can only be used in uracil-tolerant bacteria (ung gene knockout), and each detection requires fusion expression with specific transcription factors, which limits its throughput and application in eukaryotic cells. Summary of the Invention

[0008] To overcome the above defects of the prior art, the present inventors have developed a method for detecting the interaction between chromatin-related proteins and DNA based on targeted double-stranded DNA deaminases. The present inventors fused and expressed Protein A and Protein G, which have specific affinity for antibodies, with double-stranded DNA deaminases. The antibodies have specific affinity for chromatin-related proteins or chromatin-related protein modifications. Utilizing the property of Protein A-Protein G proteins binding to antibodies, the double-stranded DNA deaminase is guided to the position where a specific chromatin-related protein or chromatin-related protein modification binds to DNA. By deaminating the DNA at this site with the double-stranded DNA deaminase, the localization of chromatin-related proteins or chromatin-related protein modifications on the double-stranded DNA sequence is achieved. This process not only provides a more precise means of modification detection but also avoids the DNA fragmentation problem that may exist in common modification detection methods, making it an ideal DNA-binding protein detection technology. This detection method of the present invention is named ANDIE (Antibody-associated DeamInasE).

[0009] To achieve the above object, the present invention provides a fusion protein in a first aspect, the fusion protein comprising, from the N-terminus to the C-terminus: a molecule having specific affinity for an antibody, a linker peptide, and a double-stranded DNA deaminase, wherein the molecule having specific affinity for the antibody is Protein A and / or Protein G, and the antibody is an antibody having specific affinity for a chromatin-related protein or a chromatin-related protein modification.

[0010] In one embodiment of the first aspect of the present invention, the double-stranded DNA deaminase is the active domain DddAtox of the double-stranded DNA deaminase.

[0011] In one embodiment of the first aspect of the present invention, the Protein A sequence is Seq ID No. 1; the Protein G protein sequence is Seq ID No. 2; the linker peptide sequence is Seq ID No. 3; and / or the double-stranded DNA deaminase sequence is Seq ID No. 4.

[0012] In one embodiment of the first aspect of the present invention, the chromatin-related protein is at least one protein selected from DNA-binding proteins, transcription factors, and histones, or a protein that binds to at least one protein among DNA-binding proteins, transcription factors, and histones.

[0013] The present invention also provides a polynucleotide encoding the fusion protein of the present invention described above.

[0014] The present invention also provides a nucleic acid vector comprising the polynucleotide of the present invention described above.

[0015] The invention also provides a host cell comprising the polynucleotide of the present invention described above or the nucleic acid vector of the present invention described above.

[0016] The present invention provides a method for detecting the position of a chromatin-related protein or a chromatin-related protein modification on a double-stranded DNA sequence in a second aspect, the method comprising:

[0017] Step a. Connect a molecule having specific affinity for an antibody to a double-stranded DNA deaminase, the antibody having specific affinity for a chromatin-related protein or a chromatin-related protein modification, and through the binding of the antibody to the chromatin-related protein or the chromatin-related protein modification, enabling the double-stranded DNA deaminase to targetedly perform a deamination reaction on the DNA sequence near the chromatin-related protein or the chromatin-related protein modification bound by the antibody.

[0018] Step b. Detect the position of the chromatin-related protein or the chromatin-related protein modification bound by the antibody on the double-stranded DNA sequence by determining the position of the DNA deamination site.

[0019] Among them, the molecule with specific affinity for the antibody is Protein A and / or Protein G with specific affinity for the Fc domain of the antibody.

[0020] In one embodiment of the second aspect of the present invention, the double-stranded DNA deaminase refers to an enzyme that can deaminate cytosine and / or adenine on double-stranded DNA.

[0021] In one embodiment of the second aspect of the present invention, the molecule with specific affinity for the antibody is connected to the double-stranded DNA deaminase for protein fusion and expression.

[0022] In one embodiment of the second aspect of the present invention, the connection between the molecule with specific affinity for the antibody and the double-stranded DNA deaminase is an affinity connection.

[0023] In one embodiment of the second aspect of the present invention, the connection between the molecule with specific affinity for the antibody and the double-stranded DNA deaminase is a covalent connection.

[0024] In one embodiment of the second aspect of the present invention, the chromatin-related protein is at least one protein selected from DNA-binding proteins, transcription factors, and histones, or a protein that binds to at least one of DNA-binding proteins, transcription factors, and histones.

[0025] In one embodiment of the second aspect of the present invention, the DNA deamination site is determined by detecting the bases that have undergone sequence conversion after PCR amplification and sequencing.

[0026] In one embodiment of the second aspect of the present invention, the binding sequence of the antibody to DNA is determined by analyzing the position of the deamination site.

[0027] In one embodiment of the second aspect of the present invention, the chromatin-related protein modification includes trimethylation of lysine at position 4 on histone H3, or acetylation of lysine at position 27 on histone H3.

[0028] Beneficial effects

[0029] Different from DNA cleavage-based methods such as CUT&Tag, ANDIE of the present invention keeps genomic DNA unfragmented after deamination reaction. Different from current non-cleavage methods such as DiMeLo-seq, ANDIE of the present invention can retain cytosine conversion information during PCR amplification. This characteristic enables ANDIE of the present invention to be compatible with other genomic analysis methods and support subsequent joint analysis. To verify this advantage, by seamlessly combining ANDIE of the present invention with single-cell Hi-C, the inventors of the present invention developed ANDIE-binding Hi-C technology, achieving simultaneous mapping of histone modifications and three-dimensional genomic structures in single cells.

