Compositions and methods for altering DNA polymerase α and primase

Abstract

Disclosed are methods of decreasing lagging strand synthesis comprising interfering with or altering DNA polymerase α (DNA polα) or primase activity. Disclosed are methods of reprogramming a progenitor cell comprising administering a DNA polα inhibitor or primase inhibitor to the progenitor cell, thereby reprogramming the progenitor cell to form a reprogrammed progenitor cell. Disclosed are methods of reducing DNA polα and / or primase levels or activity in a cell comprising contacting a cell with a DNA polα inhibitor and / or primase inhibitor. Disclosed are methods of altering chromatin in a progenitor cell comprising contacting a progenitor cell with a DNA polα inhibitor and / or a primase inhibitor to the cell. Disclosed are methods of increasing fertility of a subject comprising administering a therapeutically effective amount of a DNA polα inhibitor and / or a primase inhibitor to the subject. Disclosed are methods of increasing reproductive lifespan of a subject comprising administering a therapeutically effective amount of a DNA polα inhibitor and / or a primase inhibitor to the subject.

Description

ATTORNEY DOCKET NO.36406.0038P1 COMPOSITIONS AND METHODS FOR ALTERING DNA POLYMERASE α AND PRIMASE CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 565,742, filed March 15, 2024, which is incorporated by reference herein in its entirety. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with government support under R35 GM127075 and R01 HD102474 awarded by the National Institute of Health (NIH). The government has certain rights in the invention. BACKGROUND

[0003] During metazoan development, an outstanding question is how cells take on distinct fates and have diverse functions even though they derive from one zygote. Cell fate is determined by selectively expressing a subset of the genome at the proper time, in the right place, and at the precise level. The unique gene expression program for each cell type is typically regulated by the epigenetic mechanisms, which refer to chromatin changes without alteration of the DNA sequences. Epigenetic mechanisms comprise DNA methylation, histone modifications, histone variants, as well as non-coding RNAs, among others. However, except for DNA methylation, how the epigenetic information is transferred through the active cell cycle in multicellular organisms remains largely unclear. Notably, this transfer could not only be responsible for maintaining epigenetic memory but also allow for epigenetic changes to diversify cell fates, which are essential for development, homeostasis and regeneration. One paradigmatic model to study cell fate decision is asymmetric cell division (ACD), through which one mother cell gives rise to two distinct daughter cells. Through ACD, the genetic codes inherited by the two daughter cells are identical, whereas their epigenetic information can vary, allowing them to appear and function differently. Recently, it has been revealed that ACD can be induced by DNA damage in otherwise symmetrically dividing human cells, indicating that changes on DNA strands per se could guide the cell division mode and potentially the epigenetic inheritance pattern.

[0004] To investigate the histone inheritance pattern in ACD, a tag-switch strategy to differentially label preexisting (old) versus newly synthesized (new) histones has been developed and used to study the Drosophila adult stem cell systems. These studies reveal thatATTORNEY DOCKET NO.36406.0038P1 old histones are selectively retained in the self-renewing stem cell, whereas new histones are enriched in the differentiating daughter cell during ACDs of male germline stem cells (GSCs) and intestinal stem cells. Notably, in the male germline lineage, old and new histones are inherited symmetrically during the symmetric divisions of the progenitor spermatogonial cells (SGs). Asymmetric histone inheritance has been proposed to involve a process with at least three steps in which old and new histones are first asymmetrically incorporated on the replicative sister chromatids during DNA replication. Then, the epigenetically distinct sister chromatids are differentially recognized and segregated during mitosis, leading to distinct “read-outs” in the resulting two daughter cells, such as their asynchronous S-phase initiation and distinct interchromosomal interactions at a key “stemness” gene.

[0005] A series of studies have extensively explored the roles of DNA replication components in establishing the epigenomes in unicellular organisms, such as yeast, and symmetrically dividing cells, such as cultured mouse embryonic stem cells and human cell lines. However, the focuses of these studies are how epigenetic information can be equally partitioned between sister chromatids and inherited symmetrically by the daughter cells. Nevertheless, little is known about this process in asymmetrically dividing cells in multicellular organisms. Studies in mouse development demonstrate that asymmetric inheritance of H3R26me2 or maternal chromosome-bound H3.3 and H3K9me2 are essential for early embryogenesis, in contrast to the negative effects of asymmetric histone inheritance in yeast and mouse embryonic stem cells, emphasizing the importance to study this phenomenon in an organism- and context-dependent manner. BRIEF SUMMARY

[0006] Disclosed are methods of decreasing lagging strand synthesis comprising interfering with or altering DNA polymerase α (DNA polα) or primase activity.

[0007] Disclosed are methods of decreasing lagging strand synthesis comprising contacting a cell with a DNA polα inhibitor or primase inhibitor.

[0008] Disclosed are methods of reprogramming a progenitor cell comprising administering a DNA polα inhibitor or primase inhibitor to the progenitor cell, thereby reprogramming the progenitor cell to form a reprogrammed progentior cell.

[0009] Disclosed are methods of reprogramming a progenitor cell comprising genetically altering a cell to reduce DNA polα or primase levels (or activity), thereby reprogramming the progenitor cell to form a reprogrammed progenitor cell.ATTORNEY DOCKET NO.36406.0038P1

[0010] Disclosed are methods of reducing DNA polα and / or primase levels or activity in a cell comprising contacting a cell with a DNA polα inhibitor and / or primase inhibitor.

[0011] Disclosed are methods of reducing DNA polα and / or primase levels or activity in a cell comprising genetically altering a cell to reduce DNA polα or primase levels (or activity).

[0012] Disclosed are methods of altering chromatin in a progenitor cell comprising contacting a progenitor cell with a DNA polymerase α (DNA polα) inhibitor and / or a primase inhibitor to the cell.

[0013] Disclosed are methods of altering chromatin in a progenitor cell comprising genetically altering a cell to reduce DNA polα or primase levels (or activity).

[0014] Disclosed are methods of increasing fertility of a subject comprising administering a therapeutically effective amount of a DNA polymerase α (DNA polα) inhibitor and / or a primase inhibitor to the subject.

[0015] Disclosed are methods of increasing reproductive lifespan of a subject comprising administering a therapeutically effective amount of a DNA polα inhibitor and / or a primase inhibitor to the subject.

[0016] Disclosed are methods of treating a subject in need thereof comprising administering a therapeutically effective amount of a DNA polymerase α (DNA polα) inhibitor or primase inhibitor to the subject.

[0017] Disclosed are methods of treating comprising genetically altering a cell in the subject to reduce DNA polα or primase levels (or activity).

[0018] Additional advantages of the disclosed method and compositions will be set forth in part in the description which follows, and in part will be understood from the description, or may be learned by practice of the disclosed method and compositions. The advantages of the disclosed method and compositions will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention as claimed. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several embodiments of the disclosed method and compositions and together with the description, serve to explain the principles of the disclosed method and compositions.ATTORNEY DOCKET NO.36406.0038P1

[0020] FIG.1A and FIG.1B show distinct expression patterns of different replication machinery components in the Drosophila male germline stem cell lineage. FIG.1A) Images of expression of 3xHA tagged endogenous DNA polymerases Pol^, Pol^^^Pol^^ as well as the RPA 70-EGFP expressed from the transgene under its own promoter. Somatic cell-enriched histone modification H4K20me2 / 3, DAPI, and the respective replication proteins. Representative GSCs are indicated by the white dotted circle while SGs are indicated by the yellow dotted circle. FIG 1B) Quantification of the relative expression levels of different replication proteins, using a batch-based normalization to GSCs from the corresponding testis sample followed by log2 transformation.

[0021] FIGs.2A-F show reduced primase level reduced Polα activity. Results from both treatments show increased old histone versus new histone separation in S-phase nuclei of non- stem progenitor cells. FIG.2A) Regime testing old vs. new histone localization pattern following heat shock-induced tag switch. FIG.2B) Airyscan images of representative control wild-type (WT) GSC, WT SG, polα50+ / -GSC, and polα50+ / -SG, respectively, in S-phase nuclei wherein excess nucleoplasmic histones are washed off using a stringent clearance buffer. In all merged images: old H3, new H3, as well as EdU, Arm. Asterisk: hub. Scale bars: 1 ^m. FIG. 2C) Quantification of the correlation between old H3 and new H3 signals in S-phase nuclei using a batch-based normalization to control GSCs. All images and quantifications for SGs use the 4-cell spermatogonial stage. FIG.2D) Regime testing old vs. new histone localization pattern in response to Polα180 inhibitor. FIG.2E) Airyscan images of representative GSCs and SGs treated with vehicle or Pol^180 (or Pol A1) inhibitor for 4 hours prior to clearance buffer treatment and fixation. FIG.2F) Quantification of the correlation between old H3 and new H3 signals in S-phase nuclei following inhibitor treatment using a batch-based normalization to vehicle-treated GSCs. All images and quantifications for SGs use the 4-cell spermatogonial stage. All ratios: Mean ± SEM. Mann-Whitney test, ****: P< 10-4, ns: not significant.

[0022] FIG.3A-3F show reduced Drosophila Polα levels enhanced asymmetric old histone recycling at the replication fork in non-stem progenitor cells. FIG.3A-3E) Airyscan images of chromatin fibers isolated from testes with the following genotypes: nos-Gal4ΔVP16; FIG.3A) bam-Gal80>UAS-H3-EGFP, FIG.3B), bam-Gal4>UAS-H3-EGFP, FIG.3C), nanos- Gal4>UAS-H3-EGFP; polα50+ / -, FIG.3D), nos-Gal4ΔVP16; bam-Gal80>UAS-H3-EGFP; polα50+ / -, FIG.3E), bam-Gal4>UAS-H3-EGFP; polα50+ / -, respectively. FIG.3F) Quantification of the H3K27me3 signals on chromatin fibers in log2 scale. Scale bars: 1 ^m. AllATTORNEY DOCKET NO.36406.0038P1 ratios: Mean± SE. Mann-Whitney test, ****: P< 10-4, **: P< 0.01, *: P< 0.05, ns: not significant. It is important to note that Polα is the Drosophila gene name that corresponds to primase in most other organisms. Thus, these experiments reduced primase levels. This applies for all of the genetic manipulation experiments throughout.

[0023] FIG.4A-4L shows dedifferentiated spermatogonial cells with reduced Polα levels tend to have properly oriented centrosomes. FIG.4A) Cartoon depicting the apical tip of the Drosophila testis showing early-stage germ cells, including GSCs, GBs and SGs, respectively. FIG.4B) cartoon depicting the genetic ablation by overexpression of Grim (Grim-OE) that results in depletion of early-stage germ cells. FIG.4C) cartoon depicting recovery from genetic ablation, a model to investigate de-differentiation and re-differentiation. FIG.4D) Regime of temperature controlled Grim expression (at 31°C) and recovery after inhibiting Grim expression (at 18°C, Grim-Inh) in adult flies. FIG.4E) A control (Ctrl) y,w testis tip showing early-staged germ cells. FIG.4F) representative image of the control (Ctrl, UAS-grim) testis tip showing a complete ablation of early-staged germ cells upon Grim-OE for four days at 31°C. FIG.4G) Representative image of the polα50+ / -; UAS-grim testis tip showing significantly reduced GSCs number upon Grim-OE for four days at 31°C. FIG.4I-4H) Upon recovery for four days at 18°C, a representative image of the Ctrl UAS-grim FIG.4H) and a representative image of the polα50+ / -; UAS-grim FIG.4I) testes tips showing GSCs with oriented centrosomes (cyan double-arrowed line pointing to the two centrosomes) and GSCs with misoriented centrosomes (yellow double-arrowed line pointing to the two centrosomes), respectively. FIG.4J) Quantification of the number of GSCs per testis in the Ctrl y,w fly and upon recovery in the polα50+ / -; UAS-grim after genetic ablation and upon recovery ; ****P < 10-4, Mann Whitney test. FIG.4K) A cartoon depicting the quantification of oriented versus misoriented centrosomes in GSCs. FIG.4L) Quantification of the percentage of GSCs with misoriented centrosome in the Ctrl y,w flies without genetic ablation, in the Ctrl UAS-grim after genetic ablation and upon recovery; in the polα50+ / -; UAS-grim after genetic ablation and upon recovery. ****P < 10-4, Chi-square test.

[0024] FIG.5A-5K show male flies with compromised Polα have sustainable fertility during aging. FIG.5A-5B) Representative images of the apical tips of 35-day old (D35) male testes, showing GSCs with oriented and misoriented centrosomes in the control (Ctrl) y,w (FIG.5A) and polα50+ / -(FIG.5B) backgrounds. FIG.5C) Quantification of the Stat92E immunostaining signals for both the Ctrl and polα50+ / -GSCs. ****P < 10-4by Mann-Whitney test. FIG.5D)ATTORNEY DOCKET NO.36406.0038P1 Quantification of the number of GSCs in D1-3 Ctrl testes and D35 Ctrl or polα50+ / -testes. ****P < 10-4by Mann-Whitney test. FIG.5E) Quantification of the percentage of GSCs with misoriented centrosomes in D1-3 Ctrl testes and D35 Ctrl or polα50+ / -testes. ****P < 10-4, Chi- square test. FIG.5F-5G) Representative images of the apical tips of D35 male testes, showing hub location, which is displaced from FIG.5F) the testis tip in the Ctrl but retained at FIG.5G) the testis tip in the polα50+ / -testes. FIG.5H) Quantification of the percentage of testes with displaced hub in D35 Ctrl testes (100%, n=14) or in the polα50+ / -testes (40%, n=22). **P < 0.01, Chi-square test. FIG.5I) Quantification of fertility at different time points during aging (D0 to D49) for either the Ctrl or polα50+ / -males. ****P < 10-4by Multiple Mann-Whitney t test. FIG.5J) Lifespan of control and polα50+ / -flies. P = 0.84 by Chi square test. FIG.5K) Model depicting the dedifferentiation and redifferentiation processes during aging. All values = Average ±SE.

[0025] FIG.6A-6I show asynchronous leading strand versus lagging strand syntheses. FIG. 6A) Regime with a 10-min EdU pulse followed by a 5-min wash out, then a 10-min BrdU pulse to label DNA fibers. FIG.6B) Airyscan images of DNA fibers: The two line-plots correspond to a representative symmetric replicative region with EdU labeling both strands and an asymmetric region where EdU and BrdU are on the opposing strands. FIG.6C) Log2-scale 2D plot shows the distribution of BrdU and EdU on the DNA fibers with both signals. Most fibers display EdU and BrdU on the opposing DNA strands. FIG.6D) Quantification of EdU distribution, introduced by a 15-minute EdU pulse labeling, on chromatin fibers without strandedness information. Scale bars: 1 ^m. All ratios: Mean± SE. Mann-Whitney test, **: P< 0.01, ns: not significant. FIG.6E-6F) Visualization of delayed lagging strand synthesis: Airyscan image of representative chromatin fibers labeled with endogenous Cdc45-mcherry, EdU, Hoechst, along with FIG.6E) Polε-HA or FIG.6F) Polα-HA. FIG.6G) Quantification of the distance along chromatin fibers from the center of the Cdc45 signal to the most distal corresponding DNA polymerase signal. Scale bars: 1 ^m. All ratios: Mean± SE. Mann-Whitney test, ****: P< 10-4. (FIG.6H-GI) Models depicting how reducing lagging strand polymerase levels could drive increased histone asymmetry at the replication fork.

[0026] FIG.7A-7E show expression pattern of a replication machinery component CTF4 and distinct expression patterns of an H3-GFP reporter by different drivers in the Drosophila male germline stem cell lineage. FIG.7A) Image of endogenous CTF4-GFP using knock-in strategy: DAPI, Arm, CTF4-GFP, and the somatic marker Tj. FIG.7B-7D) Images of FIG.7B)ATTORNEY DOCKET NO.36406.0038P1 nanos-Gal4> UAS-H3-EGFP, FIG.7C) bam-Gal4> UAS-H3-EGFP, FIG.7D) nos-Gal4ΔVP16; bam-Gal80> UAS-H3-EGFP, Arm, H3-EGFP, and the somatic marker Tj. FIG.7E) Quantification of the relative expression levels of H3-eGFP using each driver.

[0027] FIG.8A-8C show low concentrations of Polα180 (or PolA1) inhibitor partially inhibit Polα while permitting DNA replication. FIG.8A) PyMOL structural alignment of human DNA Polα with DNA (structure: PDB 5IUD) and the AlphaFold prediction of Drosophila Polα180. The Polα180 inhibitor is shown at the predicted binding location. The Polα180 residues predicted to interact with the inhibitor are conserved between mammals and Drosophila. FIG.8B) Quantification of total EdU incorporation in early-stage germ cells at S- phase, treated with vehicle or Polα180 inhibitor for four hours, normalized to the mean of vehicle-treated cells. Vehicle treated cells, 10μM inhibitor treated cells. Median with first and third quartile shown. Student’s t-test, ns: not significant. FIG.8C) Representative images of testes treated with vehicle or Polα180 inhibitor. In merged images: Hoechst, endogenous Vasa- mApple, EdU.

