Genes & Development

While many studies of developmental control have focused on gene activation, less is known about the extent to which regulatory programs are actively repressed in progenitor cells. We previously showed that trimethylation of histone H3 at lysine 9 (H3K9me3) is a repressive mark that is remodeled on protein-coding genes when endodermal progenitors transition to liver and pancreatic {beta} cell fates. Yet whether H3K9me3 is dynamic at promoters and enhancers has not been determined. Here we find that promoters of liver-specific genes are strongly enriched for H3K9me3 in undifferentiated progenitors, whereas such enrichment is not observed at promoters of more broadly expressed liver genes. We further show that enhancers specific to differentiated tissues--including liver, islet, and cerebral cortex--are strongly enriched for H3K9me3 in their corresponding tissue stem and progenitor cells. In hepatoblasts, H3K9me3 contributes to maintaining the undifferentiated state by restricting FOXA2 and HNF4 from binding to most enhancers, while there remain thousands of H3K9me3-marked enhancers where the factors are not restricted from binding. Our findings illustrate how H3K9me3-mediated heterochromatinization can restrict transcription factor engagement in progenitor cells to prevent inappropriate activation during early development. H3K9me3 at enhancers that allow transcription factor binding may reflect developmental competence.

While DNA replication forks initiate in S phase, they do not necessarily complete and terminate prior to cell entry into mitosis. How mitotic proteins regulate leftover replication forks is not well-understood. Using reconstituted DNA replication forks with purified proteins, we show that the budding yeast mitotic kinases Clb2-CDK (M-CDK) and Cdc5 (Plk1 homolog) phosphorylate and regulate several replication elongation proteins. Mrc1 phosphorylation by both kinases results in slower replication, and Pol phosphorylation by M-CDK results in less lagging strand initiation. We further show that a phospho-resistant mutant of Pol bypasses M-CDK inhibition of Pol activity in reconstituted replication reactions. Yeast cells expressing the phospho-resistant mutant exhibit faster cell cycle progression revealing a potential negative feedback mechanism between DNA replication forks and mitotic progression.

Neuronal maturation is associated with extensive changes in gene expression and chromatin organization. However, the molecular mechanisms that control the epigenetic landscape in terminally differentiated neurons remain poorly understood. Here, we show that maturing cerebellar granule cells undergo a striking and specific increase in the levels of the repressive histone modification H3K27me3 across different genomic regions, including individual genes, broad intergenic regions, and gene clusters. The accumulation of H3K27me3 coincides with a developmental switch from EZH2 to EZH1 and colocalizes with H3K36me2 and DNA non-CpG methylation. Using mice with a conditional deletion in the catalytic domain of EZH1, we demonstrate that the maintenance of H3K27me3 in mature neurons depends on EZH1. Unexpectedly, an almost complete loss of H3K27me3 in postmitotic GCs induces minimal changes in gene expression and chromatin accessibility at 7 months of age. Using single-nucleus RNA sequencing (snRNAseq) from the mouse neocortex, we show that, similarly to GCs, the loss of EZH1-mediated H3K27me3 also has a minimal impact on cortical neuron gene expression. The amino acid composition of EZH1 suggests reduced sensitivity to H3K36 methylation, providing a potential basis for its activity in chromatin contexts that are not permissive for EZH2. Together, our results show that a postmitotic switch from EZH2 to EZH1 establishes novel chromatin domains in neurons with a minimal role in transcriptional maintenance.

25S nonfunctional RNA decay (NRD) eliminates 60S ribosomal subunits carrying inactivating mutations in the RNA. However, how cells identify defective subunits has not been described. We recently showed that the zinc-finger protein Reh1 is the last assembly factor to be released from a nascent 60S subunit. We now show that in yeast Reh1 is required for the degradation of 25S NRD substrates. 25S rRNAs carrying mutations in the catalytic center, A2820G or U2954A (A2451 and U2585, respectively in E coli numbering), are unstable in wildtype cells but are fully stabilized when REH1 is deleted. However, not all 25S rRNA mutations are recognized by Reh1. Ribosomes with a truncated L1 stalk engage in translation but cannot support viability. These ribosomes display a half-life indistinguishable from wild-type rRNA, suggesting that yeast does not have a robust surveillance system for such mutant ribosomes. Deletion of REH1 also has no impact on the levels of defective 18S rRNA. These results indicate that Reh1 and 25S NRD are specific for mutations in or near the catalytic center of the ribosome.