[0030] Moreover, the present invention can be more flexibly applied to various antibodies or detection targets containing antibody fragments by fusing double-stranded DNA deaminase with Protein A and / or Protein G. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 Schematic illustration of the nucleic acid vector pETDuet-1::pagdddA + dddAI for ProteinAG-DddA protein expression. The inserted pagdddA gene and dddAI gene are driven by two independent lac promoters respectively.

[0032] Figure 2 Showing Coomassie blue staining on the SDS–PAGE gel of purified pAG-DddA protein.

[0033] Figure 3 Showing the nucleotide frequency sequence logo map around the cytosine converted by pAG-DddA in genomic DNA.

[0034] Figure 4 Schematically showing that lysed cell nuclei are incubated with specific antibodies conjugated with pAG-DddA, which promotes targeted deamination of cytosines near the antibody-binding regions. Subsequently, through library construction and high-throughput sequencing, the conversion sites can be accurately determined, thereby achieving precise localization and mapping of histone modifications.

[0035] Figure 5 Showing the conversion ratio (y-axis) around the centers of H3K4me3 (left) and H3K27ac (right) modification peaks. The x-axis represents the distance from the center of the ChIP-seq peak. The gray line is the IgG control group. From these data, the conversion enrichment of H3K4me3 and H3K27ac can be observed.

[0036] Figure 6Shows the conversion ratio (y-axis) of H3K4me3 (black) and H3K27ac (gray) modifications within the transcription start site (TSS) region. The x-axis represents the distance from the center of the transcription start site.

[0037] Figure 7 Shows the conversion ratios of H3K4me3 and H3K27ac modifications across the genome. The heatmap reflects the deamination efficiency in different DNA sequence contexts, where rows and columns represent adjacent bases before and after, respectively.

[0038] Figure 8 Shows: The distribution plot shows the TC conversion ratios for H3K4me3 (left) and H3K27ac (right). The dark curve represents positive reads, and the light curve represents negative reads. The average TC conversion ratio for positive reads of H3K4me3 is 0.218, while for negative reads it is 0.046 (P < 0.0001, two-sided Mann-Whitney U rank test); the average ratio for positive reads of H3K27ac is 0.229, while for negative reads it is 0.051 (P < 0.0001, two-sided Mann-Whitney U rank test).

[0039] Figure 9 Are aggregation enrichment plots and heatmaps showing the distribution of H3K4me3 signals at transcription start sites (TSSs), comparing the results of three methods: ANDIE, CUT&Tag, and ChIP-seq. All three methods show significant enrichment and consistent distribution characteristics at TSSs.

[0040] Figure 10 Is a Pearson correlation heatmap showing the correlation of H3K4me3 (left panel) and H3K27ac (right panel). The results show that ANDIE exhibits high correlation in replicate experiments and high correlation with CUT&Tag and ChIP-seq, while the correlation of the IgG control group is very low.

[0041] Figure 11 Is a principal component analysis (PCA) plot showing the results of clustering samples based on the genome-wide histone modification maps, with data generated by ChIP-seq, CUT&Tag, and ANDIE. Each point represents a biological replicate, where H3K4me3 is dark (upper left) and H3K27ac is light (upper right). The PCA plot clearly shows the separation of H3K4me3 and H3K27ac marks mapped by the three methods on the principal components.

[0042] Figure 12The genomic browser visualization shows the H3K4me3 (upper panel) and H3K27ac (lower panel) signals at representative genomic loci, which is a software screenshot. ANDIE accurately reproduces the peaks observed by CUT&Tag and ChIP-seq at different loci, showing a high degree of consistency.

[0043] Figure 13 It is a schematic diagram of the ANDIE combined with Hi-C workflow. Antibody-directed deamination occurs near the targeted histone modification. Subsequently, restriction enzyme digestion and proximity ligation to capture chromatin interactions in the Hi-C experimental steps are carried out, followed by library construction and sequencing.

[0044] Figure 14 It is the H3K4me3 profile in the Chr8 (37 - 40 Mb) region, which is a software screenshot, showing the genomic visualization from ChIP-seq, combined ANDIE combined with Hi-C data, and single-cell ANDIE combined with Hi-C and single-cell sortChIC data.

[0045] Figure 15 Show the enrichment analysis of ANDIE combined with Hi-C and ChIP-seq signals of H3K4me3 around transcription start sites (TSSs).

[0046] Figure 16 Show the comparison of the number of H3K4me3 detections in single cells between single-cell ANDIE combined with Hi-C, sortChIC, and Paired-Tag.

[0047] Figure 17 Show the correlation of A / B compartment scores between ANDIE combined with Hi-C and scMicro-C(25). Detailed implementation

[0048] As used herein, the term "fusion protein" refers to a hybrid polypeptide containing protein domains from at least two different proteins. The "fusion protein" of the present invention contains: a molecule having specific affinity for an antibody, and a double-stranded DNA deaminase, wherein, for example, the molecule having specific affinity for an antibody or the double-stranded DNA deaminase can be located in the amino-terminal (N-terminal) portion of the fusion protein; another protein, such as the double-stranded DNA deaminase or the molecule having specific affinity for an antibody, can be located in the carboxyl-terminal (C-terminal) of the fusion protein.

[0049] There may be a linker peptide between the molecule having specific affinity for an antibody and the double-stranded DNA deaminase.

[0050] The linker peptide only needs to ensure sufficient space for the N-terminal protein and the C-terminal protein to exert their activities. For example, sequences commonly used in the art can be adopted, such as GSGGSSGGSSGSGDP, SGGSSGGSSGSETPGTSESATPESSGGSSGGS, GGGGSGGGGS, GGSGGSDDDKEF, GGSDDDKEF, DDDKEF, EAAAKEAAAK, AAAKEAAAKA, EAAAK, SGGSGGSGGS, GGGGS, GGGGGG, and SGGS.