[0028] FIG.9A-9F show reduced Polα or enhanced RPA levels increased old histone- enriched H3K27me3 asymmetries at the replication fork. FIG.9A) An Airyscan image of representative asymmetric nanos-Gal4>UAS-H3-EGFP chromatin fiber. FIG.9B) An Airyscan image of representative symmetric nanos-Gal4>UAS-H3-EGFP chromatin fiber. In merged images (FIG.9A-9B): H3K27me3, H3-EGFP, PCNA, and EdU. Images in are also accompanied by line plots showing the spatial distribution of H3K27me3 and PCNA signals from the indicated regions, respectively (box with white dotted outline). FIG.9C) Quantification of H3K27me3 asymmetry using chromatin fibers with nanos-Gal4>UAS-H3-EGFP, bam- Gal4>UAS-H3-EGFP, nos-Gal4ΔVP16; bam-Gal80>UAS-H3-EGFP, and an in silico combination of bam-Gal4>UAS-H3-EGFP and nos-Gal4ΔVP16; bam-Gal80>UAS-H3-EGFP in log2 scale. FIG.9D) Quantification of additional replication protein manipulations using a nanos-Gal4 driven overexpression of UAS-rpa70-HA transgene and a P-element insertion allele of another Polα subunit gene (polα180) at a heterozygous background (polα180+ / -) in log2 scale. FIG.9E) Airyscan image of hs-flp; nanos-Gal4>UAS-FRT-H3-EGFP-FRT-H3-mCherry; polα180+ / -: H3K27me3, old H3, new H3, and EdU in the merged image. FIG.9F) Airyscan image of nanos-Gal4>UAS-rpa70-HA chromatin fiber: EdU, RPA, and H3K27me3 in the merged image. Images are also accompanied by line plots showing the spatial distribution of FIG.9E) H3K27me3 and new H3 signals in, as well as FIG.9F) H3K27me3 and RPA signals inATTORNEY DOCKET NO.36406.0038P1 from the indicated regions, respectively (box with white dotted outline). Scale bar: 1 ^m. All ratios: Mean± SE. All statistics: Mann-Whitney test, **: P< 0.01, *: P< 0.05, ns: not significant.

[0029] FIG.10A-10E show reducing Polα levels resulted in increased old H3 versus new H3 separation in M-phase nuclei of progenitor SG cells. FIG.10A-10B) Representative images of an M-phase GSC (FIG.10A) showing more compact old H3-enriched regions than new H3- enriched regions (positive with a mitotic marker anti-H3S10ph, H3S10P or S10P), while an M- phase 8-cell SG (FIG.10B) showing equally compact old H3-enriched regions and new H3- enriched regions (positive with S10P) in the control wild-type testes. (FIG.10C-10D) Representative images of an M-phase GSC (FIG.10C) an M-phase 8-cell SG (FIG.10D) in the pola50+ / -testes, both showing more compact old H3-enriched regions than new H3-enriched regions. (FIG.10E) Compaction index in log2scale. All ratios: Mean± SE. Mann-Whitney test, ****: P< 10-4, ns: not significant

[0030] FIG.11A-11C show design and results of the fertility assay in males with reduced Polα levels. FIG.11A) Regime of aging adult male flies at 25°C after eclosion (D0) up to 56 days. (FIG.11B-11C) DAPI staining of the spermatid elongation and individualization regions of the FIG.11B) control and FIG.11C) polα50+ / -flies at D35 (35 days after eclosion).

[0031] FIG.12A-12F show visualization and quantification of delayed lagging strand synthesis. FIG.12A) Assessment of observed patterns on DNA fibers, wherein symmetric fibers refer to fibers with EdU on both strands, weak asymmetric fibers have both EdU and BrdU but less than a 2-fold asymmetry on both strands, while strong asymmetric fibers have a greater than 2-fold difference for at least one of the signals (i.e., either EdU or BrdU). The percentages of each category are: 60% symmetric, 23% weak asymmetric, 17% strong asymmetric. FIG.12B) Log2-scale 1D quantification of EdU and BrdU from the DNA fibers with both signals, where the positive side is the strand with higher BrdU and the negative side is the strand with higher EdU. ****: P< 10-4, Mann-Whitney test for the comparison between two groups, one tailed t- test with a null hypothesis of log2= 0 (symmetric pattern). FIG.12C) Assessment of EdU asymmetries wherein ≥ 2-fold are considered strong asymmetry, < 2-fold are considered weak asymmetry. Here are the percentages of each category: 11% strong asymmetry toward the leading strand, 33% weak asymmetry toward the leading strand, 15% weak asymmetry toward the lagging strand, 41% strong asymmetry toward the lagging strand. FIG.12D) Quantification of H3K27me3, PCNA, and EdU asymmetry from nos>H3-EGFP labeled chromatin fibers using log2scale. ****: P< 10-4, one tailed t-test with a null hypothesis of log2= 0 (symmetric pattern).ATTORNEY DOCKET NO.36406.0038P1 FIG.12E) A model of replication patterns that could explain the observed EdU patterns. FIG. 12F) Quantification of EdU distribution on chromatin fibers across all conditions reported with regard to the strandedness using log2. All ratios: Mean± SE, one tailed t-test with a null hypothesis of log2= 0 (symmetric pattern), ***: P< 10-3, **: P< 0.01, *: P< 0.05, ns: not significant.

[0032] FIG.13 shows titration of PolA1 inhibitor in human embryonic dermal fibroblast cells.

[0033] FIG.14A-14D show PolA1 inhibition enhanced the human iPSCs reprogramming efficiency. (FIG.14A) Schematic of workflow of CytoTuneTM-iPS 2.0 Sendai virus reprogramming used to generate human iPSCs with and without PolA1 inhibitor. (FIG.14B) Images of the human dermal fibroblast cells at day 0 with and without Pola1 inhibitor. After 15 days’ reprogramming induction, more colonies are generated in the Pola1 inhibitor-treated cells. (FIG.14C) Whole dish imaging of human iPSCs with and without Pola1 inhibitor shows more number and larger size of colonies until Day 18. The cells are stained with DNA dye Hoechst 33342. (FIG.14D) Immunofluorescence staining shows strong expression of pluripotency marker Sox2 in both control and Pola1 inhibited human iPSCs.

[0034] FIG.15A-D shows PolA1 inhibition enhanced efficiency of reprogramming human PBMCs into iPSCs. 15A) The CytoTuneTM-iPS 2.0 Sendai virus reprogramming method used to generate human iPSCs from PBMCs.15B) Titration of PolA1 inhibitor in human PBMCs was performed to determine the optimal concentration. EdU was pulsed for 1 hour to label newly synthesized DNA during replication. Scale bar: 200 µm.15C) Brightfield images of human PBMC-derived iPSCs on Day 9 and Day 11 after Sendai virus transduction. Scale bar: 100 µm. 15D) Live staining of TRA1-60 in cells on Day 12 during reprogramming with and without PolA1 inhibitor. Scale bar: 100 µm. DETAILED DESCRIPTION

[0035] The disclosed method and compositions may be understood more readily by reference to the following detailed description of particular embodiments and the Example included therein and to the Figures and their previous and following description.

[0036] It is to be understood that the disclosed method and compositions are not limited to specific synthetic methods, specific analytical techniques, or to particular reagents unless otherwise specified, and, as such, may vary. It is also to be understood that the terminologyATTORNEY DOCKET NO.36406.0038P1 used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0037] Disclosed are materials, compositions, and components that can be used for, can be used in conjunction with, can be used in preparation for, or are products of the disclosed method and compositions. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutation of these compounds may not be explicitly disclosed, each is specifically contemplated and described herein. For example, if a peptide is disclosed and discussed and a number of modifications that can be made to a number of molecules including the amino acids are discussed, each and every combination and permutation of the peptide and the modifications that are possible are specifically contemplated unless specifically indicated to the contrary. Thus, if a class of molecules A, B, and C are disclosed as well as a class of molecules D, E, and F and an example of a combination molecule, A-D is disclosed, then even if each is not individually recited, each is individually and collectively contemplated. Thus, in this example, each of the combinations A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are specifically contemplated and should be considered disclosed from disclosure of A, B, and C; D, E, and F; and the example combination A-D. Likewise, any subset or combination of these is also specifically contemplated and disclosed. Thus, for example, the sub-group of A-E, B-F, and C-E are specifically contemplated and should be considered disclosed from disclosure of A, B, and C; D, E, and F; and the example combination A-D. This concept applies to all aspects of this application including, but not limited to, steps in methods of making and using the disclosed compositions. Thus, if there are a variety of additional steps that can be performed it is understood that each of these additional steps can be performed with any specific embodiment or combination of embodiments of the disclosed methods, and that each such combination is specifically contemplated and should be considered disclosed. A. Definitions

[0038] It is understood that the disclosed method and compositions are not limited to the particular methodology, protocols, and reagents described as these may vary. It is also to be understood that 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 will be limited only by the appended claims.ATTORNEY DOCKET NO.36406.0038P1

[0039] It must be noted that as used herein and in the appended claims, the singular forms "a ", "an", and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, reference to "a DNA polα inhibitor" includes a plurality of such inhibitors, reference to "the DNA polα inhibitor” is a reference to one or more DNA polα inhibitors and equivalents thereof known to those skilled in the art, and so forth.

[0040] The word “or” as used herein means any one member of a particular list and also includes any combination of members of that list.

[0041] As used herein, the term "therapeutically effective amount" means an amount of a therapeutic, prophylactic, and / or diagnostic agent that is sufficient, when administered to a subject suffering from or susceptible to a disease, disorder, and / or condition, to treat, alleviate, ameliorate, relieve, alleviate symptoms of, prevent, delay onset of, inhibit progression of, reduce severity of, and / or reduce incidence of the disease, disorder, and / or condition.

[0042] As used herein, the term "treating" refers to partially or completely alleviating, ameliorating, relieving, delaying onset of, inhibiting progression of, reducing severity of, and / or reducing incidence of one or more symptoms or features of a particular disease, disorder, and / or condition. For example, "treating" infertility may refer to increasing fertility in a subject or increasing reproductive lifespan in a subject. Treatment may be administered to a subject who does not exhibit signs of a disease, disorder, and / or condition and / or to a subject who exhibits only early signs of a disease, disorder, and / or condition for the purpose of decreasing the risk of developing pathology associated with the disease, disorder, and / or condition.

[0043] As used herein, “subject” refers to the target of administration, e.g. an animal. Thus the subject of the disclosed methods can be a vertebrate, such as a mammal. For example, the subject can be a human. The term does not denote a particular age or sex. Subject can be used interchangeably with “individual” or “patient”.

[0044] Ranges may be expressed herein as from "about" one particular value, and / or to "about" another particular value. When such a range is expressed, also specifically contemplated and considered disclosed is the range from the one particular value and / or to the other particular value unless the context specifically indicates otherwise. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another, specifically contemplated embodiment that should be considered disclosed unless the context specifically indicates otherwise. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, andATTORNEY DOCKET NO.36406.0038P1 independently of the other endpoint unless the context specifically indicates otherwise. Finally, it should be understood that all of the individual values and sub-ranges of values contained within an explicitly disclosed range are also specifically contemplated and should be considered disclosed unless the context specifically indicates otherwise. The foregoing applies regardless of whether in particular cases some or all of these embodiments are explicitly disclosed.

[0045] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of skill in the art to which the disclosed method and compositions belong. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present method and compositions, the particularly useful methods, devices, and materials are as described. Publications cited herein and the material for which they are cited are hereby specifically incorporated by reference. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such disclosure by virtue of prior invention. No admission is made that any reference constitutes prior art. The discussion of references states what their authors assert, and applicants reserve the right to challenge the accuracy and pertinency of the cited documents. It will be clearly understood that, although a number of publications are referred to herein, such reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art.

[0046] Throughout the description and claims of this specification, the word “comprise” and variations of the word, such as “comprising” and “comprises,” means “including but not limited to,” and is not intended to exclude, for example, other additives, components, integers or steps. In particular, in methods stated as comprising one or more steps or operations it is specifically contemplated that each step comprises what is listed (unless that step includes a limiting term such as “consisting of”), meaning that each step is not intended to exclude, for example, other additives, components, integers or steps that are not listed in the step. B. Methods

[0047] DNA replication comprises leading strand synthesis and lagging strand synthesis. Lagging strand synthesis occurs opposite to the direction of the growing replication fork of DNA. Because of its orientation, replication of the lagging strand is more complicated as compared to that of the leading strand. As a consequence, the DNA polymerase on this strand is seen to "lag behind" the other strand. The lagging strand is synthesized in short, separated segments. On the lagging strand template, a primase "reads" the template DNA and initiatesATTORNEY DOCKET NO.36406.0038P1 synthesis of a short complementary RNA primer. A DNA polymerase extends the primed segments, forming Okazaki fragments. The RNA primers are then removed and replaced with DNA, and the fragments of DNA are joined by DNA ligase. 1. Methods of Decreasing Lagging Strand Synthesis

[0048] DNA polymerase alpha also known as DNA polα is an enzyme complex found in eukaryotes that is involved in initiation of DNA replication. The DNA polymerase alpha complex consists of 4 subunits: POLA1, POLA2, PRIM1, and PRIM2. Pol α has limited processivity and lacks 3′ exonuclease activity for proofreading errors. Thus, it is not well suited to efficiently and accurately copy long templates (unlike Pol Delta and Epsilon). Instead, it plays a more limited role in replication. DNA polα is responsible for the initiation of DNA replication at origins of replication (on both the leading and lagging strands) and during synthesis of Okazaki fragments on the lagging strand. The DNA polα complex (pol α-DNA primase complex) consists of four subunits: the catalytic subunit POLA1, the regulatory subunit POLA2, and the small and the large primase subunits PRIM1 and PRIM2 respectively. Once primase has created the RNA primer, Pol α starts replication elongating the primer with ~20 nucleotides.

[0049] DNA primase is an enzyme involved in the replication of DNA and is a type of RNA polymerase. Primase catalyzes the synthesis of a short RNA (or DNA in some living organisms) segment called a primer complementary to a ssDNA (single-stranded DNA) template. After this elongation, the RNA piece is removed by a 5' to 3' exonuclease and refilled with DNA. In some aspects, human primase corresponds to Drosophila polα.

[0050] Disclosed are methods of decreasing lagging strand synthesis comprising interfering with or altering DNA polymerase α (DNA polα) or primase activity. In some aspects, interfering with or altering DNA polα or primase activity comprises using a DNA polα inhibitor or primase inhibitor. In some aspects, interfering with or altering DNA polα or primase activity comprises using genetic manipulation.

[0051] Disclosed are methods of decreasing lagging strand synthesis comprising contacting a cell with a DNA polα inhibitor or primase inhibitor.

[0052] In some aspects, the DNA polα inhibitor can be, but is not limited to, POLA1 inhibitor 1, Aphidicolin, 18 β-Glycyrrhetinic Acid, 1-β-D-Arabinofuranosylcytosine, Adarotene, CD437, or derivatives thereof.

[0053] In some aspects, the primase inhibitor can be, but is not limited to, amenamevir,ATTORNEY DOCKET NO.36406.0038P1 pritelivir, BILS 22 BS, T157602, or derivatives thereof.

[0054] In some aspects, the cell is in culture, thus the methods can be in vitro methods.

[0055] In some aspects, the cell is in a subject, thus the methods can be in vivo methods. Thus, disclosed are methods of decreasing lagging strand synthesis in a cell in a subject comprising administering a therapeutically effective amount of a DNA polα inhibitor or primase inhibitor to the subject.

[0056] In some aspects, the DNA polα inhibitor or primase inhibitor decreases DNA polα or primase activities and / or levels in the cell. In some aspects, the inhibition of activity or levels of DNA polα or primase is a partial inhibition. Thus, in some aspects of the disclosed methods there can be 10, 20, 30, 40, 50, 60, 70, 80, 90, or 95% inhibition of DNA polα or primase activity or levels.

[0057] The S-phase index (SPI), is a measure of cell growth and viability, especially the capacity of tumor cells to proliferate. It is defined as the number of BrdU-incorporating cells relative to the volume of DNA staining determined from whole mount confocal analyses. In some aspects, the DNA polα inhibitor or primase inhibitor does not affect the percentage of S- phase cells (namely S-phase index) within a population of cells. In some aspects, this indicates that the treatment with the DNA polα inhibitor or primase inhibitor does not change the overall cell cycle progression in the cell. In some aspects, the DNA polα inhibitor or primase inhibitor does not substantially affect the S phase of the cell cycle of a cell. In some aspects, not substantially affecting the S phase of the cell cycle can mean that at least 50, 60, 70, 80, or 90% of cells do not have the S phase affected.

[0058] In some aspects, the DNA polα inhibitor or primase inhibitor does not cause cell cycle arrest in the cell or cell death of the cell. Thus, in some aspects, replication still occurs in a cell contacted with a DNA polα inhibitor or primase inhibitor and the cell is not killed.