The discovery of neuromesodermal progenitors (NMPs) -- a bipotent progenitor population in the tailbud that gives rise to traditionally ectodermal and mesodermal tissues -- has disrupted the classical view that progenitors of the three distinct germ layers are exclusively segregated during gastrulation. However, until now the notion of lineage restriction of the endoderm to traditional gastrointestinal and respiratory tissues has largely remained intact. Here, we describe our discovery of a unique subpopulation in the chick endoderm that initially lines the ventral surface of the posterior organizer (Hensens node), but at the trunk-to-tail developmental switch, undergoes an FGF-dependent epithelial-to-mesenchymal transition, invading the tailbud and subsequently differentiating into a remarkably broad range of cell types including somites, notochord, and neural tube. Strikingly, ablation of this endodermal cell population results in a severe ([~]50%) reduction in axis elongation rate. Through single cell RNA sequencing and in situ hybridization chain reaction, we conclude that these cells lose their endodermal identity upon ingression, giving rise to NMPs that are biased toward mesodermal fates. Lineage tracing reveals that the node endoderm harbors a mixed multipotent population of progenitor cells capable of generating progeny that span endoderm and mesoderm or endoderm and ectoderm. These findings illustrate a previously unappreciated endodermal source of NMPs, and further demonstrates the breakdown of traditional lineage restriction of germ layers in the posterior embryo.

Although MTBP is essential for replication origin firing, we show here that strong depletion of MTBP can have minor effects on DNA replication rates. This suggests an adaptive process in the DNA replication program, so we examined mechanisms underlying this plasticity. Using an auxin-inducible degron to deplete MTBP, we found that acute suppression of MTBP blocked DNA replication, but that replication rates recovered over time. The timing of this recovery paralleled S phase expression of Cyclin B1, and inhibition of CDK1-Cyclin B1 prevented the recovery. Recovery did not involve restoration of origin firing; instead, replication recovered through accelerated fork progression. Consistent with CDK1 driving this acceleration, ATR inhibition, which activates CDK1, stimulated DNA replication in MTBP-depleted cells through CDK1-dependent increased fork progression rather than increased origin firing. Knockdown of RIF1, a known CDK1 target, phenocopied this effect. Although RIF1 is best known for opposing DDK-dependent MCM phosphorylation at origins, we find that RIF1 knockdown stimulates replication even when DDK is inhibited. Furthermore, RIF1 loss increased replication by accelerating fork progression rather than increasing origin firing. Together, these findings reveal a CDK1-RIF1-dependent mechanism that promotes fork speed during S phase and defines a form of replication plasticity in which fork rate compensates for reduced origin firing. SIGNIFICANCE STATEMENTAccurate genome duplication requires thousands of replication origins to fire and replication forks to complete DNA synthesis on schedule. When origin firing is compromised, it is unclear how cells avoid replication failure. We show that cells adapt to persistent loss of the origin-firing factor MTBP by accelerating replication fork progression through a CDK1-RIF1-dependent mechanism, partially compensating for reduced initiation. This adaptive response defines a form of replication plasticity in which cells rebalance origin usage and fork speed to sustain DNA synthesis. This mechanism may be especially relevant in cancer cells or other contexts where replication initiation is chronically stressed.

Faithful genome inheritance during meiosis relies on crossover repair of double-strand DNA breaks (DSBs) to connect homologous chromosomes and direct their proper segregation. The formation of crossover-specific recombination intermediates and accumulation of pro-crossover factors occurs at an extremely limited subset of DSB sites, necessitating that the subset of recombination sites designated to become crossovers reliably mature into crossovers. Here we identify C. elegans disordered protein COSA-2 as crucial for meiotic crossover maturation. COSA-2 abruptly concentrates at crossover intermediates in late pachytene nuclei, where it colocalizes and associates with other pro-crossover factors. COSA-2 is dispensable for early loading of crossover factors and for crossover designation, but is required for maintenance of pro-crossover factors at crossover-designated sites and for focal enrichment of factors initially distributed throughout the synaptonemal complex. We define a COSA-2 execution point during late pachytene wherein crossover intermediates transition from a vulnerable state (in which they require COSA-2 to avoid being dismantled) to a state where COSA-2 and local crossover-factor enrichment are no longer required to connect homologs. We propose that COSA-2 scaffolds privileged DNA repair compartments that promote crossover-factor accumulation and protect crossover intermediates until completion of repair, thereby ensuring that crossover-designated sites reliably mature into crossovers.