[0051] Molecules with specific affinity for antibodies are selected from Protein A and Protein G. Both Protein A and Protein G are proteins that bind to the Fc region of antibodies, but there are differences in the types of antibody spectra they bind. Those skilled in the art can flexibly select Protein A and Protein G according to needs.

[0052] The present invention preferably uses Protein A and Protein G simultaneously to cover more types of antibodies, so that the fusion protein of the present invention and its application method can bind a wider range of antibodies, and thus have a wider application scenario.

[0053] In the present invention, the double-stranded DNA deaminase can catalyze the cytosine deamination reaction in double-stranded DNA, converting cytosine into uracil. After the conversion, uracil is detected as thymine during PCR amplification and next-generation sequencing, so as to determine the action position of the double-stranded DNA deaminase.

[0054] In the prior art, DddA has high toxicity in living cells and can only be used in uracil-tolerant bacteria (ung gene knockout), and each detection requires fusion expression with a specific transcription factor, which limits its throughput and application in eukaryotic cells. In view of this, the present invention overcomes its toxicity by expressing the double-stranded DNA deaminase and the double-stranded DNA deaminase inhibitory protein in living cells.

[0055] In the embodiments of the present invention, DddAtox, that is, the active domain of the double-stranded DNA deaminase, is adopted, which is convenient for expression and operation. In the following examples of the present invention, the active domain DddAtox of the double-stranded DNA deaminase is actually used. Those skilled in the art understand that DddAtox has the same activity as the wild-type double-stranded DNA deaminase and can be used interchangeably in the technical solutions of the present invention.

[0056] In an embodiment of the first aspect of the present invention, the double-stranded DNA deaminase is the active domain DddAtox of the double-stranded DNA deaminase, and the sequence is shown in SEQ ID No. 4.

[0057] In one embodiment of the present invention, the Protein A sequence can be the sequence of its wild type or known variants, such as sequences from databases such as GenBank, EMBL, UniProt, and PIR, or sequences having a sequence homology of 80%, 85%, 90%, 95%, 98% or more than 99% therewith, as long as its ability to bind to the Fc segment of the antibody is retained. For example, in the embodiments of the present application, the antibody-binding domain of Protein A with the sequence of SEQ ID No. 1 is used.

[0058] The Protein G protein sequence can be the sequence of its wild type or known variants, such as sequences from databases such as GenBank, EMBL, UniProt, and PIR, or sequences having a sequence homology of 80%, 85%, 90%, 95%, 98% or more than 99% therewith, as long as its ability to bind to the Fc segment of the antibody is retained. For example, in the embodiments of the present application, the antibody-binding domain B1 of Protein G with the sequence of SEQ ID No. 2 is used.

[0059] Regarding the information on the antibody-binding domains of Protein A and Protein G, for example, see Chimeric IgG-binding receptors engineered from staphylococcal protein A and streptococcal protein G, which is hereby incorporated by reference herein. 。

[0060] The linker peptide sequence is SEQ ID No. 3, or a sequence having a sequence homology of 80%, 85%, 90%, 95%, 98% or more than 99% therewith, as long as it does not affect the activities of Protein A and / or Protein G and the double-stranded DNA deaminase activity.

[0061] The double-stranded DNA deaminase sequence is SEQ ID No. 4 or a sequence having a sequence homology of 80%, 85%, 90%, 95%, 98% or more than 99% therewith, as long as its ability to catalyze the deamination reaction of cytosine in double-stranded DNA is retained.

[0062] In one embodiment of the present invention, the chromatin-related protein is at least one protein selected from DNA-binding proteins, transcription factors, and histones, or a protein that binds to at least one protein among DNA-binding proteins, transcription factors, and histones.

[0063] A protein that binds to at least one of a DNA-binding protein, a transcription factor, and a histone refers to a protein that can specifically bind to at least one of a DNA-binding protein, a transcription factor, and a histone. In the case where the chromatin-related protein is a protein that binds to at least one of a DNA-binding protein, a transcription factor, and a histone, the antibody of the present invention indirectly binds to at least one of a DNA-binding protein, a transcription factor, and a histone.

[0064] In one embodiment of the present invention, a polynucleotide is provided that encodes the fusion protein of the present invention. Due to the degeneracy of codons, those skilled in the art can know that there are multiple nucleic acid sequences capable of encoding the fusion protein provided in this application, which are not limited herein. Those skilled in the art can perform appropriate codon optimization and select a suitable nucleic acid sequence for expressing the fusion protein provided in this application according to different expression purposes or hosts.

[0065] In one embodiment of the present invention, a nucleic acid vector is provided that contains the polynucleotide of the present invention.

[0066] In some embodiments, the nucleic acid vector includes a cloning vector and an expression vector. The expression nucleic acid vector can be used in prokaryotic cell expression systems, eukaryotic cell (mammalian cells and insect cells) expression systems, and cell-free expression systems. Both virus-based and non-virus-based expression vectors can be used to produce deaminase fusion proteins in eukaryotic host cells. Non-viral vectors and systems include plasmids, episomal vectors (typically having an expression cassette for expressing proteins or RNAs), and human artificial chromosomes. Virus-derived vectors include, but are not limited to, lentiviral vectors, retroviral vectors, adenoviral vectors, adeno-associated viral vectors, poxviral vectors, and herpesviral vectors.

[0067] In one embodiment of the present invention, a host cell is provided that contains the polynucleotide of the present invention or the nucleic acid vector of the present invention.

[0068] The host cell can be selected according to the type of the expression vector. Any suitable method well known in the art can be used to deliver the polynucleotide and / or the expression vector into the host cell, which is not limited herein.