[0059] In some aspects, leading strand synthesis is not altered in the cell. In some aspects, leading strand synthesis is not substantially altered in the cell. In some aspects, not substantially altering leading strand synthesis can mean that at least 50, 60, 70, 80, 90, or 99% of leading strand synthesis is not altered. In some aspects, DNA polα and primase act on the leading strand to start replication, but this is a single enzymatic reaction. However, for the lagging strand, there can be many repeated reactions to continually initiate Okazaki fragment synthesis, therefore because DNA Polα and primase work significantly more on the lagging strand than the leading strand, in some aspects, the leading strand synthesis is either not altered or only slightly orATTORNEY DOCKET NO.36406.0038P1 substantially altered while the lagging strand synthesis.

[0060] In some aspects, the cell is a stem cell or a progenitor cell. In some aspects, a progenitor cell is known as a dividing cell with reprogramming potential. In some aspects, the progenitor cell is a fibroblast cell, a male gonialblast or spermatogonial cell, a female cystoblast or cystocyte, or an intestinal cell.

[0061] Disclosed are methods of decreasing lagging strand synthesis comprising genetically altering a cell to reduce DNA polα or primase levels (or activity). In some aspects, genetically altering a cell decreases DNA polα or primase levels in the cell. In some aspects, genetically altering a cell can comprise contacting a cell with a double-strand RNA or siRNA to regulate (e.g., decrease) DNA polα or primase gene expression post-transcriptionally. Thus, in some aspects, the double-strand RNA can target a DNA polα or primase gene or upstream of a gene that regulates DNA polα or primase expression. In some aspects, CRIPSR / Cas9-mediated somatic cell genome engineering can be used to knock out one copy of a DNA polα or primase gene or generate loss-of-function versions of a DNA polα or primase gene in specific cell types. Thus, in some aspects, guide RNAs designed to target the DNA polα or primase gene or the upstream gene regulating the DNA polα or primase gene expression can be used. In some aspects, CRIPSR / Cas9-mediated somatic cell genome engineering can target DNA polα or primase gene or the upstream gene regulating the DNA polα or primase gene expression.

[0062] In some aspects, the disclosed methods of decreasing lagging strand synthesis can further comprise administering a composition that increases Replication Protein-A 70 (RPA70) levels in the cell. In some aspects, a composition that increases RPA70 levels can be a composition that genetically alters a cell to increase RPA70 or can be a composition that induces activation of RPA70 transcription. 2. Methods of Reprogramming a Progenitor Cell

[0063] The process of inducing a desired cell fate, by converting somatic cells from one lineage to another without transitioning through an intermediate pluripotent or multipotent state, has been described as 'direct reprogramming', also known as 'transdifferentiation'.

[0064] Disclosed are methods of reprogramming a progenitor cell comprising administering a DNA polymerase α (DNA polα) inhibitor or primase inhibitor to the progenitor cell, thereby reprogramming the progenitor cell to form a reprogrammed progenitor cell. In some aspects, a reprogammed progenitor can be understood to refer to a progenitor cell that has been reprogrammed. In some aspects, reprogramming of cells refers to the regression of a specializedATTORNEY DOCKET NO.36406.0038P1 cell to a simpler state, resulting in cells with stem-like properties. In some aspects, reprogramming refers to returning cells to an earlier stage of development, such as the stem cell stage. Thus, in some aspects, a reprogrammed progenitor cell is a cell with stem cell-like properties.

[0065] In some aspects, the cell is in culture, thus the methods can be in vitro methods.

[0066] In some aspects, the cell is in a subject, thus the methods can be in vivo methods. Thus, disclosed are methods of reprogramming a progenitor cell in a subject comprising administering a DNA polα inhibitor or primase inhibitor to the subject, thereby reprogramming the progenitor cell in the subject to form a reprogrammed progenitor cell.

[0067] In some aspects, the reprogramming of a progenitor cell can be determined based on the pattern of old histones versus new histones. In some aspects, the reprogrammed progenitor cell comprises separable old histone versus new histone pattern during DNA replication and cell division. In some aspects, the separable old histone versus new histone pattern is a stem cell- unique pattern. In some aspects, the old histone versus new histone pattern is an old histone H3 versus new histone H3 pattern. In some aspects, old histone versus new histone displays separable patterns during DNA replication. In some aspect, old histone versus new histone enriched chromosomal regions display differential condensation in the dividing reprogrammed progenitor cell.

[0068] In some aspects, the cell is a stem cell or a progenitor cell. In some aspects, a progenitor cell is known as a dividing cell with reprogramming potential. In some aspects, the progenitor cell is a fibroblast cell, a male gonialblast or spermatogonial cell, a female cystoblast or cystocyte, or an intestinal cell.

[0069] In some aspects, the DNA polα inhibitor or primase inhibitor decreases DNA polα or primase activities and / or levels in the cell. In some aspects, the inhibitor of activity or levels DNA polα or primase is a partial inhibition. Thus, in some aspects of the disclosed methods there can be 10, 20, 30, 40, 50, 60, 70, 80, 90, or 95% inhibition of DNA polα or primase activity or levels.

[0070] In some aspects, the DNA polα inhibitor or primase inhibitor does not affect the percentage of S-phase cells (namely S-phase index) within a population of cells. In some aspects, this indicates that the treatment with the DNA polα inhibitor or primase inhibitor does not change the overall cell cycle progression in the cell. In some aspects, the DNA polα inhibitor or primase inhibitor does not substantially affect the S phase of the cell cycle of a cell. In someATTORNEY DOCKET NO.36406.0038P1 aspects, not substantially affecting the S phase of the cell cycle can mean that at least 50, 60, 70, 80, or 90% of cells do not have the S phase affected.

[0071] In some aspects, the DNA polα inhibitor or primase inhibitor does not cause cell cycle arrest in the cell or cell death of the cell. Thus, in some aspects, replication still occurs in a cell contacted with a DNA polα inhibitor or primase inhibitor and the cell is not killed.

[0072] In some aspects, the DNA polα inhibitor can be, but is not limited to, POLA1 inhibitor 1, Aphidicolin, 18 β-Glycyrrhetinic Acid, 1-β-D-Arabinofuranosylcytosine, Adarotene, CD437, or derivatives thereof.

[0073] In some aspects, the primase inhibitor can be, but is not limited to, amenamevir, pritelivir, BILS 22 BS, T157602, or derivatives thereof.

[0074] Disclosed are methods of reprogramming a progenitor cell comprising genetically altering a cell to reduce DNA polα or primase levels (or activity), thereby reprogramming the progenitor cell to form a reprogrammed progenitor cell. In some aspects, genetically altering a cell decreases DNA polα or primase levels in the cell. In some aspects, genetically altering a cell can comprise contacting a cell with a double-strand RNA or siRNA to regulate (e.g., decrease) DNA polα or primase gene expression (post transcriptionally). Thus, in some aspects, the double-strand RNA can target a DNA polα or primase gene or upstream of a gene that regulates DNA polα or primase expression. In some aspects, CRIPSR / Cas9-mediated somatic cell genome engineering can be used to knock out one copy of a DNA polα or primase gene or generate loss-of-function versions of a DNA polα or primase gene in specific cell types. Thus, in some aspects, guide RNAs designed to target the DNA polα or primase gene or the upstream gene regulating the DNA polα or primase gene expression can be used. In some aspects, CRIPSR / Cas9-mediated somatic cell genome engineering can target DNA polα or primase gene or the upstream gene regulating the DNA polα or primase gene expression 3. Methods of Reducing DNA Polymerase α or Primase Levels or Activity

[0075] Disclosed are methods of reducing DNA polymerase α (DNA polα) and / or primase levels or activity in a cell comprising contacting a cell with a DNA polα inhibitor and / or primase inhibitor.

[0076] In some aspects, the cell is in culture, thus the methods can be in vitro methods.

[0077] In some aspects, the cell is in a subject, thus the methods can be in vivo methods. Thus, disclosed are methods of reducing DNA polα and / or primase levels or activity in a cell in a subject comprising administering a therapeutically effective amount of a DNA polα inhibitorATTORNEY DOCKET NO.36406.0038P1 and / or primase inhibitor to the subject.

[0078] In some aspects, the DNA polα inhibitor can be, but is not limited to, POLA1 inhibitor 1, Aphidicolin, 18 β-Glycyrrhetinic Acid, 1-β-D-Arabinofuranosylcytosine, Adarotene, CD437, or derivatives thereof.

[0079] In some aspects, the primase inhibitor can be, but is not limited to, amenamevir, pritelivir, BILS 22 BS, T157602, or derivatives thereof.

[0080] In some aspects, the DNA polα inhibitor or primase inhibitor decreases DNA polα or primase activities and / or levels in the cell. In some aspects, the inhibition of activity or levels of DNA polα or primase is a partial inhibition. Thus, in some aspects of the disclosed methods there can be 10, 20, 30, 40, 50, 60, 70, 80, 90, or 95% inhibition (or reduction) of DNA polα or primase activity or levels.

[0081] In some aspects, the cell is a stem cell or a progenitor cell. In some aspects, a progenitor cell is known as a dividing cell with reprogramming potential. In some aspects, the progenitor cell is a fibroblast cell, a male gonialblast or spermatogonial cell, a female cystoblast or cystocyte, or an intestinal cell.

[0082] In some aspects, the reduction of DNA polymerase α or primase levels or activity reprograms the cell. In some aspects, the reduction of DNA polymerase α or primase levels or activity disrupts lagging strand synthesis in the cell. In some aspects, the reduction of DNA polymerase α or primase levels or activity calibrates the activity of components contributing to lagging strand synthesis in the cell.

[0083] In some aspects, the DNA polα inhibitor or primase inhibitor does not affect the percentage of S-phase cells (namely S-phase index) within a population of cells. In some aspects, this indicates that the treatment with the DNA polα inhibitor or primase inhibitor does not change the overall cell cycle progression in the cell. In some aspects, the DNA polα inhibitor or primase inhibitor does not substantially affect the S phase of the cell cycle of a cell. In some aspects, not substantially affecting the S phase of the cell cycle can mean that at least 50, 60, 70, 80, or 90% of cells do not have the S phase affected.

[0084] In some aspects, the DNA polα inhibitor or primase inhibitor does not cause cell cycle arrest in the cell or cell death of the cell. Thus, in some aspects, replication still occurs in a cell contacted with a DNA polα inhibitor or primase inhibitor and the cell is not killed.

[0085] Disclosed are methods of reducing DNA polα and / or primase levels or activity in a cell comprising genetically altering a cell to reduce DNA polα or primase levels (or activity). InATTORNEY DOCKET NO.36406.0038P1 some aspects, genetically altering a cell decreases DNA polα or primase levels in the cell. In some aspects, genetically altering a cell can comprise contacting a cell with a double-strand RNA or siRNA to regulate (e.g., decrease) DNA polα or primase gene expression (post transcriptionally). Thus, in some aspects, the double-strand RNA can target a DNA polα or primase gene or upstream of a gene that regulates DNA polα or primase expression. In some aspects, CRIPSR / Cas9-mediated somatic cell genome engineering can be used to knock out one copy of a DNA polα or primase gene or generate loss-of-function versions of a DNA polα or primase gene in specific cell types. Thus, in some aspects, guide RNAs designed to target the DNA polα or primase gene or the upstream gene regulating the DNA polα or primase gene expression can be used. In some aspects, CRIPSR / Cas9-mediated somatic cell genome engineering can target DNA polα or primase gene or the upstream gene regulating the DNA polα or primase gene expression. 4. Methods of Altering Chromatin in a Progenitor Cell

[0086] Disclosed are methods of altering chromatin in a progenitor cell comprising contacting a progenitor cell with a DNA polymerase α (DNA polα) inhibitor and / or a primase inhibitor to the cell. In some aspects, altering chromatin is observed by the separability of old and new histones. In some aspects, altering chromatin is observed by a change in the chromatin compaction regulated by histone density, based on an area-based assay to measure the chromatin regions occupied by old versus new histones.

[0087] In some aspects, the cell is in culture, thus the methods can be in vitro methods.

[0088] In some aspects, the cell is in a subject, thus the methods can be in vivo methods. Thus, disclosed are methods of altering chromatin in a progenitor cell in a subject comprising administering a therapeutically effective amount of a DNA polα inhibitor to the subject.

[0089] In some aspects, the DNA polα inhibitor can be, but is not limited to, POLA1 inhibitor 1, Aphidicolin, 18 β-Glycyrrhetinic Acid, 1-β-D-Arabinofuranosylcytosine, Adarotene, CD437, or derivatives thereof.

[0090] In some aspects, the primase inhibitor can be, but is not limited to, amenamevir, pritelivir, BILS 22 BS, T157602, or derivatives thereof.

[0091] In some aspects, the cell is a stem cell or a progenitor cell. In some aspects, a progenitor cell is known as a dividing cell with reprogramming potential. In some aspects, the progenitor cell is a fibroblast cell, a male gonialblast or spermatogonial cell, a female cystoblast or cystocyte, or an intestinal cell.ATTORNEY DOCKET NO.36406.0038P1

[0092] In some aspects, altering chromatin of a progenitor cell can be determined based on the pattern of old histones versus new histones. In some aspects, the altering chromatin comprises separable old histone versus new histone pattern during DNA replication and cell division. In some aspects, the separable old histone versus new histone pattern is a stem cell- unique pattern. In some aspects, the old histone versus new histone pattern is an old histone H3 versus new histone H3 pattern. In some aspects, old histone versus new histone displays separable patterns during DNA replication. In some aspect, old histone versus new histone enriched chromosomal regions display differential condensation in the dividing reprogrammed progenitor cell.

[0093] In some aspects, altering chromatin comprises altering the orientation of centrosomes in the progenitor cell. In some aspects, the orientation of centrosomes in the progenitor cell recapitulates the stem cell-unique orientation. For example, the stem cell-unique orientation can be centrosomes perpendicular to the GSC-Hub cell interface.

[0094] In some aspects, the DNA polα inhibitor or primase inhibitor does not affect the percentage of S-phase cells (namely S-phase index) within a population of cells. In some aspects, this indicates that the treatment with the DNA polα inhibitor or primase inhibitor does not change the overall cell cycle progression in the cell. In some aspects, the DNA polα inhibitor or primase inhibitor does not substantially affect the S phase of the cell cycle of a cell. In some aspects, not substantially affecting the S phase of the cell cycle can mean that at least 50, 60, 70, 80, or 90% of cells do not have the S phase affected.

[0095] In some aspects, the DNA polα inhibitor or primase inhibitor does not cause cell cycle arrest in the cell or cell death of the cell. Thus, in some aspects, replication still occurs in a cell contacted with a DNA polα inhibitor or primase inhibitor and the cell is not killed.

[0096] Disclosed are methods of altering chromatin in a progenitor cell comprising genetically altering a cell to reduce DNA polα or primase levels (or activity). In some aspects, genetically altering a cell decreases DNA polα or primase levels in the cell. In some aspects, genetically altering a cell can comprise contacting a cell with a double-strand RNA or siRNA to regulate (e.g., decrease) DNA polα or primase gene expression (post transcriptionally). Thus, in some aspects, the double-strand RNA can target a DNA polα or primase gene or upstream of a gene that regulates DNA polα or primase expression. In some aspects, CRIPSR / Cas9-mediated somatic cell genome engineering can be used to knock out one copy of a DNA polα or primase gene or generate loss-of-function versions of a DNA polα or primase gene in specific cell types.ATTORNEY DOCKET NO.36406.0038P1 Thus, in some aspects, guide RNAs designed to target the DNA polα or primase gene or the upstream gene regulating the DNA polα or primase gene expression can be used. In some aspects, CRIPSR / Cas9-mediated somatic cell genome engineering can target DNA polα or primase gene or the upstream gene regulating the DNA polα or primase gene expression. 5. Methods of Increasing Fertility and Reproductive Lifespan

[0097] Disclosed are methods of increasing fertility of a subject comprising administering a therapeutically effective amount of a DNA polymerase α (DNA polα) inhibitor and / or a primase inhibitor to the subject. In some aspects, methods to identify or determine an increase in fertility include, but are not limited to, counting all offspring (e.g., flies and mice) a subject can have, test sperm for amount and quality.

[0098] In some aspects, human sperm can decline in quality during aging, thus therapeutically effective amount of a DNA polymerase α (DNA polα) inhibitor and / or a primase inhibitor to a subject could increase fertility.

[0099] Disclosed are methods of increasing reproductive lifespan of a subject comprising administering a therapeutically effective amount of a DNA polα inhibitor and / or a primase inhibitor to the subject. In some aspects, increasing reproductive lifespan can mean maintaining the ability to reproduce for a longer period of time.

[0100] In some aspects, the DNA polα inhibitor and / or primase inhibitor increases the number of reprogrammed progenitor cells to be germline stem cells having oriented centrosomes and normal activity and differentiation potential

[0101] In some aspects, the subject is male. In some aspects, the subject is female.