In animals, plants, and some fungi, Polycomb Repressive Complex 2 (PRC2) catalyzes trimethylation of histone H3 lysine 27 (H3K27me3) to establish transcriptionally repressed chromatin. Here, we identify the histone acetyltransferase RTT109 as a key regulator of PRC2-repressed domains in the model fungus Neurospora crassa. Although RTT109 interacts with the VPS75 homolog Nucleosome Assembly Factor 2 (NAF-2), we show that proper structure and function of PRC2-methylated chromatin require RTT109 catalytic activity but are independent of NAF-2 and H3K56 acetylation. We further demonstrate that H3K27me3 can be stably propagated over multiple rounds of mitosis in the absence of sequence-specific PRC2 targeting, and that RTT109 is essential for maintenance of the repressed state. These findings uncover a replication-linked mechanism for epigenetic memory and establish RTT109 as a key regulator of Polycomb-mediated chromatin inheritance.

In mammals, a small population of spermatogonial stem cells (SSCs) is established shortly after birth. These cells self-renew and produce sperm for the entirety of a males reproductive lifespan, passing the genome on to the next generation. Thus, establishment of a population of SSCs with high genomic integrity is essential. SSCs are derived from a much larger precursor population of male germ cells (MGCs) that differentiate during fetal life. During the last third of gestation, MGCs undergo a prolonged period of G0 cell cycle arrest during which they sustain high levels of transcription and acquire epigenetic programming for SSC fate. Although these differentiation steps can cause cellular and genomic damage, it has been unclear whether selection for germ cell quality occurs during G0 arrest since no classic markers of cell death have been detected. In this study, we utilize a mouse model to characterize a population of MGCs that begin to accumulate markers if cell death, such as AnnexinV (AnV) and propidium iodide (PI), at E16.5. The AnV- and PI-positive MGC population is characterized by low expression of the RNA-binding protein, Dead End 1 (DND1), and exhibit dsDNA breaks and mitochondrial dysfunction. Interestingly, we do not see evidence of an active cell death cascade until the time of birth, where we see phosphorylation of MLKL, a hallmark of a necroptotic cell death mechanism. Based on these findings, we propose that variable cellular health is an important basis for selection of the SSC precursors. Significance StatementSpermatogonial stem cells (SSCs) are essential for reproductive fitness, yet how their precursors are selected during development is not known. Utilizing a mouse model, this study describes high levels of cellular damage within a subset of male germ cells (MGCs) during G0 arrest. The damaged MGC population was marked by low expression of the RNA-binding protein, DND1, and was strongly associated with mitochondrial dysfunction and dsDNA breaks. We observed signs of non-apoptotic cell death by embryonic day (E)16.5 and the appearance of necroptotic markers in MGCs at the time of birth. This study uncovers previously unknown heterogeneity in the MGC pool and points to MGC health as an important source of selection during G0 arrest.