[0069] [Method for Detecting the Position of Chromatin-Related Proteins or Chromatin-Related Protein Modifications on Double-Stranded DNA Sequences]

[0070] In one embodiment of the present invention, a method for detecting the position of chromatin-related proteins or chromatin-related protein modifications on double-stranded DNA sequences is provided, the method comprising:

[0071] Step a. Connect a molecule with specific affinity for an antibody to a double-stranded DNA deaminase. The antibody has specific affinity for a chromatin-associated protein or a chromatin-associated protein modification. Through the binding of the antibody to the chromatin-associated protein or the chromatin-associated protein modification, the double-stranded DNA deaminase is targeted to perform a deamination reaction on the DNA sequence near the chromatin-associated protein or the chromatin-associated protein modification bound by the antibody.

[0072] Step b. Detect the position of the chromatin-associated protein or the chromatin-associated protein modification bound by the antibody on the double-stranded DNA sequence by determining the location of the DNA deamination site.

[0073] The molecule with specific affinity for the antibody is Protein A and / or Protein G, or a fragment thereof with specific affinity for the antibody.

[0074] The chromatin-associated protein is at least one protein selected from DNA-binding proteins, transcription factors, and histones, or a protein that binds to at least one of DNA-binding proteins, transcription factors, and histones.

[0075] In step a, due to the binding of the molecule with specific affinity for the antibody to the antibody and the binding of the antibody to the chromatin-associated protein or the chromatin-associated protein modification, the double-stranded DNA deaminase connected to the molecule with specific affinity for the antibody is guided by the antibody to the chromatin-associated protein or the chromatin-associated protein modification, and a deamination reaction is performed on the DNA sequence near the chromatin-associated protein or the chromatin-associated protein modification.

[0076] In one embodiment of the present invention, a molecule with specific affinity for an antibody is connected to a double-stranded DNA deaminase. Since the antibody has specific affinity for a chromatin-associated protein or a chromatin-associated protein modification, through the binding of the antibody to the chromatin-associated protein or the chromatin-associated protein modification and the binding of the molecule with specific affinity for the antibody to the antibody, the double-stranded DNA deaminase is targeted to the DNA sequence bound by the chromatin-associated protein or the chromatin-associated protein modification, and a deamination reaction is performed on the DNA sequence bound by the chromatin-associated protein or the chromatin-associated protein modification.

[0077] In one embodiment of the present invention, the deamination reaction occurs on the DNA sequence bound by the chromatin-associated protein or the chromatin-associated protein modification, and the DNA sequences within the ranges of 300 bp, 200 bp, 150 bp, 100 bp upstream and 300 bp, 200 bp, 150 bp, 100 bp downstream.

[0078] In one embodiment of the present invention, the position of the chromatin-related protein or chromatin-related protein modification bound by the antibody on the double-stranded DNA sequence is detected by determining the position of the DNA deamination site.

[0079] In one embodiment of the present invention, the DNA deamination site is determined by detecting the bases that have undergone sequence conversion after PCR amplification and sequencing.

[0080] In one embodiment of the present invention, the binding sequence of the antibody to DNA is determined by analyzing the position of the deamination site.

[0081] In one embodiment of the present invention, the deamination reaction is a reaction that catalyzes the deamination of cytosine in double-stranded DNA and converts cytosine into uracil.

[0082] In one embodiment of the present invention, a method for detecting the interaction between chromatin-related proteins and DNA by a targeted double-stranded DNA deaminase. For example, Protein A and Protein G are fused and expressed with the double-stranded DNA deaminase. When an antibody with specific affinity for a chromatin-related protein or chromatin-related protein modification binds to the chromatin-related protein or chromatin-related protein modification, the double-stranded DNA deaminase is guided to the DNA where the specific chromatin-related protein or chromatin-related protein modification is located through the binding of the Protein A-Protein G protein to the antibody, thereby volatilizing the deamination of the DNA at this site by the double-stranded DNA deaminase, and the localization of the chromatin-related protein or chromatin-related protein modification on the double-stranded DNA sequence is achieved through the recognition of the deaminated sequence.

[0083] This process not only provides a more accurate means of modification detection, but also avoids the DNA fragmentation problem that may exist in common modification detection methods, making it an ideal DNA-binding protein detection technology. This detection method of the present invention is named ANDIE.

[0084] Different from the DNA cleavage-based method (such as CUT&Tag), ANDIE of the present invention keeps the genomic DNA unfragmented after the deamination reaction. Different from the current non-cleavage methods (such as DiMeLo-seq), ANDIE of the present invention can retain the cytosine conversion information during the PCR amplification process. This characteristic enables ANDIE of the present invention to be compatible with other genomic analysis methods and supports subsequent joint analysis. To verify this advantage, by seamlessly combining ANDIE of the present invention with single-cell Hi-C, the inventors of the present invention developed the ANDIE-binding Hi-C technology, which achieved the simultaneous mapping of histone modifications and three-dimensional genomic structures in single cells.

[0085] Moreover, by fusing the double-stranded DNA deaminase with Protein A and / or Protein G, the present invention can be more flexibly applied to the detection targets against various antibodies or antibody fragment-containing substances.

[0086] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0087] Examples:

[0088] Materials and Methods:

[0089] 1. Construction of nucleic acid expression vectors

[0090] Construction of expression vectors: The expression constructs of ProteinAG-DddA (PAG-DddA) and DddAI were obtained from Genescript through gene synthesis services.

[0091] Construct a ProteinAG-DddA expression vector based on pETDuet-1, where

[0092]

[0093]

[0094] The coding sequence of ProteinAG-DddA was synthesized and cloned into MCS-1 (NcoI and NotI sites, retaining the N-terminal hexahistidine tag).