[0102] In some aspects, the DNA polα inhibitor or primase inhibitor does not affect the percentage of S-phase cells (namely S-phase index) within a population of cells. In some aspects, this indicates that the treatment with the DNA polα inhibitor or primase inhibitor does not change the overall cell cycle progression in the cell. In some aspects, the DNA polα inhibitor or primase inhibitor does not substantially affect the S phase of the cell cycle of a cell. In some aspects, not substantially affecting the S phase of the cell cycle can mean that at least 50, 60, 70, 80, or 90% of cells do not have the S phase affected.

[0103] In some aspects, the DNA polα inhibitor or primase inhibitor does not cause cell cycle arrest in the cell or cell death of the cell. Thus, in some aspects, replication still occurs in a cell contacted with a DNA polα inhibitor or primase inhibitor and the cell is not killed.

[0104] In some aspects, the DNA polα inhibitor or primase inhibitor decreases DNA polα orATTORNEY DOCKET NO.36406.0038P1 primase activities and / or levels in the cell. In some aspects, the inhibition of activity or levels of DNA polα or primase is a partial inhibition. Thus, in some aspects of the disclosed methods there can be 10, 20, 30, 40, 50, 60, 70, 80, 90, or 95% inhibition of DNA polα or primase activity or levels.

[0105] In some aspects, leading strand synthesis is not altered in the cell. In some aspects, leading strand synthesis is not substantially altered in the cell. In some aspects, not substantially altering leading strand synthesis can mean that at least 50, 60, 70, 80, 90, or 99% of leading strand synthesis is not altered. In some aspects, DNA polα and primase act on the leading strand to start replication, but this is a single enzymatic reaction. However, for the lagging strand, there can be many repeated reactions to continually initiate Okazaki fragment synthesis, therefore because DNA Polα and primase work significantly more on the lagging strand than the leading strand, in some aspects, the leading strand synthesis is either not altered or only slightly or substantially altered while the lagging strand synthesis.

[0106] In some aspects, the cell is a progenitor cell. In some aspects, a progenitor cell is known as a dividing cell with reprogramming potential. In some aspects, the progenitor cell is a a male gonialblast or spermatogonial cell, a female cystoblast or cystocyte.

[0107] In some aspects, the DNA polα inhibitor can be, but is not limited to, POLA1 inhibitor 1, Aphidicolin, 18 β-Glycyrrhetinic Acid, 1-β-D-Arabinofuranosylcytosine, Adarotene, CD437, or derivatives thereof.

[0108] In some aspects, the primase inhibitor can be, but is not limited to, amenamevir, pritelivir, BILS 22 BS, T157602, or derivatives thereof.

[0109] Disclosed are methods of increasing fertility or increasing reproductive lifespan of a subject comprising genetically altering a cell to reduce DNA polα or primase levels (or activity). In some aspects, genetically altering a cell decreases DNA polα or primase levels in the cell. In some aspects, genetically altering a cell can comprise contacting a cell with a double-strand RNA or siRNA to regulate (e.g. decrease) DNA polα or primase gene expression (post transcriptionally). Thus, in some aspects, the double-strand RNA can target a DNA polα or primase gene or upstream of a gene that regulates DNA polα or primase expression. In some aspects, CRIPSR / Cas9-mediated somatic cell genome engineering can be used to knock out one copy of a DNA polα or primase gene or generate loss-of-function versions of a DNA polα or primase gene in specific cell types. Thus, in some aspects, guide RNAs designed to target the DNA polα or primase gene or the upstream gene regulating the DNA polα or primase geneATTORNEY DOCKET NO.36406.0038P1 expression can be used. In some aspects, CRIPSR / Cas9-mediated somatic cell genome engineering can target DNA polα or primase gene or the upstream gene regulating the DNA polα or primase gene expression. 6. Methods of Treating

[0110] Disclosed are methods of treating a subject in need thereof comprising administering a therapeutically effective amount of a DNA polymerase α (DNA polα) inhibitor or primase inhibitor to the subject.

[0111] In some aspects, a subject in need thereof is a subject having infertility problems or gut inflammation. In some aspects, a subject in need thereof is a subject that needs to have certain tissues regenerated from using induced pluripotent stem cells, such as neuron degeneration, skin injury or burning, blood cell disorder or hair loss. Thus, in some aspects, the result of treating the subject by altering DNA polα or primase (with an inhibitor or genetically) can result in increased fertility, longer reproductive lifespan such as higher quality of sperm at older ages, decreased gut inflammation or better recovery from gut inflammation.

[0112] In some aspects, the DNA polα inhibitor can be, but is not limited to, POLA1 inhibitor 1, Aphidicolin, 18 β-Glycyrrhetinic Acid, 1-β-D-Arabinofuranosylcytosine, Adarotene, CD437, or derivatives thereof.

[0113] In some aspects, the primase inhibitor can be, but is not limited to, amenamevir, pritelivir, BILS 22 BS, T157602, or derivatives thereof.

[0114] In some aspects, the DNA polα inhibitor or primase inhibitor decreases DNA polα or primase activities and / or levels in a cell in a subject. In some aspects, the inhibition of activity or levels of DNA polα or primase is a partial inhibition. Thus, in some aspects of the disclosed methods there can be 10, 20, 30, 40, 50, 60, 70, 80, 90, or 95% inhibition of DNA polα or primase activity or levels.

[0115] In some aspects, the DNA polα inhibitor or primase inhibitor does not affect the percentage of S-phase cells (namely S-phase index) within a population of cells in a subject. In some aspects, this indicates that the treatment with the DNA polα inhibitor or primase inhibitor does not change the overall cell cycle progression in the cell. In some aspects, the DNA polα inhibitor or primase inhibitor does not substantially affect the S phase of the cell cycle of a cell. In some aspects, not substantially affecting the S phase of the cell cycle can mean that at least 50, 60, 70, 80, or 90% of cells do not have the S phase affected.

[0116] In some aspects, the DNA polα inhibitor or primase inhibitor does not cause cellATTORNEY DOCKET NO.36406.0038P1 cycle arrest in a cell or cell death of a cell in the subject. Thus, in some aspects, replication still occurs in a cell contacted with a DNA polα inhibitor or primase inhibitor and the cell is not killed.

[0117] In some aspects, the DNA polα inhibitor or primase inhibitor decreases lagging strand synthesis in a cell in the subject. In some aspects, leading strand synthesis is not altered in a cell in the subject. In some aspects, leading strand synthesis is not substantially altered in a cell in the subject. In some aspects, not substantially altering leading strand synthesis can mean that at least 50, 60, 70, 80, 90, or 99% of leading strand synthesis is not altered. In some aspects, DNA polα and primase act on the leading strand to start replication, but this is a single enzymatic reaction. However, for the lagging strand, there can be many repeated reactions to continually initiate Okazaki fragment synthesis, therefore because DNA Polα and primase work significantly more on the lagging strand than the leading strand, in some aspects, the leading strand synthesis is either not altered or only slightly or substantially altered while the lagging strand synthesis.

[0118] In some aspects, the DNA polα inhibitor or primase inhibitor can reprogram a progenitor cell in the subject. In some aspects, the reprogrammed progenitor cell comprises separable old histone versus new histone pattern during DNA replication and cell division. In some aspects, the separable old histone versus new histone pattern is a stem cell-unique pattern. In some aspects, the old histone versus new histone pattern is an old histone H3 versus new histone H3 pattern. In some aspects, old histone versus new histone displays separable patterns during DNA replication. In some aspect, old histone versus new histone enriched chromosomal regions display differential condensation in the dividing reprogrammed progenitor cell.

[0119] In some aspects, the cell is a stem cell or a progenitor cell. In some aspects, a progenitor cell is known as a dividing cell with reprogramming potential. In some aspects, the progenitor cell is a fibroblast cell, a male gonialblast or spermatogonial cell, a female cystoblast or cystocyte, or an intestinal cell.

[0120] Disclosed are methods of treating comprising genetically altering a cell in the subject to reduce DNA polα or primase levels (or activity). In some aspects, genetically altering a cell decreases DNA polα or primase levels in the cell. In some aspects, genetically altering a cell can comprise contacting a cell with a double-strand RNA or siRNA to regulate (e.g. decrease) DNA polα or primase gene expression (post transcriptionally). Thus, in some aspects, the double-strand RNA can target a DNA polα or primase gene or upstream of a gene that regulatesATTORNEY DOCKET NO.36406.0038P1 DNA polα or primase expression. In some aspects, CRIPSR / Cas9-mediated somatic cell genome engineering can be used to knock out one copy of a DNA polα or primase gene or generate loss-of-function versions of a DNA polα or primase gene in specific cell types. Thus, in some aspects, guide RNAs designed to target the DNA polα or primase gene or the upstream gene regulating the DNA polα or primase gene expression can be used. In some aspects, CRIPSR / Cas9-mediated somatic cell genome engineering can target DNA polα or primase gene or the upstream gene regulating the DNA polα or primase gene expression.

[0121] In some aspects, the disclosed methods of treating a subject can further comprise administering a composition that increases Replication Protein-A 70 (RPA70) levels in a cell in the subject. In some aspects, a composition that increases RPA70 levels can be a composition that genetically alters a cell to increase RPA70 or can be a composition that induces activation of RPA70 transcription. Examples A. Example 1 1. Summary

[0122] In the Drosophila male germline stem cell (GSC) lineage, GSCs demonstrate asymmetric histone inheritance while progenitor germ cells exhibit an overall symmetric pattern. Here, we report that an essential mechanism underlying this phenomenon is delayed lagging strand synthesis. Components involved in lagging-strand synthesis, such as DNA display significantly reduced expression in GSCs compared to non-stem progenitor cells. Compromising Polα genetically induces the replication-coupled histone incorporation pattern in progenitor cells to be indistinguishable from that in GSCs, which can be recapitulated using a Polα inhibitor in a concentration-dependent manner. Furthermore, GSC-derived chromatin fibers show a significantly higher degree of old histone recycling by the leading strand than the progenitor cell-derived fibers. However, upon reducing Polα levels in progenitor cells, the chromatin fibers now display asymmetric old histone recycling just like GSC-derived fibers, and such an asymmetry is comparable between GSCs and progenitor cells at both S-phase and M-phase. Importantly, these altered chromatin features in progenitor cells allow them to act like bona fide GSCs under both pathological and physiological conditions. Together, these results indicate that developmentally programmed expression of key DNA replication components could beATTORNEY DOCKET NO.36406.0038P1 manipulated to promote progenitor cell dedifferentiation and re-differentiation, indicating a new pathway for cell reprogramming in stem cell lineages. 2. Results i. Reduced expression of the lagging strand enriched replication components in gscs

[0123] To identify which factors could be responsible for stem cell-specific asymmetric histone inheritance, a candidate gene screen was performed using a series of CRISPR / Cas9- mediated knock-in lines with the hemagglutinin (HA) tag at individual genes that encode different key replication machinery components. Endogenously tagged fly strains were generated by CRISPR-Cas9 with the genome editing service provided by Fungene Inc. (Beijing, China). The following strains were generated and used in this study: cdc45-mCherry (internally tagged between D163 and Q164), cdc45-3xHA (internally tagged between D163 and Q164), DNA polymerase ε 255kD subunit-3xHA (tagged at the C-terminus), DNA polymerase α 180kD- 3xHA (tagged at the C-terminus), DNA polymerase δ-3xHA (tagged at the C-terminus), ctf4- eGFP (tagged at the C-terminus).

[0124] Immunostaining experiments were performed using standard procedure. Primary antibodies used were DE-cadherin (1:200; DHSB AB_528120), γ-Tubulin (1:200; Sigma- Aldrich AB_T6557), Stat92E (1:500; from Denise Montell, University of Santa Barbara, CA, USA), VASA (1:500; from Ruth Lehmann, Whitehead Institute, USA) Armadillo (1:100; DSHB N27A1), Traffic Jam (1:100, from Mark Van Doren, Johns Hopkins University, USA), anti- PCNA (1:100; Santa Cruz sc-56), anti-GFP (1:1,000; Abcam ab 13970), anti-HA (1:200; Sigma- Aldrich H3663), anti-mCherry (1:1,000; Invitrogen M11217), anti-H3K27me3 (1:400; Millipore 07-449), anti- H4K20me2 / 3 (1:400; Abcam ab78517), anti-H3S10ph (1:2000; Cell Signaling Technology 9701), and anti-BrdU (1:200; Abcam ab6326). BrdU analog was Invitrogen B23151 5-bromo-2′-deoxyuridine (BrdU). Secondary antibodies were the Alexa Fluor-conjugated series (1:1,000; Molecular Probes). Confocal images were taken on the Zeiss LSM800 (with Airyscan mode) with a 63x oil objective lenses or on the Leica SPE with 63x oil immersion lenses.

[0125] Images were analyzed using the ImageJ software FIJI. Germline cyst stages were identified using Arm signal to label the two cyst cells encapsulating each cyst. Average intensity values were recorded for the center Z-slice of each cell / nucleus of interest. For germ cells within one cyst, only one germline nucleus from the entire cyst was measured as one data point. For the comparison of protein levels of endogenously tagged proteins, immunostaining signals in GSCs,ATTORNEY DOCKET NO.36406.0038P1 4-cell and 8-cell SGs were measured, and a background was subtracted using the post-mitotic hub cells, which are devoid of any of these replication components’ signals. Signal intensity from 4-cell and 8-cell SGs were then normalized to the average intensity of GSCs from the same batch of testes. For the batch-based normalization, within one experimental batch, each data point was normalized to the average of WT GSCs in this corresponding batch.

[0126] The levels of proteins involved in lagging-strand synthesis, (Pol^), differed significantly between GSCs and SGs, with substantially reduced levels in GSCs compared to SGs. In contrast, a component for leading-strand synthesis, DNA polymerase ^ (Pol^), exhibited comparable levels between GSCs and SGs). On the other hand, the single-stranded DNA (ssDNA) binding protein Replication Protein-A 70 (RPA70), the largest subunit of the ssDNA- binding heterotrimeric complex, displayed the opposite trend with higher RPA levels in GSCs compared to SGs (FIG.1A).

[0127] Quantification of these results revealed that GSCs have approximately 58% of the levels of Pol^ as compared to SGs, while Pol^ levels are insignificantly different between GSCs and SGs (FIG.1A and FIG.1B). However, other replication machinery components, such as the replication fork progression Cell Division Cycle protein 45 (Cdc45), showed no significant difference between GSCs and SGs (FIG.1B). Another component whose yeast homolog has been shown to have histone chaperoning activities, Chromosome Transmission Fidelity 4 (Ctf4), has no significant difference between GSCs and SGs, either (FIG.1B and FIG.7A).

[0128] These results show that decreased Pol^^and increased RPA could cooperatively contribute to delayed lagging strand synthesis in the GSCs, which could also underlie the longer cell cycle length of GSCs. ii. Reducing Polα levels or inhibiting Polα activities increase old versus new histone separation in S-phase nuclei of progenitor cells

[0129] To investigate how delayed lagging strand synthesis could bias old histone recycling to the leading strand, a dual color labeling system was used to examine the distribution of old histones (eGFP) versus new histones (mCherry) in intact nuclei in the mail germline. To visualize potentially differential histone incorporation during S-phase, a clearance buffer which effectively removes nucleoplasmic protein was applied. Briefly, the clearance buffer was prepared by mixing 989µls of the clearance buffer stock solution (8.4 mM HEPES, 100 mM NaCl, 3 mM MgCl2, 1 mM EGTA, 300 mM Sucrose, 2% Triton X-1000, and 2% BSA in ddH2O) with 1 µl DTT and 10 µl protease inhibitor (100x Leupeptin). After dissection, tissueATTORNEY DOCKET NO.36406.0038P1 samples were incubated in 10 μM EdU (Invitrogen Click-iT EdU Imaging Kit, catalog # C10340) for 15 minutes in Schneider’s media at room temperature. At the end of the 15 minutes, the Schneider’s media were drained and the clearance buffer was added for two minutes at 4°C in darkness. Samples were then fixed in 4% PFA, washed with 1xPBST, and then blocked in 3% BSA for 30 minutes. For robust signals, both the old H3-eGFP and new H3-mCherry were immunostained with antibodies (e.g., anti-eGFP and anti-mCherry) using standard procedures. The CLICK reaction was performed according to manufacturer’s instructions to label EdU. The DNA dye Hoechst was also added at this step.

[0130] Images were acquired on the Zeiss LSM800 using Airyscan mode on a 63x oil immersion objective. All samples were imaged using the identical settings. GSCs were identified by their proximity to the hub region. When 4-cell stage SGs were used, only one SG per cyst was analyzed to represent one data point. All images were analyzed using FIJI software. The Pearson score was recorded using the Coloc2 plugin for each nucleus, which was cropped to include just the nucleus as much as possible as delineated by the Hoechst signals. For each batch of images, the average measurement of the control GSCs was set to one (1) and the other treatments are normalized to control GSCs, in order to avoid batch variability. The resulting values are then used to calculate mean and standard error (Mean± SE).