Cellular homeostasis relies on the organization of RNA and protein into membrane-less organelles. Stress granules (SGs) are well-known hubs for translational repression during cellular stress. Similar condensates formed by RNA-binding proteins such as FUS-- mutations in which cause amyotrophic lateral sclerosis (ALS)--remain poorly characterized relative to canonical SGs. Here, we develop Granule-seq, a microcapillary-based granule-resolved RNA sequencing approach, and demonstrate that SGs and FUS condensates are functionally distinct RNA compartments rather than variants of a unified granule class. Granules were individually aspirated, analyzed in small pooled sets, and validated through overlap with known SG components (794/2,759 genes, 28.8%, p < 0.001). Gene Ontology analysis revealed that G3BP-positive SGs sequester transcripts for stress responses and translational control, while FUS condensates are enriched for transcripts essential for synaptic function and neuronal development. Direct comparison showed that 63% of FUS-enriched and 82% of G3BP-enriched genes were granule-specific, with only 493 genes shared. Exploratory sequence analysis revealed modest contributions from compositional features, with functional identity providing primary selectivity. As a proof-of-concept study with limited biological replication, our results suggest that distinct granule identities are established through functionally specialized transcriptomes, a process that may be disrupted in ALS, and provide a framework for understanding RNA sorting. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=112 SRC="FIGDIR/small/726389v1_ufig1.gif" ALT="Figure 1"> View larger version (55K): org.highwire.dtl.DTLVardef@b867dforg.highwire.dtl.DTLVardef@ab89d9org.highwire.dtl.DTLVardef@1e71c78org.highwire.dtl.DTLVardef@1fe23e0_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphic abstract.C_FLOATNO Granule-seq workflow and key findings.(Left) Individual cytoplasmic granules are isolated by microcapillary aspiration, pooled ([~]10 granules per replicate), and processed for RNA sequencing alongside total-cell input. (Middle) Venn diagram showing the overlap between FUS condensate-enriched (1,334 genes) and G3BP1 stress granule-enriched (2,759 genes) transcripts. FUS condensates preferentially recruit neuronal and synaptic transcripts; G3BP1 stress granules are enriched for stress-response and proteostasis transcripts. (Right) Two-step recruitment model: shared sequence features (long 3'UTR, GC-rich composition, stable RNA structure) provide permissiveness for granule entry (Step 1), while RBP interactome composition and functional context determine granule-type specificity (Step 2). C_FIG

Liver injury induces rapid transcriptional responses in hepatocytes, yet the chromatin features that distinguish injured hepatocytes from healthy hepatocytes remain poorly understood. Using an integrated functional genomics approach combining bulk RNA-seq, ATAC-seq, and CUT&Tag profiling of H3K27ac and H3K27me3, we define the transcriptional and chromatin landscape of Sox9-expressing hepatocytes, which exhibit gene expression consistent with both hepatocyte and biliary identity. Under homeostatic conditions, Sox9+ hybrid hepatocytes (HybHeps) are rare and confined to the periportal space, while chronic injury induces an expansion of Sox9+ metaplastic hepatocytes (MetHeps). We identify three classes of differentially expressed genes associated with injury-responsive, state-associated, or shared regulatory programs and demonstrate that these classes are governed by distinct chromatin mechanisms. Injury-responsive transcription is driven primarily by dynamic chromatin accessibility remodeling at NF-{kappa}B- and AP-1-enriched regulatory elements, while state-associated and shared programs are reinforced through selective H3K27ac and H3K27me3 modification with comparatively stable accessibility. Relative to conventional hepatocytes, HybHeps encode a permissive chromatin landscape at injury-responsive loci under homeostatic conditions, consistent with epigenetic priming that facilitates rapid inflammatory activation. Projection of mouse-derived gene programs onto a human liver single-cell atlas encompassing both healthy and diseased hepatocytes confirms that SOX9-expressing hepatocytes preferentially engage injury-associated inflammatory modules while attenuating hepatocyte metabolic identity programs. Together, these findings define a chromatin-based regulatory dichotomy between inflammatory responsiveness and hybrid hepatocyte cell state stability, providing mechanistic insight into how differentiated epithelial cells integrate inflammatory signals while preserving cell state.