[0095] In PAG-DddA, the protein sequence HHHHHH is the hexahistidine tag,

[0096]

[0097] The coding sequence of the immune protein DddAI was synthesized and cloned into MCS-2 (NdeI and XhoI sites, removing the C-terminal S tag). The pETDuet-1::pagdddAtox + dddAI vector was transformed into Escherichia coli DH5α and BL21 strains and stored at -20°C.

[0098] 2. Bacterial strain culture conditions

[0099] The Escherichia coli (E. coli) strains transformed with nucleic acid vectors were cultured in LB medium at 37 °C or on LB solid medium supplemented with agar (LBA, 1.5% w / v). If necessary, ampicillin (100 μg / mL) or IPTG (0.5 mM) was added to the medium. E. coli DH5α and BL21 were used for nucleic acid vector preservation and protein expression, respectively.

[0100] 3. Purification of PAG-DddAtox

[0101] Briefly, to purify His-tagged PAG-DddAtox complexed with DddAI, 2 L of LB medium was inoculated with E. coli BL21(pETDuet-1::pagdddA + dddAI) at an inoculation ratio of 1:100 and cultured overnight at 37 °C. When the OD600 of the bacterial culture reached approximately 0.6, 0.5 mM IPTG was added and the culture was shaken at 18 °C for 16 h. Bacterial cell pellets were collected by centrifugation at 4000 g for 30 min and resuspended in 50 ml of lysis buffer (50 mM Tris-HCl pH 8.0, 500 mM NaCl, 10 mM imidazole, 1 mg / mL lysozyme, and protease inhibitor mixture). The bacterial cell pellets were lysed by sonication (five pulses of 10 s each), and the cell debris was removed from the sonicated supernatant by centrifugation at 25,000 g for 30 min. The His-tagged PAG-DddAtox-DddAI complex was purified from the supernatant using a nickel column. The DddAtox-DddAI complex was eluted using elution buffer (50 mM Tris-HCl pH 7.5, 300 mM imidazole, 500 mM NaCl, 30 mM imidazole, 1 mM DTT). The eluted PAG-DddA-DddAI complex was denatured by adding 50 ml of 8 M urea denaturing buffer (50 mM Tris-HCl pH 7.5, 300 mM imidazole, 500 mM NaCl, 1 mM DTT) at 4 °C for 16 h to completely separate PAG-DddA from DddAI. The denatured protein was separated again using a nickel column in 8 M urea denaturing buffer, and PAG-DddA could bind to the column due to the His-tag. The column was washed with 50 ml of 8 M urea denaturing buffer to remove residual DddAI. Subsequently, the column was washed successively with denaturing buffers with gradually decreasing urea concentrations (6 M, 4 M, 2 M, 1 M), and finally with a washing buffer without urea. The PAG-DddAtox bound to the column was eluted with 5 ml of elution buffer. The eluted PAG-DddA was separated by size exclusion chromatography using fast protein liquid chromatography (FPLC) and gel filtration on a Superdex 200 column (GE Healthcare) using size buffer (20 mM Tris-HCl pH 7.5, 200 mM NaCl, 1 mM DTT, 5% (w / v) glycerol). The purity of the eluted PAG-DddA was evaluated by SDS-PAGE gel and Coomassie Brilliant Blue staining. The purified protein was stored at -80 °C.

[0102] 4. Eukaryotic cell culture and treatment conditions

[0103] Drosophila Schneider 2 (S2) cells were cultured in Schneider's Drosophila medium (Gibco, product number: 21720024), supplemented with 10% fetal bovine serum (FBS) (Cellmax, product number: SA301.02) and 1% penicillin-streptomycin (Pen-Strep) (Gibco, Thermo Fisher Scientific, product number: 15140122), and incubated at 25 °C without CO2. For use, S2 cells were collected by centrifugation at 300 g for 5 minutes and resuspended in 1×PBS. When fixation was required (paraformaldehyde, PFA), 3 million S2 cells were resuspended in 500 μL of 0.5% PFA and fixed for 10 minutes. Fixed cells were collected by centrifugation at 600 g for 5 minutes, resuspended in 300 μL of DPBS (product number: 14040117, Thermo Fisher)-1% BSA (product number: A1933, Sigma), washed once, allowed to stand for 5 minutes to eliminate the effect of PFA, and finally washed three times with 1% BSA-PBS.

[0104] The human cell line GM12878 cells were cultured in RPMI 1640 medium (product number: 11875093, Gibco), supplemented with 10% fetal bovine serum (FBS) (product number: 10099141C, Gibco), in a 5% CO2 incubator at 37 °C. If needed, 1 million cells were crosslinked with 1 ml of freshly prepared 0.5% PFA fixation buffer at room temperature (RT) for 10 minutes. To terminate the fixation reaction, the cells were resuspended in 1 ml of 1% BSA / PBS, allowed to stand for 5 minutes to eliminate the effect of PFA, and finally washed three times with 1% BSA-PBS.