[0131] Flies with UASp-FRT-H3-EGFP-FRT-H3-mCherry along with any relevant genotypes were crossed with hs-flp; nanos-Gal4 and raised at 25°C. Within two days of eclosure, adult male flies were transferred to a vial and the vial was submerged underwater at 37°C for 90 minutes. Flies were then recovered at 29°C for 18 hours prior to dissection for experiments, with the exception of experiments using the PolA1 inhibitor.

[0132] Polα180 inhibitor (MedChemExpress Cat# HY-147812) was prepared in DMSO as stock and stored at -20°C (for short term) and -80°C (for long term) according to manufacturer’s instructions. Drug incubation was performed on testes in “live cell media” containing Schneider’s insect medium with 200 μg / ml insulin, 15% FBS by volume, and 0.6x pen / strep. Prior to experiments, incubation media was prepared by diluting inhibitor solution (or DMSO vehicle) to the proper concentration in live cell media. Testes were dissected and placed in 100μl of incubation media as quickly as possible following dissection. Incubated testes were left in open tubes in darkness at room temperature (RT) for 4 hours. Because 4 hours is longer than the standard S-phase of the early male germline, all S-phase cells at the end of the incubation should have been exposed to the inhibitor for the entirety of their current S-phase.ATTORNEY DOCKET NO.36406.0038P1

[0133] For S-phase colocalization experiments using the inhibitor, flies were heat shocked as described above and left at 29°C to recover for 14 hours. Testes were then dissected and placed in incubation media for 4 hours to result in 18 total hours of heat shock recovery. After incubation with the inhibitor at the designated concentrations, these tissues were processed for S-phase colocalization analysis.

[0134] The dual color label strategy combined with high spatial resolution Airyscan microscopy allowed visualization of separable old H3-eGFP versus new H3-mCherry enriched regions in the control wild-type (WT) GSCs at S-phase, labeled with a pulse of thymidine analog 5-ethynyl-2´-deoxyuridine (EdU) (FIG.2A and FIG.2B). In contrast, the degree of separation between old and new H3 was less in the WT SGs than that in the WT GSCs during S-phase (FIG.2B). Quantification using a relative Pearson colocalization measurement revealed significantly higher degree of colocalization between old H3-eGFP and new H3-mCherry in WT SGs than in WT GSCs (FIG.2C), consistent with the idea that old and new histones are asymmetrically incorporated in WT GSCs.

[0135] To evaluate if compromising lagging strand synthesis in SGs could recapitulate GSC-like features, heterozygous male flies (pol^50+ / -) were generated by introducing null allele of the pol^50 gene, which encodes the DNA Primase Subunit 1 (or Prim1). When the Primase levels are reduced using the pola50+ / -males, SGs display much more separable patterns between old and new H3, to a level indistinguishable from the WT GSCs as well as the pola50+ / -GSs (FIG 2B and FIG.2C).

[0136] Additionally, a pharmacological strategy was implemented, utilizing a Pol^ inhibitor that prevents the DNA binding ability and primer elongation activity of DNA Polymerase^^ subunit 1 (PolA1 or Pol^180, FIG.8A) At a high concentration (e.g., 100^M), this inhibitor completely blocked DNA replication, indicated by the absence of EdU incorporation in different staged germ cells. However, when using this inhibitor at a low concentration (e.g., 10^M), normal DNA replication could proceed with proper EdU incorporation compared to the control sample treated with vehicle (FIG.2D, FIG.8B, and FIG.8C). With this inhibitor treatment, SGs exhibited separable old versus new H3 patterns similar to those detected in the pol^50+ / -cells (FIG.2B and FIG.2E). The inhibitor-treated SGs showed more separation than the vehicle- treated SGs, but display patterns comparable to either inhibitor-treated GSCs or vehicle-treated GSCs (FIG.2C). Quantification confirmed these patterns and further revealed that this inhibitor induces old versus new H3 separation in SGs in a dosage-dependent manner but causesATTORNEY DOCKET NO.36406.0038P1 insignificant changes in GSCs (FIG.2F). Notably, the presence of intermediate histone separation patterns at decreasing concentrations of inhibitor (e.g., 2.5^M and 5.0^M) indicated that the asymmetric histone incorporation pattern is tunable and scales to the inhibition of lagging strand synthesis. iii. Reducing Polα levels enhances asymmetric old histone recycling at the replication fork in non-stem progenitor cells

[0076] To directly visualize the dynamic histone incorporation patterns at the actively replicating regions, a short pulse of EdU was introduced in combination with a single-molecule chromatin fiber technique. Briefly, after adding EdU to the testis samples and incubating for 15 minutes, lysis buffer was added (100 mM NaCl, 25 mM Tris-base, 0.2% Joy detergent, pH=10). The testis tip was then micro-dissected on the slide and the rest of the testis was removed. Cells were allowed to fully lyse for approximately 5 minutes and then a Sucrose / Formalin (1M sucrose; 10% formaldehyde) solution was added and left for 2-minute to incubate, before a cover slip was gently placed on the top. The slide was then transferred to liquid nitrogen for 2 minutes before the cover slip was removed. The slide was then transferred to 95% EtOH for 10 min at -20°C in a freezer. Afterwards, the slide was fixed in 1% PFA for 1 minute. Samples were washed 3x in a Coplin jar with 1xPBST followed by blocking the sample with 3% BSA in 1xPBST for 30 minutes. Primary antibodies were then added for overnight incubation in a humidity chamber at 4°C. To assess histone asymmetry, anti-PCNA, anti-H3K27me3, and anti- GFP primary antibodies were added to chromatin fibers from the testes from the males with the following genotypes: each of the drivers (nos-Gal4 itself, nos-Gal4ΔVP16; bam-Gal80 combination, or bam-Gal4 itself) crossed with UASp-FRT-H3-EGFP-FRT-H3-mcherry without hs-flp. For cdc45-mCherry; DNA Polymerase-HA fibers, mCherry and HA primary antibodies were used. After the incubation with the primary antibodies, the slides are washed in a coplin jar with 1xPBS. Then the secondary antibodies were added and incubated for 2 hours at room temperature in a humidity chamber. The click chemistry was performed to label EdU following the manufacturer’s instruction. When DNA needs to be labeled, Hoechst is included at 1:1,000 to stain the samples. Additionally, for samples that need DNA labeling, ProLong™ Gold Antifade Mountant with DNA Stain DAPI (Thermo Fisher catalog # P36931) was used. For samples that do not need DNA labeling, ProLong Diamond mounting media without DAPI (Thermo Fisher catalog# P36961) was used.

[0077] To precisely label chromatin fibers derived from GSCs versus non-stem progenitor SGs,ATTORNEY DOCKET NO.36406.0038P1 Gal4 transcription activator controlled by the early germline-specific nanos driver (nos- Gal4ΔVP16) was paired with the Gal80 transcription repressor under the control of the bag of marbles promoter (bam-Gal80), which turns on expression from 2-cell to late stage SGs. This combination restricted the transgene expression almost exclusively in GSCs with some detectable expression in the gonialblasts (GBs) but almost undetectable signals in the SGs (FIG. 7D), which differed from the early-stage germ cell expression pattern driven solely by nos-Gal4 (FIG.7B) and late-stage germ cell expression pattern driven solely by bam-Gal4 (FIG.7C). These germline stage-specific expression patterns were confirmed by quantification using a H3- eGFP reporter (FIG.1E).

[0078] Using the H3-eGFP expressed by different drivers, chromatin fibers derived from early- stage germ cells including GSCs (nos>H3-eGFP), from very early-stage germ cells enriched with almost exclusive GSCs (nos-Gal4ΔVP16; bam-Gal80>H3-eGFP) and from late-stage SGs (bam>H3-eGFP) were labeled. Next, old histone recycling patterns at the eGFP-labeled and EdU-positive chromatin fibers using the old H3-enriched H3K27me3 histone modification were observed. The strandedness with a lagging-strand-enriched component Proliferating Cell Nuclear Antigen (PCNA) was distinguished. Together, chromatin fibers carrying all four markers (i.e., H3-eGFP, EdU, anti-H3K27me3, and anti-PCNA) were analyzed from fly testes carrying the H3-eGFP reporter with the following drivers: nos-Gal4 only, nos-Gal4ΔVP16; bam-Gal80 combination, and bam-Gal4 only. While nos>H3-eGFP-labeled chromatin fibers showed a relatively wide distribution of H3K27me3 between replicative sister chromatids (FIG. 9A and FIG.9B) with an overall biased distribution toward the PCNA-depleted leading strand (FIG.3F and FIG.9C), nos-Gal4ΔVP16; bam-Gal80>H3-eGFP-labeled chromatin fibers showed consistently more asymmetric H3K27me3 distribution toward the leading strand (FIG. 3A, FIG.3F, and FIG.9C). In contrast, bam>H3-eGFP-labeled fibers displayed a more symmetric H3K27me3 distribution pattern (FIG.3B, FIG.3F, and FIG.9C).

[0079] To quantify old histone incorporation patterns, H3K27me3 was used as a proxy for old histones and plotted its ratio on the PCNA-depleted leading strand to the PCNa-enriched lagging strand (FIG.3F, FIG.9C, and FIG.9D). The nos-Gal4ΔVP16; bam-Gal80-labeled chromatin fibers and the bam-labeled ones were not only statistically distinguishable from each other (P< 10-4, FIG.3F), but also statistically different from the nos-labeled group (FIG.3F). Interestingly, combining the nos-Gal4ΔVP16; bam-Gal80-labeled and bam-labeled groups in silico generated a data set indistinguishable from the nos-labeled group (FIG.9C), indicating that theATTORNEY DOCKET NO.36406.0038P1 heterogeneity of both GSC-derived and SG-derived fibers could underlie the detected H3K27me3 variation among the nos-labeled chromatin fibers.

[0080] Furthermore, the nos-labeled fibers from pol^50+ / -testes showed more asymmetric H3K27me3 distribution toward the leading strand than the nos-labeled fibers from the control (FIG.3C and FIG.3F). Consistently, the nos-labeled fibers from heterozygotes of the polα180 gene, which encodes DNA Polymerase^^ subunit 1 (or PolA1), also exhibited a more asymmetric H3K27me3 distribution pattern toward the leading strand than those from the control (FIG.9D and FIG.9E). In contrast, compromising Pol^50 had little effect on nos- Gal4ΔVP16; bam-Gal80-labeled chromatin fibers (FIG.3D and FIG.3F). Notably, bam>H3- eGFP-labeled chromatin fibers displayed significantly more asymmetric patterns in the pol^50+ / -samples than in the control (FIG 3E and FIG.3F), in accordance with the results shown in intact S-phase nuclei (FIG.2B and FIG.2C). These results demonstrated that compromising Polα affects SGs with normally high levels of Polα more than GSCs that already have low levels of Polα (FIG.2 and FIG.3).

[0081] To test whether RPA also contributes to asymmetric histone incorporation, rpa70 cDNA was overexpressed using nos-Gal4 (nos>rpa70-HA). Likewise, the overexpression of RPA70 results in enhanced asymmetric H3K27me3 incorporation at the replicative regions, indicating that increased levels of RPA lead to enhanced asymmetric old histone recycling (FIG.9D and FIG.9F). In summary, the chromatin fiber results demonstrated that SGs with relatively high levels of Polα and low levels of RPA can be induced to have GSC-like asymmetric old histone incorporation at the replicative regions by reducing Polα levels or by enhancing RPA expression. iv. Reducing Polα induces differential condensation of old histone- versus new histone-enriched regions in M-phase progenitor cells

[0082] An area-based method was used to monitor the chromosomal condensation using a dual- color histone transgene UASp-FRT-histone-eGFP -FRT-histone-mCherry. A maximum intensity projection was generated for old H3- (eGFP) and new H3- (mCherry) enriched areas. The intensity of each pixel was determined and scaled individually, setting the minimum intensity to 0 and the maximum to 65,535 (a 16-bit range). The pixels were monitored across the image with a threshold of 35% of the maximum intensity. Condensation kinetic profiles were generated to compare old H3- versus new H3-enriched regions by calculating the percentage of pixels above the threshold (the condensation parameter). Relative compaction index was measured andATTORNEY DOCKET NO.36406.0038P1 plotted by taking a ratio of the percentage of pixels of the new H3-enriched to the old H3- enriched regions.

[0083] To determine if old H3- versus new H3-enriched chromosomal regions display differential condensation in the M-phase GSCs but overlapping pattern om SGs, a chromatin compaction index was used to measure the differential condensation between old H3- and new H3-enriched regions. The control GSCs and SGs showed marked differences (FIG.10A and FIG.10B), while the pol^50+ / -SGs showed GSC-like patterns (FIG.10C and FIG.10D). Using a relative condensation index as reported previously, significant difference was detected between GSCs and SGs in the control testes but not between GSCs and SGs in the pol^50+ / -testes (FIG. 10E). Importantly, in the pol^50+ / -testes, both GSCs and SGs displayed similar patterns compared to the control GSCs but significantly distinct patterns compared to the control SGs (FIG.10E). Collectively, these results demonstrated that by compromising a single lagging- strand-enriched component, primase, differential condensation of the regions enriched with old H3 versus new H3 in M-phase cells, a GSC-specific feature, could be recapitulated in the SGs. v. Dedifferentiated spermatogonial cells with reduced Polα levels tend to have properly oriented centrosomes

[0084] GSCs and early-stage germ cells (up to 4-cell spermatogonia) were depleted using genetic manipulation of the pro-apoptotic gene, grim. The UAS-grim flies (were crossed with nanos-Gal4^VP16; tubulin-Gal80tsand grown at 18°C to prevent grim expression, which is only turned on when shifting flies to 31°C to inactivate Gal80. The nanos-Gal4^VP16; tubulin- Gal80ts>UAS-grim flies were kept at 31°C for four days, which ablated early-stage germ cells (FIG.4B). After 4-day ablation, flies were recovered at 18°C for another four days, when grim expression was inhibited again (FIG.4C). After recovery, all GSCs including dedifferentiated GSC-like cells and bona fide GSCs were evaluated by immunostaining using anti-γ-Tubulin as the centrosome marker and anti-Stat92E as the stemness marker. Centrosome misorientation is defined as neither of the two centrosomes being within the 90ohub–GSC interface (FIG.4K). Centrosomes were scored

[0085] To detect more efficient SG dedifferentiation during regeneration from genetically manipulated depletion of GSCs, the grim gene was ectopically expressed gene in the early-stage germ cells using a temporally controllable condition (nos-Gal4ΔVP16; tubulin-Gal80ts; UAS- grim) followed by recovery to allow for SG dedifferentiation (FIG.4A-D). Grim functions to inhibit apoptotic antagonists, so its overexpression promotes apoptosis. This genetic ablationATTORNEY DOCKET NO.36406.0038P1 effectively caused GSC death in both the control and polα50+ / -testes, shown by greatly reduced GSC number after turning on Grim expression (FIG.4E-F, and FIG.4J). Upon recovery, the dedifferentiation process occured, leading to a significantly increased number of GSC-like cells [i.e., dedifferentiated GSCs (FIG.4H-J0]. Therefore, ectopic expression of an apoptosis activator, Grim, ablated GSCs and promoted SGs to dedifferentiate. However, dedifferentiated SGs often carried misoriented centrosomes (FIG.4H, FIG.4i, FIG.4K, and FIG.4L). Dedifferentiated polα50+ / -SGs had significantly fewer incidence of misoriented centrosomes than dedifferentiated control SGs (FIG.4H-J and FIG.4L). Notably, even though the number of GSCs doubled after the recovery in polα50+ / -testes (FIG.4J), there was almost no change for the percentage of GSCs with misoriented centrosomes (FIG.4L), indicating that almost all dedifferentiated polα50+ / -SGs had oriented centrosomes. In summary, these results demonstrated that dedifferentiated polα50+ / -SGs tend to have more bona fide GSC-like cellular features, such as properly oriented centrosomes, than dedifferentiated control SGs. vi. Male flies with reduced Polα levels have sustainable fertility during aging

[0086] To determine whether pola+ / -SGs could replace GSCs more effectively than the control SGs, cellular features and redifferentiation capabilities of the dedifferentiated SGs in both WT and pola+ / -males during aging were investigated. Control and the polα50+ / -male flies were examined over a 56-day period of time after eclosure (FIG.11A). GSC cellular features and testicular morphology in males from different age groups was examined. In D35 control males, the number of GSCs did not decrease but the percentage of GSCs with misoriented centrosomes significantly increased compared to D1-3 young males (FIG.5A, FIG.5D, and FIG.5E). In contrast, in polα50+ / -males at the same age (i.e., D35), there was a significant reduction of the percentage of GSCs displaying misoriented centrosomes (FIG.5B, FIG.5D, and FIG.5E). Additionally, a critical “stemness” transcription factor, Stat92E, displays significantly higher levels in the polα50+ / -GSCs than the levels in the control GSCs from D35 testes (FIG.5C, FIG. 5F, and FIG.5G). Morphologically, testes from the D35 control males frequently showed abnormal niche anatomy such as the displaced hub structure (FIG.5A, FIG.5F, and FIG.5H), as well as the much thinner overall testicular structure, including the terminal differentiating regions with elongating and mature sperm (FIG.5F and FIG.11B). In contrast, testes from D35 polα50+ / -males displayed more normal hub structure at the testis apical tip (FIG.5B, FIG.5G, and FIG.5H) and at the terminal differentiating regions (FIG.11C). The displaced hub structure could be due to age-dependent declined expression of the adhesion molecules, which accountedATTORNEY DOCKET NO.36406.0038P1 for the increased number of GSCs, counted as germ cells surrounding the hub (FIG.5A, FIG. 5D, and FIG.5F).