Centromeres are essential for chromosome segregation, yet in many genomes they are composed entirely of rapidly evolving repetitive DNA, embedded in other repetitive DNA that forms pericentromeric heterochromatin. Due to the difficulties of manipulating these repeat-rich regions, how the relative size of pericentromeric repeat regions influences chromosome segregation remains an open question. Here, we take advantage of the tractable Schizosaccharomyces pombe system by combining population-level analysis, complete long-read assemblies, and engineered near-isogenic strains to test how pericentromeric repeat copy number affects chromosome biology in its native context. We find that pericentromeric dh/dg arrays on chromosome 3 vary almost tenfold in size among natural S. pombe isolates, ranging from 35 to 265 kb. We converted this natural diversity into an experimental system of nearly isogenic strains that primarily differ in pericentromere size (35 to >350 kb). We found that pericentromere size does not alter baseline growth under standard conditions. However, larger pericentromeres alter transcriptional output and sensitize cells to spindle stress. We show that this spindle-stress phenotype depends on heterochromatin: loss of the H3K9 methyltransferase Clr4 abolishes size-dependent differences, whereas artificial targeting of the Chromosomal Passenger Complex to heterochromatin partially rescues the defect. Thus, we find that larger pericentromeres act as sinks for limiting regulatory factors, weakening their effective concentration at centromeres and compromising faithful chromosome segregation under stress. These results establish that naturally occurring copy-number variation within repetitive pericentromeric DNA is not merely noise, but a functional source of variation in chromosome segregation and gene regulation. Our work provides an experimentally tractable framework for understanding how repeat expansion in centromere-proximal heterochromatin influences chromosome behavior across eukaryotes.

KLF14 acts as a master regulator of gene expression in adipose tissue and variants at the human KLF14 gene show strong and reproducible association with type 2 diabetes and metabolic syndrome. Risk alleles are only pathologic when maternally inherited, consistent with the observation that KLF14/Klf14 is a maternally-expressed imprinted gene in human and mouse. However, how genomic imprinting is regulated at this important locus is currently unknown. In both species, KLF14/Klf14 is located [~]200 kb away from the paternally imprinted gene MEST/Mest, which is regulated by a maternal gametic differentially methylated region (gDMR) at its CpG island (CGI) promoter. Although the Klf14 CGI is kept unmethylated in most tissues, maternally-inherited DNA methylation (DNAme) marks are paradoxically required for Klf14 expression in mouse. Here, we show that Mest and Klf14 reside within the same topologically associating domain (TAD) in both mouse embryonic stem cells (ESCs) and differentiated cells, defined by sites of biallelic CTCF binding at the boundaries. Using allele-specific 4C-seq in F1 hybrid ESCs, we show that CTCF binding specifically to the unmethylated Mest gDMR generates a paternal allele-specific sub-TAD encompassing Klf14. We further show through CRISPR-Cas9-mediated mutagenesis, that deletions of the paternal Mest promoter region in ESCs as well as in vivo in mutant mice, results in both loss of Mest expression and acquisition of biallelic expression at Klf14. By analyzing epigenetic marks and chromatin looping in the Klf14-expressing pituitary cell line AtT-20, we identify a putative enhancer element shared by Mest and Klf14, providing a mechanistic model for the regulation of Klf14 imprinting. This work defines a new role for the Mest maternally methylated gDMR, revealing that it exerts long-range effects via allele-specific modulation of TAD structures and consequently act as an imprinting control region (ICR) in the regulation of Klf14 imprinting. Conservation of this TAD organisation at the human locus suggests that a similar regulatory mechanism operates at KLF14.

The ISWI chromatin remodeler regulates nucleosome spacing using one of two ATPase subunits Snf2h (Smarca5) and Snf2l (Smarca1). While Snf2h stable knockout (KO) is known to markedly reduce genomic binding of CTCF, an architectural protein organizing the 3D genome, ISWIs role in regulating genomic binding and function of lineage-determining transcription factors (LDTFs) during cell fate transition remains largely unclear. Using conditional KO mice and derived cells, we show Snf2h and Snf2l are partially redundant and are required for embryonic development of muscle and adipose tissue as well as myogenesis and adipogenesis in culture. Stable KO of ISWI impairs LDTF-stimulated cell differentiation and disrupts de novo binding of the myogenic LDTF MyoD and the cBAF chromatin remodeler. Surprisingly, acute depletion of ISWI leaves de novo MyoD binding landscape largely intact while disrupting MyoD-dependent recruitment of cBAF and CTCF, with minimal effects on constitutive genomic binding of cBAF and CTCF. Together, our findings identify ISWI as an important mediator connecting LDTF binding to cBAF recruitment and chromatin organization during cell fate transition. Bullet points- ISWI ATPases Snf2h and Snf2l are partially redundant and essential for muscle and adipose development - ISWI is required for MyoD, C/EBP, and PPAR{gamma}-driven cell fate transition - Stable KO of ISWI disrupts genomic binding of MyoD, while acute depletion does not - Acute ISWI deletion disrupts MyoD-dependent, but not constitutive, genomic binding of cBAF and CTCF