[0105] 5. Solution preparation

[0106] Table 1. Components of the deaminase incubation buffer

[0107]

[0108] Table 2. Components of the wash buffer

[0109]

[0110] Table 3. Components of the antibody buffer

[0111]

[0112] Table 4. Components of the high-salt buffer

[0113]

[0114] Table 5. Components of 1% BSA / PBS

[0115]

[0116] Table 6. 0.5% PFA Composition

[0117]

[0118] 6. Detection of specific histone modifications in Drosophila cells using pAG-DddA (ANDIE)

[0119] Rinse S2 cells with PBS and resuspend them in PBS. Use a cell counter to count the cells. Next, centrifuge 3×10^6 cells from each sample at 300×g for 5 minutes. To slightly fix the cells, resuspend approximately 3×10^6 S2 cells in 500 μL of 0.5% PFA, rotate for 5 minutes or manually invert to mix well to ensure full contact between the fixative and the cells. Then, add 300 μL of 1% BSA / PBS and mix. Centrifuge at 500×g for 4 minutes at 4°C and remove the supernatant. Next, resuspend the cells in 300 μL of 1% BSA / PBS, incubate with rotation for 5 minutes, and resuspend in 200 μL of PBS and count the cells. Take 5×10^5 S2 cells into a PCR tube, centrifuge at 800×g for 5 minutes at 4°C and remove the supernatant. Then, add 1 μL of 20% Np-40 to 200 μL of incubation buffer (to make the final concentration 0.1%). Place the S2 cells in the PCR tube, add 100 μL of the resulting liquid to resuspend, and incubate on ice for 5 minutes. Immediately, centrifuge at 800×g for 5 minutes at 4°C and remove the supernatant. Next, wash once with 100 μL of wash buffer, centrifuge at 800×g for 5 minutes at 4°C and remove the supernatant. Subsequently, add 1 μL of antibody to 50 μL of antibody buffer (set up one IgG control group, one H3K4me3 experimental group, and one H3K27ac experimental group) and mix well. Resuspend the precipitate and incubate with rotation at room temperature for 2 hours. After that, centrifuge the S2 cells at 300×g for 5 minutes, wash once more with 100 μL of cold antibody buffer, and gently pipette about 10 times. After resuspension, place on a rotator at 4°C and rotate for 5 minutes, then centrifuge at 300×g to remove the supernatant. Gently dissolve the precipitate in 50 μL of high-salt buffer containing 2.5 μL of PA / G-DddA and place on a rotator for 1 hour, rotate and mix in a cold room at 4°C. Centrifuge at 300×g for 5 minutes and remove the supernatant. Then, wash twice with 150 μL of cold high-salt buffer, gently pipette about 10 times. After resuspension, place on a rotator at 4°C and rotate for 5 minutes, then centrifuge at 300×g to remove the supernatant. For each sample, gently dissolve the precipitate in 100 μL of incubation buffer and incubate at 37°C for 1 hour. Finally, centrifuge the S2 cells at 800×g for 5 minutes, remove the supernatant, and add 100 μL of 0.2% SDS-PBS at room temperature (i.e., add 2 μL of 10% SDS to 98 μL of PBS) and 10 μL of proteinase K, and let stand overnight at 55°C. After overnight incubation, extract and purify genomic DNA using Zymo Research D4010 Genomic DNA Clean & Concentrator®-10 (or 0.5× Vazyme beads), and measure the concentration of the resulting DNA using Qubit.The Vazyme TD502 (TD503) TruePrep DNA Library Preparation Kit was used to prepare the sequencing library.

[0120] 7. Co-detection of histone modifications and chromatin structure in single cells using pAG-DddA (ANDIE combined with Hi-C)

[0121] The present inventors performed the ANDIE experiment as described above using 5×10^4 0.5% PFA-fixed GM12878 cells. After the antibody-directed deamination reaction, the cells were fixed again with 1.5% PFA and processed according to the previously reported Dip-C cell fixation protocol. 。

[0122] [Construction, Expression, and Deamination Detection of Fusion Vectors]

[0123] Constructed according to "1. Construction of Expression Nucleic Acid Vectors" above: ProteinAG-DddAtox (PAG-DddAtox) expression vector and DddAI expression construct.

[0124] Among them, the active region DddAtox of the wild-type DddA protein was used. The present inventors, through gene synthesis, placed the His tag, Protein A, Protein G, and a linker sequence GSGGSSGGSSGSGDP in the same open reading frame (ORF) with the coding gene of DddAtox, and placed the coding gene of DddAI (the active neutralizing protein of DddA) in another open reading frame, driven by two independent promoters ( Figure 1 ). After protein induction expression, through renaturation treatment, DddDddAtoxA was separated from DddAI to obtain purified protein ( Figure 2 ). DddAtox (pAG-DddAtox) fused with Protein A and Protein G still has deamination activity on double-stranded DNA, and its deamination sites are mainly TC ( Figure 3 ), which is consistent with the previous reports.

[0125] [Detection of Genome-wide Histone Modifications - ANDIE]

[0126] The detection process for genome-wide histone modifications using PAG-DddA is as follows: First, permeabilized cell nuclei are incubated with specific antibodies to identify histone modification sites. Then, PAG-DddA is added and incubated at low temperature to enable PAG-DddA to bind to the antibodies and reduce non-specific deamination reactions. Subsequently, unbound antibodies and PAG-DddA in the cell nuclei are washed away under low-temperature conditions. The PAG-DddA bound to the antibodies then catalyzes DNA deamination at 37 degrees Celsius, achieving antibody-guided targeted DNA deamination. The DNA then undergoes PCR library construction and amplification. During the amplification process, cytosine deamination to uracil can be converted to thymine, enabling the detection of deamination conversion sites after sequencing. DNA regions with a relatively high conversion ratio can be determined as the binding regions on DNA occupied by histone modifications( Figure 4 ).