[0087] To test the redifferentiation capabilities of the dedifferentiated SGs during aging, a time- course male fertility assay using males from different age groups was performed. polα50 P- element insertion flies were maintained over a balancer on the 3rdchromosome. The polα50+ / -flies are from outcrossing the polα50 / Balancer stock with y,w flies at 25°C to generate the F1 progenies, where the polα50 P-element insertion allele is over a wild-type chromosome. The F1 polα50+ / -males were aged at 25°C for the mentioned period of time with females. Vials were flipped every five days to prevent the generation of the F2 progenies. As a control, newly enclosed y,w males were collected and aged simultaneously in separate vials. When male flies (polα50+ / -and y,w) reached the desired age, one male was put into a new vial with three virgin y,w females. These flies were allowed to mate for five days, and then parents were tossed away. Only those vials with all four live parents (1 male and 3 female) were retained for fertility assay with the 5-day mating period. If any of the four parental flies died during the 5-day mating period, those vials were excluded from data collection and analyses. All F1 progenies were counted for 15 days after tossing the parent flies as one data point.

[0088] Very intriguingly, the polα50+ / -males displayed persistent fertility over time from D0 to D42 and only show reduced fertility at D49. In a drastic comparison, the control males showed continuously reduced male fertility from D7 to D49. Notably, both genotyped males showed indistinguishable fertility as young males from D0 to D14. The pola+ / -males still retained relatively high fertility 49 days after eclosion (D49), significantly higher than that of the control males at D49 (FIG.5I).

[0089] To assess the lifespan of polα50+ / -flies, a longevity assay was performed. Groups of 20 newly eclosed flies (10 males and 10 females) were placed in vials with fly food and yeast. The day the flies eclosed was considered Day 0, and flies were kept in a temperature-controlled 25°C incubator for their entire lifespan. Every 2-3 days, flies were flipped onto fresh fly food with dry yeast, and deceased flies were removed and counted. Prior to Day 30, if all the males or all the females in a vial died (leaving all remaining flies as the same sex), then new flies of the missing sex were added, but not counted in the lifespan assay, to maintain the effects of mating in the remaining flies. Because the polα50+ / -flies were made by outcrossing a balanced polα50P-element / Balancer line to y,w, the control flies used were y,w flies crossed to another wild-type strain Oregon R flies in order to approximate similar heterozygosity between the control andATTORNEY DOCKET NO.36406.0038P1 experimental flies.

[0090] Remarkably, the pola50+ / -flies displayed comparable lifespan compared to the control flies derived from outcrossing two wild-type strains (FIG.5J), in order to eliminate the potential fitness benefit resulting from outcrosses. Furthermore, control versus pola50+ / -lifespans showed insignificant differences when considering just males, just females, and males and females combined (FIG.5J). These results confirmed that the sustained pola+ / -male fertility is not at the expense of shortened lifespan, indicating a reproductive longevity in pola+ / -males. vii. asynchronous leading versus lagging strand syntheses

[0091] It has been reported that the temporal differences in replicating leading strand versus lagging strand biases old histone inheritance to the strand more closely coupled to the replication fork progression in S. cerevisiae. To investigate whether this temporal difference exists and is detectable in the Drosophila testis, it was necessary to determine whether leading strand versus lagging strand syntheses can be differentially labeled by using distinct nucleoside analogs introduced in a sequential order (FIG.6A). Brifely, after sample dissection, 10 μM EdU was added for a 10-minute incorporation, followed by washing out EdU. BrdU was subsequently added for another 10 minutes. After this sequential labeling, DNA fibers were prepared using the same procedure as described above for chromatin fibers, with the exception of using a different lysis buffer to strip proteins from the DNA (200 mM Tris–HCl, pH 7.5, 50 mM EDTA, 0.5% SDS). The fibers were then treated with 1M HCl for 30 minutes at room temperature to expose the incorporated BrdU. After washing with 1xPBST, BrdU antibodies were added for incubation overnight at 4°C in a humidity chamber. Secondaries antibodies against the BrdU primary antibodies were then added for 2 hours at room temperature in a humidity chamber. The click reaction to recognize EdU was performed subsequently along with Hoechst incubation at 1:1000. Samples were then mounted in ProLong Diamond mounting media with DAPI. The EdU-positive DNA fibers representing regions that undergo DNA replication during EdU pulse (and thus have EdU on at least one side) were used for subsequent analyses (FIG.6D).

[0092] Using this experimental regime, DNA fibers where both strands are co-labeled with just one nucleoside (e.g., EdU) should represent regions where both strands are replicated within the time window of the EdU pulse (FIG.6B). However, DNA fibers with EdU and BrdU on opposing strands likely capture the uncoupled syntheses of the two strands (FIG.6B). Indeed, the DNA fibers derived from the apical testis tips display the latter pattern in approximately 40% of the fibers (FIG.12A), indicating that asynchronous leading versus lagging strand synthesesATTORNEY DOCKET NO.36406.0038P1 can be detected. On average, DNA fibers carried both EdU and BrdU displayed a 2.35-fold BrdU enrichment toward one strand whereas a 1.91-fold EdU enrichment toward the opposing strand (FIG.6C and FIG.12B). On the other hand, when a short EdU pulse was introduced by itself on early germline-derived chromatin fibers, 52% of them displayed a strong bias (>2-fold) toward one of the two strands, with 79% of them displaying strong asymmetry toward the lagging strand (FIG.12C, FIG.12D, and FIG.9A), likely due to a longer time for synthesizing lagging strand and thus a higher opportunity for the lagging strand to be labeled (FIG.12E). Consistently, PCNA signals often displayed asymmetric distribution on early germline-derived chromatin fibers, along with EdU, toward the H3K27me3-depleted lagging strand (FIG.3A, FIG.9A, and FIG.12D).

[0093] To further test whether delayed lagging strand synthesis was mechanistically responsible for asymmetric histone incorporation, the EdU signals of all chromatin fibers that had been analyzed for the H3K27me3 patterns (FIG.3) were reexamined. Indeed, very early germline- derived (nanos-Gal4ΔVP16; bam-Gal80) chromatin fibers showed a high degree of asymmetric EdU patterns, while the late germline-derived (bam-Gal4) chromatin fibers primarily showed symmetric EdU distribution between sister chromatids. Notably, EdU asymmetry was substantially enhanced in the late germline-derived (bam-Gal4) chromatin fibers from the polα50+ / -testes than that in the control (FIG.6D and FIG.12F). The small incidence of leading strand- but a large population of lagging strand-biased EdU incorporation (FIG.12D and FIG. 12F) were consistent with the temporal asynchrony between leading strand and lagging strand syntheses, with the lagging side synthesizing DNA more slowly on average. Overall, these results showed that compromising Polα is sufficient to increase asymmetric H3K27me3 incorporation by the leading strand, likely by enhancing the temporal asynchrony between sister chromatid syntheses.

[0094] Finally, to directly visualize delayed lagging strand synthesis, an endogenously tagged cdc45 gene was used. This resulted in the Cdc45-mCherry fusion protein as a marker for the Cdc45-MCM-GINS (CMG) complexes, in order to label actively progressing replication forks. Immunostaining was performed to label Polα-HA for lagging strand polymerase and Polε-HA for leading strand polymerase, using the endogenously tagged genes, respectively. Remarkably, while Polε was always tightly associated with Cdc45 at EdU-labeled replicative chromatin fibers (FIG.6E and FIG.6G), Polα was found in tracts extending away from Cdc45 (FIG.6F and FIG. 6G), indicating Polα was spatially decoupled from the opening of the replication fork. Together,ATTORNEY DOCKET NO.36406.0038P1 these results indicated that the temporal separation between leading strand and lagging strand syntheses can be visualized by nucleoside analogs introduced at different time points during DNA replication or with different replisome components.

[0095] Sfdf viii. Titration of Polx1 inhibitor in human dermal fibroblast cells and polx1 inhibition enhances human ipscs reprogramming efficiency

[0096] To perform the Pola1 inhibition during the iPSC reprogramming of human dermal fibroblast cells, the CytoTuneTM-iPS 2.0 Sendai Reprogramming Kit was used. The working concentration of Pola1 inhibitor in human dermal fibroblast cells was optimized with minimal effects on the DNA replication in order to determine a working concentration of the Pola1 inhibitor that does not change the percentage of replicating cells among the population of cells (FIG.13). This concentration of Pola1 inhibitor was utilized to treat fibroblast cells during the iPSC induction process. Subsequently, the colony-forming efficiency of human iPSCs was detected and characterized.

[0097] Compared to the control reprogramming condition (overexpression of Oct3 / 4, Sox2, Klf4, and C-Myc), cells treated with the Pola1 inhibition (FIG.14A) produced iPSCs with higher efficiency, characterized by iPSC colonies and larger colony size. iPSC colonies also formed at an earlier time point during this reprogramming time course in Pola1 inhibitor-treated cells (FIG.14B and 14C). Furthermore, immunostaining of Sox2 transcription factor, a well- known stem cell marker, validated the pluripotent state of the induced human iPSCs in both the control and Pola1 inhibitor-treated group (FIG.14D). This indicated that the iPSCs with Pola1 inhibition induced stemness status. These results indicated that the Pola1 inhibition can be applied to significantly enhance the cellular reprogramming with faster dynamics and higher efficiency in human fibroblasts. 3. Materials and Methods

[0137] Fly strains and husbandry. Fly strains were raised on standard Bloomington media. All flies were raised at 25°C unless noted otherwise. The following fly strains were used: hs-flp on the X chromosome (Bloomington Stock Center BL-26902), nos-Gal4 (with VP16) on the 2ndchromosome, nos-Gal4 (without VP16 or ^VP16) on the 2ndchromosome [(from Yukiko Yamashita, Whitehead Institute, USA)], bam-Gal4 on the 3rdchromosome, bam-Gal80 on the 3rdchromosome (from Juliette Mathieu and Jean-René Huynh, Collège de France, France), UASp-FRT-H3-EGFP-FRT-H3-mCherry on the 2ndchromosome, polα50 P-element insertionATTORNEY DOCKET NO.36406.0038P1 (BL-27205), polα180 P-element insertion (BL-31805), pcna>EGFP-pcna and rpa>rpa-EGFP [from Eric Wieschaus, Princeton University, USA]. The UAS-grim flies (from Erika Matunis, Johns Hopkins School of Medicine, USA) were crossed with nanos-Gal4^VP16; tubulin- Gal80ts(from Yukiko Yamashita, Whitehead Institute, USA) for genetic ablation experiments to induce dedifferentiation (see below).

[0138] The polα50 P-element insertion (BL-27205) was verified by sequencing to be a null allele using the following primers: 5’-AGCTCCAATCGTGTATCTCTCT- 3’ (specific to the 5’ UTR of the polα50 gene locus) and 5’-CAATCATATCGCTGTCTCACTC- 3’ (specific to the P-element sequences of the EP insertion) were used to amplify the genomic sequences corresponding to the 5’ end of the polα50 gene locus, where the P-element insertion was located based on the Flybase ( / / flybase.org / ). Sequencing with this pair of primers confirmed that the P- element is inserted at a position nine base pairs downstream of the start codon, resulting in the new coding sequence 5’-ATGCCCGAAcatgatgaaataacataa (lowercase sequences indicate the P- element insertion). This sequence leads to eight codons followed by a stop codon (underlined). Hence, this allele results in an early stop codon that very likely represents a null loss-of-function allele of the polα50 gene. This allele is not homozygous viable and is maintained as a heterozygous stock over a balancer chromosome. All experiments using the polα50+ / -background were outcrossing the polα50 / Balancer stock to have the polα50 P-element insertion allele over a wild-type chromosome.

[0139] Generating knock-in fly strains. Endogenously tagged fly strains were generated by CRISPR-Cas9 with the genome editing service provided by Fungene Inc. (Beijing, China). The following strains were generated and used in this study: cdc45-mCherry (internally tagged between D163 and Q164), cdc45-3xHA (internally tagged between D163 and Q164), DNA polymerase ε 255kD subunit-3xHA (tagged at the C-terminus), DNA polymerase α 180kD-3xHA (tagged at the C-terminus), DNA polymerase δ-3xHA (tagged at the C-terminus), ctf4-eGFP (tagged at the C-terminus).

[0140] Heat shock scheme. Flies with UASp-FRT-H3-EGFP-FRT-H3-mCherry along with any relevant genotypes were crossed with hs-flp; nanos-Gal4 and raised at 25°C. Within two days of eclosure, adult male flies were transferred to a vial and the vial was submerged underwater at 37°C for 90 minutes. Flies were then recovered at 29°C for 18 hours prior to dissection for experiments, with the exception of experiments using the PolA1 inhibitor, as described below.ATTORNEY DOCKET NO.36406.0038P1

[0141] Whole mount immunostaining experiments. Immunostaining experiments were performed using standard procedure7. Primary antibodies used were DE-cadherin (10:200; DHSB AB_528120), γ-Tubulin (1:200; Sigma-Aldrich AB_T6557), Stat92E (1:500; from Denise Montell, University of Santa Barbara, CA, USA), VASA (1:500; from Ruth Lehmann, Whitehead Institute, USA) Armadillo (1:100; DSHB N27A1), Traffic Jam (1:100, from Mark Van Doren, Johns Hopkins University, USA), anti-PCNA (1:100; Santa Cruz sc-56), anti-GFP (1:1,000; Abcam ab 13970), anti-HA (1:200; Sigma-Aldrich H3663), anti-mCherry (1:1,000; Invitrogen M11217), anti-H3K27me3 (1:400; Millipore 07-449), anti- H4K20me2 / 3 (1:400; Abcam ab78517), anti-H3S10ph (1:2000; Cell Signaling Technology 9701), and anti- BrdU (1:200; Abcam ab6326). BrdU analog was Invitrogen B231515-bromo-2′-deoxyuridine (BrdU). Secondary antibodies were the Alexa Fluor-conjugated series (1:1,000; Molecular Probes). Confocal images were taken on the Zeiss LSM800 (with Airyscan mode) with a 63x oil objective lenses or on the Leica SPE with 63x oil immersion lenses.

[0142] Quantification of protein levels in the early germline. Images were analyzed using the ImageJ software FIJI. Germline cyst stages were identified using Arm signal to label the two cyst cells encapsulating each cyst. Average intensity values were recorded for the center Z-slice of each cell / nucleus of interest. For germ cells within one cyst, only one germline nucleus from the entire cyst was measured as one data point. For the comparison of protein levels of endogenously tagged proteins, immunostaining signals in GSCs, 4-cell and 8-cell SGs were measured, and a background was subtracted using the post-mitotic hub cells, which are devoid of any of these replication components’ signals. Signal intensity from 4-cell and 8-cell SGs were then normalized to the average intensity of GSCs from the same batch of testes. For the batch- based normalization, within one experimental batch, each data point is normalized to the average of WT GSCs in this corresponding batch. To compare data among different batches, the resulting values were then used to calculate the relative amount of GSC protein level to SG protein level (set to one (1) to facilitate comparison), and plot on a log2scale (FIG.1B). The dataset shown in FIG.1B are from germ cells at each corresponding differentiation stages. S- phase germ cells were labeled using a EdU pulse and quantified them separately. The results using S-phase germ cells were similar to using germ cells without distinguishing S-phase from G2-phase.

[0143] For the comparison of the stage-specificity of each driver or driver combination, nanos-Gal4 by itself, nos-Gal4ΔVP16; bam-Gal80 combination, or bam-Gal4 by itself wasATTORNEY DOCKET NO.36406.0038P1 crossed to the UASp-FRT-H3-eGFP-FRT-H3-mCherry transgene without hs-flp. The EGFP signals reflecting the relative strength of each driver or driver combination were quantified in the corresponding germline cyst stages, identified using Arm to label the two encapsulating cyst cells. The central slice of a representative nucleus was taken for each cyst measured as one data point. The cytoplasmic space was used as a background for subtraction. The EGFP signals were normalized to the stage with the highest relative signal intensity: For nanos-Gal4 by itself, all quantifications were normalized to the signals in GSCs; for the nos-Gal4ΔVP16; bam-Gal80 combination, all quantifications were also normalized to the signals in GSCs; for bam-Gal4 by itself, all quantifications were normalized to the signals in the 8-cell SGs.