The classic model of dorsal spinal cord patterning proposes that roofplate-derived BMP patterns dorsal interneuron subtypes in a concentration-dependent manner. However, genetic perturbations of BMP pathway components produce variable effects, challenging this model. Here we implemented single-cell profiling, fate mapping, and mosaic perturbations to determine when BMP signaling patterns dorsal neural fates in vivo. Contrary to the classic model, we demonstrate that dorsal fates are patterned by BMP signaling during gastrulation. Following neural tube formation, BMP signaling continues but plays limited roles in domain specification and maturation. Fate mapping revealed that dorsal progenitors originate from the ventral gastrula, adopting BMP-dependent transcriptional states that prime dorsal neural fate. We propose that dorsal neural fates are initially patterned by gastrulation-stage sources of BMP, prior to roofplate induction.

In Schizosaccharomyces pombe, the conserved CHD remodeler Mit1 function within the SHREC remodeler-deacetylase complex (a homolog of the metazoan Mi-2/NuRD complex), which is essential for H3K9 methylation-dependent heterochromatin establishment. However, the mechanism by which remodeler activity promotes silencing is unknown. Current models posit a hierarchical relationship between histone modifications and remodeler activity, with Mit1 acting exclusively downstream of H3K9 methylation. Here, we challenge this model by showing that tethering Mit1 at an ectopic site within euchromatin is sufficient to initiate heterochromatin assembly and generate extended domains of de novo H3K9 methylation. This process requires the Mit1 catalytic activity but does not involve direct physical interaction with Clr4, suggesting Mit1-mediated nucleosome remodeling creates a chromatin context that enhances Clr4 function. Using a genome-wide deletion screen, we determined that Mit1-initiated silencing requires all core heterochromatin factors and is critically dependent on Clr4 dosage. Furthermore, Mit1 activity facilitates heterochromatin spreading at subtelomeric regions and promotes H3K9 methylation at novel genomic sites implicated in cellular adaptation. Together, our findings support a model in which remodeler-writer pairs, analogous to reader-writer pairs, constitute conserved regulatory modules through which nucleosome organization directs the establishment of heritable epigenetic states.

Many transcription factors (TFs) bind only a subset of their canonical binding sites in mammalian cells. To identify differences between bound and unbound sites we examined Zta(N182S), a mutant of the Epstein Barr Virus (EBV)-encoded Zta bZIP protein that binds distinct DNA sequences that are not strongly bound by any known human or viral TF, reducing the effects of selective pressure on endogenous genomic binding sites. We stably expressed Zta(N182S) in human HEK293 cells and monitored protein binding (ChIP-seq) and effects on chromatin accessibility (ATAC-seq). Zta(N182S) binds ~10% of the 14,979 genomic occurrences of the canonical 9-mer ATCACTCAT, creating stronger overall ATAC-seq signal compared to control cells, suggesting nucleosome displacement. Nucleosome occupancy, either predicted or experimentally determined (MNase), indicates that canonical Zta and Zta(N182S) sites are more strongly bound when they are ~60bp from a positioned nucleosome dyad. These data suggest that Zta and Zta(N182S) binding results in nucleosome remodeling, consistent with pioneer-like activity. Examination of amino acids across Zta and human bZIPs identifies four conserved basic amino acids, a proline, and acidic amino acids immediately N-terminal of the basic amino acids of the bZIP domain (PARRTRKPQQPESLEECDSELEIKRYKN). We term this new protein motif "BPabZIP" (Basic-Proline-acidic bZIP). Molecular structure predictions for both Zta and human Fos/Jun reveal the basic amino acids interacting with the acidic patch on the nucleosome. The acidic amino acids act as an a-helical extension of the basic region that mimics DNA by interacting with histones H2A and H2B. Taken together, our analyses of this synthetic TF reveal a pioneer-like mechanism that is present in both human and viral bZIP proteins.