[0127] The inventors named this detection method ANDIE (Antibody-directed deamination) and applied it to the detection of lysine 27 acetylation of histone H3 (H3K27ac) and trimethylation of lysine 4 (H3K4me3). Among them, H3K27ac is usually used as a marker of active enhancers, while H3K4me3 is a common marker in the promoter regions of actively transcribed genes. The inventors analyzed the ANDIE data obtained using H3K4me3 or H3K27ac antibodies. At the centers of the ChIP-seq peaks of H3K4me3 or H3K27ac, significant increases in conversion were observed in both H3K4me3 and H3K27ac ANDIE( Figure 5 ). Notably, ANDIE using the IgG control showed almost no background conversion, further verifying the specificity of this method( Figure 5 ). In addition, at transcription start sites (TSSs), ANDIE for H3K4me3 and H3K27ac also showed significant signal enrichment and had a signal periodicity of approximately 150 bp, consistent with the known distribution pattern of nucleosomes at TSS positions( Figure 6 ). These experimental results fully demonstrate the high specificity and sensitivity of ANDIE in histone modification detection.

[0128] At the genome-wide level, the global TC conversion rate of ANDIE in histone modification detection is approximately 2%( Figure 7 ). To accurately define the positive signals of histone modifications, the inventors set a criterion: within a specific histone modification region, a positive result requires at least three converted TC sites in paired-end 150-bp NGS sequencing reads. Specifically, the minimum conversion rate for H3K4me3 is 7%, while for H3K27ac it is 6%(Figure 8 )。In the histone modification positive regions, the average TC conversion rate of positive reads in the target regions (average about 20%) is about five times higher than that of negative reads in the non-target regions (average about 4% after excluding sequencing reads with zero conversion), further demonstrating the reliability of ANDIE in histone modification detection.

[0129] To verify the performance of ANDIE, the present inventors compared it with the currently widely used detection methods, CUT&Tag and ChIP-seq. In the detection of the H3K4me3 mark, all three methods showed a consistent enrichment pattern when centered on the active TSS ( Figure 9 )。To further evaluate the correlation between these methods, the present inventors calculated the correlation coefficients of ANDIE, CUT&Tag, and ChIP-seq. The results showed that the correlation coefficients of ANDIE replicate experiments for H3K4me3 all exceeded 0.95, and the correlation coefficients with the other two methods all exceeded 0.8 ( Figure 10 )。This result indicates that ANDIE is highly consistent with other mature methods in terms of repeatability and reliability. Similarly, ANDIE for H3K27ac also showed high experimental repeatability and was highly consistent with the results of CUT&Tag and ChIP-seq ( Figure 10 )。

[0130] Although H3K4me3 and H3K27ac are usually located in different regions of the genome, they often coexist in highly active regulatory elements. To deeply compare the differences in the detection of H3K4me3 and H3K27ac obtained by ANDIE, CUT&Tag, and ChIP-seq, the present inventors performed principal component analysis (PCA). The analysis results showed that the replicate experiments of ANDIE, CUT&Tag, and ChIP-seq clustered together, and the distributions of H3K4me3 and H3K27ac marks along the principal components were clearly separated ( Figure 11 )。To further verify these results, the present inventors selected two genomic regions and used ANDIE for visual comparison of histone marks ( Figure 12)。In the Chr2R (6.55 - 6.8 Mb) region, CUT&Tag and ChIP-seq generated almost identical H3K4me3 and H3K27ac peaks, and ANDIE also reproduced this pattern with specific signals. In contrast, near Chr2R (5.56 - 5.86 Mb), CUT&Tag and ChIP-seq generated sharp H3K4me3 peaks, while H3K27ac showed a broader distribution, and ANDIE was also able to reproduce this phenomenon. These experimental results indicate that ANDIE can provide sensitivity and resolution comparable to CUT&Tag and ChIP-seq in different genomic regions, with excellent signal quality and experimental reproducibility.

[0131] [ANDIE combined with Hi-C and single-cell detection]

[0132] Detection methods based on deamination reactions can maintain the integrity of genomic DNA and retain cytosine conversion information during the PCR amplification process. This feature enables ANDIE to be compatible with other genomic analysis methods (such as chromatin conformation capture technology Hi-C). The inventors named this combined method ANDIE combined with Hi-C. After antibody-directed deamination reactions in specific histone modification regions, chromatin interactions are subsequently captured by restriction enzyme digestion and proximity ligation techniques ( Figure 13 ).

[0133] The inventors conducted validation experiments on single-cell ANDIE combined with Hi-C to simultaneously detect H3K4me3 patterns and three-dimensional chromatin contacts across the genome in the same cell. The ANDIE combined with Hi-C signals at the same positions were highly consistent with the ChIP-seq results of H3K4me3 detection ( Figure 14 ). Compared with the sparse and low-resolution signals observed by the sortChIC method in single cells, single-cell ANDIE combined with Hi-C showed stronger signal intensity and lower background noise in H3K4me3-enriched regions ( Figure 14 ). Consistent with ChIP-seq, H3K4me3-positive reads from ANDIE combined with Hi-C showed significant enrichment in the transcription start site (TSS) region ( Figure 15 ). Notably, compared with other single-cell methods such as sortChIC or Paired-Tag, single-cell ANDIE combined with Hi-C increased the number of detected reads of H3K4me3 by approximately 10-fold, demonstrating the sensitivity and accuracy of this method in histone modification profiling. Figure 16)。A strong A / B compartment correlation (r = 0.967) was observed between ANDIE - combined Hi - C and high - resolution scMicro - C three - dimensional genomic data, indicating that ANDIE - combined Hi - C can accurately capture chromatin structure at the single - cell level. Figure 17 )。These results strongly demonstrate the association of histone modification profiles with chromatin structure by the ANDIE - combined Hi - C technique.

[0134] Cited references:

[0135] 1. Cramer P (2019) Organization and regulation of gene transcription. Nature 573(7772):45 - 54.

[0136] 2. Bannister AJ&Kouzarides T (2011) Regulation of chromatin by histone modifications. Cell Res 21(3):381 - 395.