[0144] S-Phase colocalization imaging and analysis. Even though mitotic GSCs display separation between old H3 and new H3 due to differential condensation of old H3- and new H3- enriched sister chromatids, their separation during interphase is more difficult to detect, due to the decondensed chromosomal status and the presence of nucleoplasmic free histones. To visualize potentially differential histone incorporation during S-phase, a clearance buffer was applied which effectively removes nucleoplasmic protein. Briefly, the clearance buffer is prepared by mixing 989µls of the clearance buffer stock solution (8.4 mM HEPES, 100 mM NaCl, 3 mM MgCl, 1 mM EGTA, 300 mM Sucrose, 2% Triton X-1000, and 2% BSA in ddH2O) with 1 µl DTT and 10 µl protease inhibitor (100x Leupeptin). After dissection, tissue samples were incubated in 10 μM EdU (Invitrogen Click-iT EdU Imaging Kit, catalog # C10340) for 15 minutes in Schneider’s media at room temperature. At the end of the 15 minutes, the Schneider’s media were drained and the clearance buffer was added for two minutes at 4°C in darkness. Samples were then fixed in 4% PFA, washed with 1xPBST, and then blocked in 3% BSA for 30 minutes. For robust signals, both the old H3-eGFP and new H3-mCherry were immunostained with antibodies (e.g., anti-eGFP and anti-mCherry) using standard procedures. The CLICK reaction was performed according to manufacturer’s instructions to label EdU. The DNA dye Hoechst was also added at this step.

[0145] Images were acquired on the Zeiss LSM800 using Airyscan mode on a 63x oil immersion objective. All samples were imaged using the identical settings. GSCs were identified by their proximity to the hub region. When 4-cell stage SGs were used, only one SG per cyst was analyzed to represent one data point. All images were analyzed using FIJI software. The Pearson score was recorded using the Coloc2 plugin for each nucleus, which was cropped to include just the nucleus as much as possible as delineated by the Hoechst signals. For each batchATTORNEY DOCKET NO.36406.0038P1 of images, the average measurement of the control GSCs was set to one (1) and the other treatments are normalized to control GSCs, in order to avoid batch variability. The resulting values are then used to calculate mean and standard error (Mean± SE).

[0146] Inhibitor treatment and analysis. For S-phase colocalization experiments using the inhibitor, flies were heat shocked as described above and left at 29°C to recover for 14 hours. Testes were then dissected and placed in incubation media for 4 hours to result in 18 total hours of heat shock recovery. After incubation with the inhibitor at the designated concentrations, these tissues were processed for S-phase colocalization analysis as described above.

[0147] Polα180 inhibitor (MedChemExpress Cat# HY-147812) was prepared in DMSO as stock and stored at -20°C (for short term) and -80°C (for long term) according to manufacturer’s instructions. Drug incubation were performed on testes in “live cell media” containing Schneider’s insect medium with 200 μg / ml insulin, 15% FBS by volume, and 0.6x pen / strep10. Prior to experiments, incubation media was prepared by diluting inhibitor solution (or DMSO vehicle) to the proper concentration in live cell media. Testes were dissected and placed in 100μl of incubation media as quickly as possible following dissection. Incubated testes were left in open tubes in darkness at room temperature (RT) for 4 hours. Because 4 hours is longer than the standard S-phase of the early male germline, all S-phase cells at the end of the incubation should have been exposed to the inhibitor for the entirety of their current S-phase.

[0148] For EdU incorporation, 20μM EdU was added to the incubation media for the last 15 minutes of the drug incubation before tissues were fixed. Only cells in early- to mid-S-phase were used for quantifications, as denoted by EdU staining covering all or most of the nucleus. Cells with focal EdU signal, indicative of late S-phase, were excluded to avoid skewing of the data. Germline nuclei were determined by endogenously tagged Vasa-mApple signals. Following imaging, EdU incorporation was quantified by measuring the mean EdU signal intensity in EdU-positive germline nuclei and subtracting the background measured from the nearby EdU-negative cells. When a cyst was considered, only one nucleus from each cyst was measured as one data point. Data shown (FIG.2B) are based on all early germline cells, as no significant difference of EdU incorporation was detected among GSCs, GBs, and SGs from the same sample.

[0149] Generation of chromatin fibers from the Drosophila male germline. Chromatin fibers were prepared as using known techniques. Briefly, after adding EdU to the testis samples and incubating for 15 minutes, lysis buffer was added (100 mM NaCl, 25 mM Tris-base, 0.2%ATTORNEY DOCKET NO.36406.0038P1 Joy detergent, pH=10). The testis tip was then micro-dissected on the slide and the rest of the testis was removed. Cells were allowed to fully lyse for approximately 5 minutes and then a Sucrose / Formalin (1M sucrose; 10% formaldehyde) solution was added and left for 2-minute to incubate, before a cover slip was gently placed on the top. The slide was then transferred to liquid nitrogen for 2 minutes before the cover slip was removed. The slide was then transferred to 95% EtOH for 10 min at -20°C in a freezer. Afterwards, the slide was fixed in 1% PFA for 1 minute. Samples were washed 3x in a Coplin jar with 1xPBST followed by blocking the sample with 3% BSA in 1xPBST for 30 minutes. Primary antibodies were then added for overnight incubation in a humidity chamber at 4°C. To assess histone asymmetry, anti-PCNA, anti- H3K27me3, and anti-GFP primary antibodies were added to chromatin fibers from the testes from the males with the following genotypes: each of the drivers (nos-Gal4 itself, nos- Gal4ΔVP16; bam-Gal80 combination, or bam-Gal4 itself) crossed with UASp-FRT-H3-EGFP- FRT-H3-mcherry without hs-flp. For cdc45-mCherry; DNA Polymerase-HA fibers, mCherry and HA primary antibodies were used. After the incubation with the primary antibodies, the slides are washed in a coplin jar with 1xPBS. Then the secondary antibodies were added and incubated for 2 hours at room temperature in a humidity chamber. The click chemistry was performed to label EdU following the manufacturer’s instruction. When DNA needs to be labeled, Hoechst is included at 1:1,000 to stain the samples. Additionally, for samples that need DNA labeling, ProLong™ Gold Antifade Mountant with DNA Stain DAPI (Thermo Fisher catalog # P36931) was used. For samples that do not need DNA labeling, ProLong Diamond mounting media without DAPI (Thermo Fisher catalog# P36961) was used.

[0150] Sequential labeling using EdU and BrdU analogs on DNA fibers. After sample dissection, 10 μM EdU was added for a 10-minute incorporation, followed by washing out EdU. BrdU was subsequently added for another 10 minutes. After this sequential labeling, DNA fibers were prepared using the same procedure as described above for chromatin fibers, with the exception of using a different lysis buffer to strip proteins from the DNA (200 mM Tris–HCl, pH 7.5, 50 mM EDTA, 0.5% SDS). The fibers were then treated with 1M HCl for 30 minutes at room temperature to expose the incorporated BrdU. After washing with 1xPBST, BrdU antibodies were added for incubation overnight at 4°C in a humidity chamber. Secondaries antibodies against the BrdU primary antibodies were then added for 2 hours at room temperature in a humidity chamber. The click reaction to recognize EdU was performed subsequently along with Hoechst incubation at 1:1000. Samples were then mounted in ProLong Diamond mountingATTORNEY DOCKET NO.36406.0038P1 media with DAPI. The EdU-positive DNA fibers representing regions that undergo DNA replication during EdU pulse (and thus have EdU on at least one side) were used for subsequent analyses as shown in FIG.6B.

[0151] Identifying and imaging replicative DNA fibers and chromatin fibers. All DNA fibers and chromatin fibers in this study were imaged with the Airyscan mode on a Zeiss LSM800 using a 63x oil immersion lens. Germline-derived chromatin fibers were identified using the H3-EGFP signal expressed with different germ cell-specific drivers or driver combination. Replicative regions were identified by both PCNA and EdU signals, or the presence of Cdc45, DNA Polymerase, and EdU. Fibers regions with detectable separation between sister chromatids were imaged and analyzed. Quality controls to select appropriate chromatin fiber regions for further analyses included fiber length, shape, and the molecular specificity of signals. For example, for quantifying old histone-enriched H3K27me3 with strandedness information, the EdU labeled fibers positive with PCNA, H3-EGFP and H3K27me3 signals were used. For analyzing the Cdc45 signals with DNA polymerases, fibers with EdU-labeling regions, clear Cdc45 and anti-HA signals were used.

[0152] For sequential EdU and BrdU labeled DNA fibers, two patterns were imaged and quantified at DNA regions that replicate during the EdU pulse (thus incorporating EdU on at least one side of the duplicated sister chromatids): First, regions with clear sister chromatid separation with Hoechst and EdU signals but no discernable BrdU signal. Second, regions with clear sister chromatid separation with clear Hoechst, EdU, and BrdU signals. For detailed description of the analyses of sister chromatids using chromatin fibers.

[0153] Quantification of DNA fibers and chromatin fibers. All images were analyzed using FIJI software. To quantify the asymmetry between sister chromatids, line plots were drawn on both strands, using the PCNA-enriched side to denote the lagging strand. Most fibers have relatively short separable regions (≤ 2^m), for which the entire fiber was used for quantification. For fibers with longer separable regions (> 2 μm), they were divided into 2μm- long non-overlapping segments along the length of the chromatin fiber and each of them was used for analyses. The region with no overlap with any of the chromatin fibers was used as background signal for subtraction from the measured signals from both strands. The ratio of signals = log2(leading strand signal̶ background signal) / (lagging strand signal̶ background signal).

[0154] For the sequential EdU- and BrdU-labeled DNA fibers, there is no strandednessATTORNEY DOCKET NO.36406.0038P1 indicator such as PCNA. As such, the strand with higher BrdU signals was used as the reference strand, allowing EdU signal to be independently measured, which could be on the same or the opposite strand. All quantifications were performed similar to the chromatin fibers, with the ratio of signals = log2 (BrdU-enriched strand signal̶ background signal) / (BrdU-depleted strand signal̶ background signal).

[0155] For the Cdc45- and DNA Polymerase-labeled fibers, the distance between Cdc45 signal and the HA signal (labeling either Pol^ or Pol^) was quantified from the center of the Cdc45 focus to the nearest HA signal.

[0156] A quantitative assay for chromosomal condensation state. An area-based method was used to monitor the chromosomal condensation state, using a dual-color histone transgene UASp-FRT-histone-eGFP -FRT-histone-mCherry. A maximum intensity projection was generated for old H3- (eGFP) and new H3- (mCherry) enriched areas. The intensity of each pixel was determined and scaled individually, setting the minimum intensity to 0 and the maximum to 65,535 (a 16-bit range). The pixels were monitored across the image with a threshold of 35% of the maximum intensity. Condensation kinetic profiles were generated to compare old H3- versus new H3-enriched regions by calculating the percentage of pixels above the threshold (the condensation parameter). Relative compaction index was measured and plotted by taking a ratio of the percentage of pixels of the new H3-enriched to the old H3- enriched regions.

[0157] Dedifferentiation assay. GSCs and early-stage germ cells (up to 4-cell spermatogonia) were depleted using genetic manipulation of the pro-apoptotic gene, grim. The UAS-grim flies (were crossed with nanos-Gal4^VP16; tubulin-Gal80tsand grown at 18°C to prevent grim expression, which is only turned on when shifting flies to 31°C to inactivate Gal80. The nanos-Gal4^VP16; tubulin-Gal80ts>UAS-grim flies were kept at 31°C for four days, which ablates early-stage germ cells (Abl in FIG.4B). After 4-day ablation, flies were recovered at 18°C for another four days, when grim expression was inhibited again (FIG.4C). After recovery, all GSCs including dedifferentiated GSC-like cells and bona fide GSCs were evaluated by immunostaining using anti-γ-Tubulin as the centrosome marker and anti-Stat92E as the stemness marker.

[0158] Centrosome orientation assay. Both dedifferentiated GSC-like cells and bona fide GSCs were evaluated using centrosome orientation as a criterion. Centrosomes were immunostained using anti-γ-Tubulin. Centrosome misorientation is defined as neither of the twoATTORNEY DOCKET NO.36406.0038P1 centrosomes being within the 90ohub–GSC interface (red in FIG.4K). Centrosomes were scored to be oriented when one of two centrosomes is within the 90ohub–GSC interface (FIG. 4K).

[0159] Male fertility assay. polα50 P-element insertion flies were maintained over a balancer on the 3rdchromosome. The polα50+ / -flies are from outcrossing the polα50 / Balancer stock with y,w flies at 25°C to generate the F1 progenies, where the polα50 P-element insertion allele is over a wild-type chromosome. The F1 polα50+ / -males were aged at 25°C for the mentioned period of time with females. Vials were flipped every five days to prevent the generation of the F2 progenies. As a control, newly enclosed y,w males were collected and aged simultaneously in separate vials. When male flies (polα50+ / -and y,w) reached the desired age, one male was put into a new vial with three virgin y,w females. These flies were allowed to mate for five days, and then parents were tossed away. Only those vials with all four live parents (1 male and 3 female) were retained for fertility assay with the 5-day mating period. If any of the four parental flies died during the 5-day mating period, those vials were excluded from data collection and analyses. All F1 progenies were counted for 15 days after tossing the parent flies as one data point.

[0160] Lifespan assay. The lifespan of polα50+ / -flies was assessed. Groups of 20 newly eclosed flies (10 males and 10 females) were placed in vials with fly food and yeast. The day the flies eclosed was considered Day 0, and flies were kept in a temperature-controlled 25°C incubator for their entire lifespan. Every 2-3 days, flies were flipped onto fresh fly food with dry yeast, and deceased flies were removed and counted. Prior to Day 30, if all the males or all the females in a vial died (leaving all remaining flies as the same sex), then new flies of the missing sex were added, but not counted in the lifespan assay, to maintain the effects of mating in the remaining flies. Because the polα50+ / -flies were made by outcrossing a balanced polα50P-element / Balancer line to y,w, the control flies used were y,w flies crossed to another wild-type strain Oregon R flies in order to approximate similar heterozygosity between the control and experimental flies. Deceased flies were counted with respect to sex, but no significant difference was observed for control versus experimental males or control versus experimental females.

[0161] Statistics and reproducibility. For all comparisons between two groups, Mann- Whitney tests were used unless otherwise noted. For one-group datasets, one sample t-test was used with a null hypothesis that the data is symmetrically distributed (e.g., ratio= 1 for datasets without logarithmic transformation, log2= 0 for logarithmically transformed data).ATTORNEY DOCKET NO.36406.0038P1 4. Discussion

[0162] A crucial molecular mechanism underlying asymmetric histone incorporation in stem cells is delayed lagging strand synthesis. In symmetrically dividing cells, comparable leading versus lagging strand syntheses give the old histone equal opportunities to be recycled by both strands (FIG.6H). In asymmetrically dividing germline stem cells, reducing lagging strand polymerase levels could delay lagging strand synthesis relative to the leading strand, which results in a pronounced temporal difference. This difference would bias old histone recycling by the leading strand whereas new histones infill to the lagging strand (FIG.6I). This model is consistent with a previous report that nucleosomes have the priority to be reincorporated by the double-stranded leading strand in vitro. The increased expression of RPA in stem cells could also facilitate this process (FIG.6I). This model is consistent with the previous results displaying abundant RPA bound to the lagging strand on chromatin fibers derived from early- stage germ cells. This mechanism alone is likely sufficient to induce asymmetric histone incorporation, as either reducing the expression of the key lagging strand polymerase or inhibiting its activity can induce stem cell-specific histone incorporation patterns even in non- stem progenitor cells. Surprisingly, these primed progenitor cells could functionally replace bona fide stem cells under both pathological and physiological conditions, indicating a new pathway for cell reprogramming in stem cell lineages.

[0163] In the Drosophila male germline, even though SGs with compromised Polα have S- phase replication-dependent histone incorporation patterns and M-phase differential chromosomal condensation patterns similar to those in GSCs, SGs do not reside in a polarized microenvironment like the “niche” for GSCs and there is no evidence that the microtubule organization centers, the centrosomes, have asymemtric activities in the wild-type SGs. Therefore, these asymmetries may not result in differences between the two daughter cells resulting from SG symmetric cell division, unlike the asymmetric GSC division. Additionally, the polα50+ / - SGs seem to undergo terminal differentiation properly, as there are no obvious germline defects detectable in the polα50+ / - testes. It is plausible that the transcriptome and other features of polα50+ / - SGs remain unchanged or with insignificant changes compared to the control SGs, despite the detectable changes of their chromatin architectures. This indicates that the epigenome potentiates cell fate change but may not be deterministic for such a decision. However, given the opportunities, such as forced or natural cell reprogramming, including both the dedifferentiation and the redifferentiation processes, a potentiated epigenome can empowerATTORNEY DOCKET NO.36406.0038P1 these cells to outperform control cells.