Ubiquitin-specific protease 10 (USP10) is a multifunctional deubiquitinating enzyme that primarily regulates cellular stress responses, including the DNA damage response. Here, we show that USP10 is required for homologous recombination (HR)-mediated repair of DNA double-strand breaks (DSBs) and for the maintenance of genomic stability. USP10-depleted cells exhibit spontaneous micronuclei, impaired DSB repair following zeocin and camptothecin treatment, and reduced sister chromatid exchange. These cells are also more sensitive to irradiation and mitomycin C and display increased chromosomal abnormalities after mitomycin C treatment. Persistent RAD51 foci formation in USP10-depleted cells suggests that USP10 functions at a step downstream of RAD51 nucleofilament formation. This function of USP10 in facilitating HR repair depends on deubiquitinase activity but is independent of G3BP1/2 and PABP binding. In addition, a newly identified nucleolar localization signal is required for USP10s function in DSB repair. Together, these findings indicate that USP10 maintains genome integrity by localizing to the nucleolus and facilitating HR-mediated repair of DSBs.

Transposable elements (TEs) pose a major threat to genome integrity in the germline, where the piRNA pathway ensures heritable TEs silencing. How the chromatin environment that enables piRNA biogenesis is first established during oogenesis remains unclear. Here, we identify the histone variant His2Av, the single H2A variant in Drosophila combining H2A.Z (transcriptional) and H2A.X (DNA repair) features, as a critical regulator of the earliest steps of piRNA pathway activation. Germline-specific depletion of His2Av disrupts transcription of piRNA pathway genes, abolishes dual-strand piRNA cluster transcription, and triggers strong TEs derepression. These defects are associated with loss of Rhino recruitment despite intact H3K9me3, suggesting that His2Av contributes to the establishment of a specialized heterochromatin permissive for noncanonical piRNA cluster transcription. His2Av depletion also causes DNA damage, replication stress, and activation of Chk2- and Claspin-dependent checkpoints, leading to oogenesis arrest. Remarkably, overexpression of RNase H, but not a catalytic-dead variant, robustly rescues oocyte development, suggesting that replication stress is a major source of DNA damage in His2Av mutants. Finally, using a separation-of-function approach with a C-terminal truncation inhibiting H2A.X-like activity, we show that the essential germline role of His2Av is transcriptional (H2A.Z-like). Together, our findings reveal that His2Av primes germline chromatin for piRNA pathway initiation while limiting transcription-replication conflicts during early oogenesis.

The spatiotemporal control of transcription and the maintenance of germline genome integrity depend on dynamic chromatin architecture. In Drosophila, the actin-related protein Arp6--a core subunit of the SWR1-like Domino chromatin remodeling complex--mediates the deposition of the histone variant H2Av. Previous studies have established H2Av as a key transcriptional regulator that modulates the +1 nucleosome barrier to promote RNA Polymerase II (Pol II) pause release and productive elongation. Conversely, H2Av is also integral to heterochromatin assembly and gene silencing. Here we demonstrate that Arp6 and H2Av are essential for female fertility and the global repression of transposable elements (TEs) in the Drosophila ovary. Rather than repressing TEs directly, we show that Arp6 and H2Av maintain genomic stability indirectly by driving the transcription of core PIWI-interacting RNA (piRNA) pathway genes. Depletion of either chromatin factor leads to a significant loss of piRNAs and reduced non-canonical transcription of dual-strand piRNA clusters. This defect stems from a failure to express the Rhino-Deadlock-Cutoff (RDC) complex, alongside the downregulation of multiple other piRNA biogenesis factors. Genomic profiling confirms that H2Av acts predominantly as an activating signal at host gene promoters. Upon H2Av or Arp6 depletion, genes that rely on H2Av for their expression exhibit a distinct upstream shift and more precise spatial localization of the Pol II peak at the TSS, indicating an impaired transition from transcription initiation into productive elongation. Together, our findings build upon the known transcriptional activation functions of the Arp6-H2Av axis, revealing that this established chromatin mechanism is critical for licensing piRNA-mediated genome defense and ensuring germline maintenance.