[0137] 3. Orlando V (2000) Mapping chromosomal proteins in vivo by formaldehyde - crosslinked - chromatin immunoprecipitation. Trends in Biochemical Sciences 25(3):99 - 104.

[0138] 4. Solomon MJ, Larsen PL,&Varshavsky A (1988) Mapping protein–DNA interactions in vivo with formaldehyde: evidence that histone H4 is retained on a highly transcribed gene. Cell 53(6):937 - 947.

[0139] 5. Johnson DS, Mortazavi A, Myers RM,&Wold B (2007) Genome - wide mapping of in vivo protein - DNA interactions. Science316(5830):1497 - 1502.

[0140] 6. Barski A , et al. (2007) High - resolution profiling of histone methylations in the human genome. Cell 129(4):823 - 837.

[0141] 7. Robertson G , et al. (2007) Genome - wide profiles of STAT1 DNA association using chromatin immunoprecipitation and massively parallel sequencing. Nature Methods 4(8):651 - 657.

[0142] 8. Mikkelsen TS , et al. (2007) Genome - wide maps of chromatin state in pluripotent and lineage - committed cells. Nature 448(7153):553 - U552.

[0143] 9. Park PJ (2009) ChIP - seq: advantages and challenges of a maturing technology. Nature Reviews Genetics 10(10):669 - 680.

[0144] 10. Meers MP, Bryson TD, Henikoff JG, & Henikoff S (2019) Improved CUT&RUN chromatin profiling tools. Elife 8.

[0145] 11. Skene PJ & Henikoff S (2017) An efficient targeted nuclease strategy for high - resolution mapping of DNA binding sites. Elife 6.

[0146] 12. Kaya-Okur HS , et al. (2019) CUT&Tag for efficient epigenomic profiling of small samples and single cells. bioRxiv .

[0147] 13. Skene PJ, Henikoff JG, & Henikoff S (2018) Targeted in situ genome-wide profiling with high efficiency for low cell numbers. Nature Protocols 13(5):1006 - 1019.

[0148] 14. Altemose N , et al. (2022) DiMeLo-seq: a long-read, single-molecule method for mapping protein-DNA interactions genome wide. Nature Methods 19(6):711 - +.

[0149] 15. Bartosovic M & Castelo-Branco G (2023) Multimodal chromatin profiling using nanobody-based single-cell CUT&Tag. Nat Biotechnol 41(6):794 - 805.

[0150] 16. Mok BY , et al. (2020) A bacterial cytidine deaminase toxin enables CRISPR-free mitochondrial base editing. Nature 583(7817):631 - +.

[0151] 17. Gallagher LA , et al. (2022) Genome-wide protein-DNA interaction site mapping in bacteria using a double-stranded DNA-specific cytosine deaminase.Nat Microbiol 7(6):844 - 855.

[0152] 18. Eliasson M , et al. (1988) Chimeric IgG - binding receptors engineered from staphylococcal protein A and streptococcal protein G. J Biol Chem 263(9):4323 - 4327.

[0153] 19. Tan L (2021) Determining the 3D genome structure of a single mammalian cell with Dip - C. STAR Protoc 2(3):100622.

[0154] 20. Tan L, Xing D, Chang CH, Li H, & Xie XS (2018) Three - dimensional genome structures of single diploid human cells. Science 361(6405):924 - 928.

Claims

1. A fusion protein, which is composed of a molecule having a specific affinity for an antibody, a connecting peptide and a double-stranded DNA deaminase from the N-terminus to the C-terminus, wherein: The molecules having specific affinity for the antibody are Protein A and Protein G, and the antibody is an antibody having specific affinity for chromatin-associated proteins or chromatin-associated protein modifications, in, The protein A sequence is SEQ ID No. 1; The protein G protein sequence is SEQ ID No. 2; The connecting peptide sequence is SEQ ID No. 3; The double-stranded DNA deaminase is a double-stranded DNA deaminase domain DddAtox with a sequence of SEQ ID No.

4.

2. The fusion protein according to claim 1, wherein The chromatin-associated protein is at least one protein selected from the group consisting of DNA binding proteins, transcription factors and histones. A polynucleotide encoding the fusion protein according to claim 1 or 2. A nucleic acid vector comprising the polynucleotide according to claim 3. A host cell comprising the polynucleotide according to claim 3 or the nucleic acid vector according to claim 4.

6. A method for detecting the position of a chromatin-associated protein or a chromatin-associated protein modification on a double-stranded DNA sequence using the fusion protein of claim 1, wherein the method is a non-diagnostic method and comprises: Step a. connecting a molecule having a specific affinity for an antibody, a connecting peptide and a double-stranded DNA deaminase to form the fusion protein, wherein the antibody has a specific affinity for a chromatin-associated protein or a chromatin-associated protein modification, and through the binding of the antibody to the chromatin-associated protein or the chromatin-associated protein modification, the double-stranded DNA deaminase performs a targeted deamination reaction on a DNA sequence near the chromatin-associated protein or the chromatin-associated protein modification bound by the antibody, Step b. Detecting the position of the chromatin-associated protein or chromatin-associated protein modification bound by the antibody on the double-stranded DNA sequence by determining the position of the DNA deamination site. 7 . The method according to claim 6 , wherein the chromatin-associated protein is at least one protein selected from the group consisting of a DNA binding protein, a transcription factor and a histone.

8. The method according to claim 6, wherein the DNA deamination site is determined by detecting the bases that undergo sequence conversion after PCR amplification and sequencing.

9. The method according to claim 6, wherein the binding sequence of the antibody to DNA is determined by analyzing the position of the deamination site.

10. The method according to claim 6, wherein the chromatin-associated protein modification comprises trimethylation of lysine 4 on histone H3, or acetylation of lysine 27 on histone H3.

Images