[0164] In this study, the Polα-primase complex was focused on because it predominantly acts on the lagging strand except the initial priming event on the leading strand and during rare re-priming events at stalled replication forks. Although Polα and RPA also play a role in chaperoning histones during replication-coupled nucleosome assembly, such activities have been demonstrated using specific mutations at their histone interacting domains. Here, the studies make use of genetic approaches that either compromise function or overexpress the full- length proteins. Additionally, the pharmacological method utilizes enzyme inhibitors whose effects are more related to their roles as replication machinery components, such as primer elongation, rather than chaperones. Notably, DNA replication inhibitors are often used to target and kill over-proliferative cancer cells. Indeed, the drug adarotene and its derivative molecule used in this study have been shown to have anti-cancer properties in mice. The finding that a low dose of lagging strand inhibitor can not only be tolerated but also confer regenerative properties for the progenitor cells could present a prudent consideration for clinical applications. For example, if Polα inhibitors are not delivered to tumors at a sufficiently high concentration, the inhibitor may promote the establishment of a cancer stem cell-like status, which could become more potent and ultimately exacerbate cancer progression. On the other hand, in a normal stem cell lineage, the results that Polα inhibitor can induce stem cell-like chromatin structure in progenitor cells in a dosage-dependent manner presents a very exciting potential pharmaceutical approach to stimulate stem cell regeneration following stem cell loss during injury or aging. Excitingly, it has been shown that DNA replication speed underlies cell fate change in early mouse embryogenesis and reducing this speed promotes cellular reprogramming toward totipotency.

[0165] Finally, DNA replication is fundamentally an inherently asymmetric process wherein the synthesizing processes of the leading strand versus the lagging strand are widely divergent. Previous studies have shown examples of uncoupled leading strand versus lagging strand syntheses in bacteria and cultured cells, particularly in cases where Polα or its priming activity is compromised. It has long been recognized that the leading strand versus the lagging strand may have the potential to differentially incorporate nucleosomes. It has been shown that the old histone-enriched H3K9me3 is recycled by the leading strand at the retrotransposon element to repress its transcription in S-phase mouse embryonic stem cells. Here, the results indicate that the inherent asymmetry of DNA replication itself could be utilized to differentially regulateATTORNEY DOCKET NO.36406.0038P1 histone incorporation and this process displays stage specificity within an endogenous adult stem cell lineage. These results point to a very exciting possibility that developmentally programmed expression of key DNA replication components could regulate the establishment of distinct epigenomes in a cell type- and stage-specific manner. Given that replication components as well as histone proteins and their respective modifications are highly conserved, exploring how this mechanism may be utilized in other developmental contexts across different multicellular organisms could be a very intriguing research direction. These elegant and efficient mechanisms could be used to balance differential versus equal epigenome establishment in asymmetrically versus symmetrically dividing cells, which could then impact plasticity versus fidelity in cell fate decisions during development, homeostasis and tissue regeneration. B. Example 2 1. Experimental Design

[0166] Previously, it was shown that the PolA1 inhibitor of the lagging strand DNA replication Polymerase α could improve the reprogramming of human dermal fibroblast cells, accelerating reprogramming and increasing efficiency. To determine whether the PolA1 inhibitor also improves reprogramming in other cell types, human peripheral blood mononuclear cells (PBMCs) were used for iPSC reprogramming (FIG.15A). The concentration of the PolA1 inhibitor was optimized in PBMCs to minimize disruption of DNA replication FIG.15B). Next, the PBMCs were treated with two doses of PolA1 inhibitor during iPSC reprogramming. The colony-forming efficiency of the resulting human iPSCs was assessed and characterized as described below. 2. Results

[0167] Compared to the control reprogramming condition with Yamanaka factors (Oct3 / 4, Sox2, Klf4, and L-Myc), it was found that PBMCs, which have a low proliferative capacity, treated with the PolA1 inhibitor produced iPSCs with higher efficiency. This was evident from the earlier formation of iPSC colonies and larger colony size at transition time points (Day 9 and Day 11) during reprogramming (FIG.15C). These results indicate that PolA1 inhibition enhances reprogramming efficiency from human PBMCs to iPSCs. Furthermore, live staining with the stem cell marker TRA1-60 validated the pluripotency of the generated human iPSCs in both the control and PolA1 inhibitor-treated groups, indicating that PolA1 inhibition does not affect stemness (FIG.15D). Dose-dependent effects were also observed on iPSC formation. Cells treated with a higher dose (100 nM) of PolA1 inhibitor generated larger and more coloniesATTORNEY DOCKET NO.36406.0038P1 compared to those treated with a lower dose (10 nM) or those with no PolA1 treatment (FIG. 15C-D).

[0168] In conclusion, PolA1 inhibition enhances iPSC generation from human PBMCs, accelerating the process and improving efficiency.

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Tarayrah, L., Li, Y., Gan, Q. & Chen, X. Epigenetic regulator Lid maintains germline stem cells through regulating JAK-STAT signaling pathway activity. Biol Open 4, 1518- 27 (2015). 20. Tulina, N. & Matunis, E. Control of stem cell self-renewal in Drosophila spermatogenesis by JAK-STAT signaling. Science 294, 2546-9 (2001). 21. Piper, M.D. & Partridge, L. Protocols to Study Aging in Drosophila. Methods Mol Biol 1478, 291-302 (2016). 22. Cincinelli, R. et al. Novel adamantyl retinoid-related molecules with POLA1 inhibitory activity. Bioorg Chem 104, 104253 (2020). 23. Xie, J. et al. Histone H3 Threonine Phosphorylation Regulates Asymmetric Histone Inheritance in the Drosophila Male Germline. Cell 163, 920-33 (2015).

Claims

ATTORNEY DOCKET NO.36406.0038P1 CLAIMS We claim:

1. A method of decreasing lagging strand synthesis in a cell comprising contacting a cell with a DNA polymerase α (DNA polα) inhibitor and / or a primase inhibitor.

2. The method of claim 1, wherein the cell is in culture.

3. The method of claim 1, wherein the cell is in a subject.

4. The method of any one of claims 1-3, wherein the DNA polα inhibitor or primase inhibitor decreases DNA polα levels or primase levels in the cell.

5. The method of any one of claims 1-3, wherein the DNA polα inhibitor or primase inhibitor inhibits DNA polα DNA synthesis activity or primase activity in the cell.

6. The method of any one of claims 1-5, wherein the DNA polα inhibitor or primase inhibitor does not affect an S phase of a cell cycle in the cell.

7. The method of any one of claims 1-6, wherein the DNA polα inhibitor or primase inhibitor does not cause cell cycle arrest in the cell or cell death of the cell.

8. The method of any one of claims 1-7, further comprising administering a composition that increases Replication Protein-A 70 levels in the cell.

9. The method of any one of claims 1-8, wherein leading strand synthesis is not altered in the cell.

10. The method of any one of claims 1-9, wherein the cell is a progenitor cell.

11. The method of claim 10, wherein the progenitor cell is a fibroblast cell or intestinal cell.

12. The method of claim 10, wherein the progenitor cell is a progenitor spermatogonial cell.

13. A method of reprogramming a progenitor cell comprising administering a DNA polymerase α (DNA polα) inhibitor and / or primase inhibitor to the progenitor cell, thereby reprogramming the progenitor cell to form a reprogrammed progenitor cell.ATTORNEY DOCKET NO.36406.0038P1 14. The method of claim 13, wherein the reprogrammed progenitor cell comprises a separable old histone versus new histone pattern.

15. The method of claim 14, wherein the old histone versus new histone pattern is an old histone H3 versus new histone H3 pattern.

16. The method of any one of claims 13-15, wherein old histone versus new histone enriched chromosomal regions display differential condensation in the dividing reprogrammed progenitor cell.

17. The method of any one of claims 13-16, wherein the progenitor cell is a fibroblast cell or intestinal cell.

18. The method of any one of claims 13-16, wherein the progenitor cell is a progenitor spermatogonial cell.

19. The method of any one of claims 13-18, wherein the DNA polα inhibitor or primase inhibitor reduces DNA polα levels and / or primase levels.

20. The method of any one of claims 13-18, wherein the DNA polα inhibitor or primase inhibitor inhibits DNA polα DNA synthesis activity.

21. The method of any one of claims 13-20, wherein the DNA polα inhibitor or primase inhibitor is administered at a concentration that does not affect an S phase of a cell cycle.

22. The method of any one of claims 13-21, wherein the DNA polα inhibitor or primase inhibitor is administered at a concentration that does not cause cell cycle arrest or cell death.

23. The method of any one of claims 13-22, wherein the cell is in culture.

24. The method of any one of claims 13-22, wherein the cell is in a subject.

25. A method of reducing DNA polymerase α and / or primase levels or activity in a cell comprising contacting a cell with a DNA polymerase α (DNA polα) inhibitor and / or a primase inhibitor.

26. The method of claim 25, wherein the cell is a progenitor cell.ATTORNEY DOCKET NO.36406.0038P1 27. The method of any one of claims 25-26, wherein the reduction of DNA polymerase α or primase levels or activity reprograms the cell.

28. The method of any one of claims 25-27, wherein the reduction of DNA polymerase α or primase levels or activity disrupts lagging strand synthesis in the cell.

29. The method of any one of claims 25-28, wherein the DNA polα inhibitor or primase inhibitor does not affect an S phase of a cell cycle in the cell.

30. The method of any one of claims 25-29, wherein the DNA polα inhibitor or primase inhibitor does not cause cell cycle arrest in the cell or cell death of the cell.

31. A method of altering chromatin in a progenitor cell comprising contacting a progenitor cell with a DNA polymerase α (DNA polα) inhibitor and / or primase inhibitor to the cell.

32. The method of claim 31, wherein altering chromatin comprises a separable old histone versus new histone pattern in the progenitor cell.

33. The method of any one of claims 31-32, wherein altering chromatin comprises altering the orientation of centrosomes in the progenitor cell.

34. The method of any one of claims 31-33, wherein the DNA polα inhibitor or primase inhibitor does not affect an S phase of a cell cycle in the progenitor cell.

35. The method of any one of claims 31-34, wherein the DNA polα inhibitor does not cause cell cycle arrest in the progenitor cell or cell death of the progenitor cell.

36. The method of any one of claims 31-35, wherein the progenitor cell is a fibroblast cell or intestinal cell.

37. The method of any one of claims 31-35, wherein the progenitor cell is a progenitor spermatogonial cell.

38. A method of decreasing lagging strand synthesis in a cell in a subject comprising administering a therapeutically effective amount of a DNA polymerase α (DNA polα) inhibitor and / or primase inhibitor to the subject.ATTORNEY DOCKET NO.36406.0038P1 39. The method of claim 38, wherein the DNA polα inhibitor or primase inhibitor decreases DNA polα levels or primase levels in the cell.

40. The method of any one of claims 38-39, wherein the DNA polα inhibitor or primase inhibits DNA polα DNA synthesis activity in the cell.

41. The method of any one of claims 38-40, wherein the DNA polα inhibitor or primase inhibitor does not affect an S phase of a cell cycle in the cell.

42. The method of any one of claims 38-41, wherein the DNA polα inhibitor or primase inhibitor does not cause cell cycle arrest in the cell or cell death of the cell.

43. The method of any one of claims 38-42, further comprising administering a composition that increases Replication Protein-A 70 levels in the cell.

44. The method of any one of claims 38-43, wherein leading strand synthesis is not altered in the cell.

45. The method of any one of claims 38-44, wherein the cell is a progenitor cell.

46. The method of claim 45, wherein the progenitor cell is a fibroblast cell or intestinal cell.

47. The method of claim 45, wherein the progenitor cell is a progenitor spermatogonial cell.

48. A method of reprogramming a progenitor cell in a subject comprising administering a therapeutically effective amount of a DNA polymerase α (DNA polα) inhibitor or primase inhibitor to the subject, thereby reprogramming the progenitor cell in the subject to form a reprogrammed progenitor cell.

49. The method of claim 48, wherein the reprogrammed progenitor cell comprises a separable old histone versus new histone pattern.

50. The method of claim 49, wherein the old histone versus new histone pattern is an old histone H3 versus new histone H3 pattern.ATTORNEY DOCKET NO.36406.0038P1 51. The method of any one of claims 48-50, wherein old histone versus new histone enriched chromosomal regions display differential condensation in the reprogrammed progenitor cell.

52. The method of any one of claims 48-51, wherein the progenitor cell is a fibroblast cell or intestinal cell.

53. The method of any one of claims 48-52, wherein the progenitor cell is a progenitor spermatogonial cell.

54. The method of any one of claims 48-53, wherein the DNA polα inhibitor or primase inhibitor reduces DNA polα levels.

55. The method of any one of claims 48-54, wherein the DNA polα inhibitor or primase inhibitor inhibits DNA polα DNA synthesis activity.

56. The method of any one of claims 48-55, wherein the DNA polα inhibitor or primase inhibitor is administered at a concentration that does not affect an S phase of a cell cycle.

57. The method of any one of claims 48-57, wherein the DNA polα inhibitor or primase inhibitor is administered at a concentration that does not cause cell cycle arrest or cell death.

58. A method of reducing DNA polymerase α and / or primase levels or activity in a cell in a subject comprising administering a therapeutically effective amount of a DNA polymerase α (DNA polα) inhibitor or primase inhibitor to the subject.

59. The method of claim 58, wherein the cell is a progenitor cell.

60. The method of any one of claims 58-59, wherein the reduction of DNA polymerase α or primase levels or activity reprograms the cell.

61. The method of any one of claims 58-60, wherein the reduction of DNA polymerase α or primase levels or activity disrupts lagging strand synthesis in the cell.

62. The method of any one of claims 58-61, wherein the DNA polα inhibitor or primase inhibitor does not affect an S phase of a cell cycle in the cell.ATTORNEY DOCKET NO.36406.0038P1 63. The method of any one of claims 58-62, wherein the DNA polα inhibitor or primase inhibitor does not cause cell cycle arrest in the cell or cell death of the cell.

64. A method of altering chromatin in a progenitor cell in a subject comprising administering a therapeutically effective amount of a DNA polymerase α (DNA polα) inhibitor or primase inhibitor to the subject.

65. The method of claim 64, wherein altering chromatin comprises a separable old histone versus new histone pattern in the progenitor cell.

66. The method of any one of claims 64-65, wherein altering chromatin comprises altering the orientation of centrosomes in the progenitor cell.

67. The method of any one of claims 64-66, wherein the DNA polα inhibitor or primase inhibitor does not affect an S phase of a cell cycle in the progenitor cell.

68. The method of any one of claims 64-67, wherein the DNA polα inhibitor or primase inhibitor does not cause cell cycle arrest in the progenitor cell or cell death of the progenitor cell.

69. The method of any one of claims 64-68, wherein the progenitor cell is a fibroblast cell or intestinal cell.

70. The method of any one of claims 64-69, wherein the progenitor cell is a progenitor spermatogonial cell.

71. A method of increasing fertility of a subject comprising administering a therapeutically effective amount of a DNA polymerase α (DNA polα) inhibitor and / or a primase inhibitor to the subject.

72. A method of increasing reproductive lifespan of a subject comprising administering a therapeutically effective amount of a DNA polymerase α (DNA polα) inhibitor and / or a primase inhibitor to the subject.

73. The method of any one of claims 71-72, wherein the DNA polα inhibitor or primase inhibitor reduces the number of germline stem cells having misoriented centrosomes.

74. The method of any one of claims 71-73, wherein the subject is male or female.ATTORNEY DOCKET NO.36406.0038P1 75. The method of any one of claims 71-74, wherein the therapeutically effective amount of a DNA polα inhibitor or primase inhibitor comprises a concentration of a DNA polα inhibitor or primase inhibitor that does not affect an S phase of a cell cycle.

76. The method of any one of claims 71-75, wherein the therapeutically effective amount of a DNA polα inhibitor or primase inhibitor comprises a concentration of a DNA polα inhibitor or primase inhibitor that does not cause cell cycle arrest or cell death.

77. The method of any one of claims 71-76, wherein the DNA polα inhibitor or primase inhibitor reduces DNA polα or primase levels.

78. The method of any one of claims 71-77, wherein the DNA polα inhibitor or primase inhibitor inhibits DNA polα DNA synthesis activity or primase activity.

79. The method of any one of claims 71-78, wherein leading strand synthesis is not altered in a cell in the subject.

80. The method of any one of claims 71-79, wherein the cell is a progenitor cell.

81. The method of claim 80, wherein the progenitor cell is a progenitor spermatogonial cell.

82. The method of any one of claims 1-811, wherein the DNA polα inhibitor is POLA1 inhibitor 1, Aphidicolin, 18 β-Glycyrrhetinic Acid, 1-β-D-Arabinofuranosylcytosine.

83. The method of any one of claims 1-82, wherein the primase inhibitor is amenamevir, pritelivir, BILS 22 BS, or T157602.

84. A method of treating a subject comprising administering a therapeutically effective amount of a DNA polymerase α (DNA polα) inhibitor or primase inhibitor to the subject.

85. The method of claim 84, wherein the subject has infertility problems or gut inflammation.

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