Polycomb repressive-deubiquitinase complex safeguards oocyte epigenome and female fertility by restraining Polycomb activity

Domain: biology
Published: 2026-03-19
Editorial outcome: major_revision

Abstract
Mouse oocytes exhibit a unique chromatin landscape characterized by broad H3K27ac and H3K27me3 domains, demarcating euchromatin and facultative heterochromatin, respectively. However, the mechanisms underlying this non-canonical landscape remain elusive. Here we report BAP1, a core component of the Polycomb Repressive-Deubiquitinase (PR-DUB) complex, as a key negative regulator of Polycomb activity during oogenesis. BAP1 restricts pervasive H2AK119ub1 accumulation and protects oocyte-specific broad H3K27ac, particularly within gene-poor regions, from ectopic H3K27me3 deposition. While PR-DUB has been linked to gene repression, in oocytes BAP1 primarily promotes transcription and contributes minimally to Polycomb-mediated silencing. BAP1-dependent transcriptional activation during oogenesis is essential for oocyte developmental competence, maternal-to-zygotic transition, and female fertility. Notably, ectopic H3K27me3 domains established in BAP1-deficient oocytes persist in preimplantation embryos but are resolved after implantation, and loss of maternal BAP1 does not impair either canonical or non-canonical genomic imprinting. Together, these findings reveal a critical role for PR-DUB in safeguarding the oocyte epigenome by protecting euchromatin from ectopic Polycomb activity, rather than enforcing transcriptional repression.

Introduction
Polycomb group (PcG) proteins play a central role in establishing and maintaining epigenetic silencing during animal development 1,2. PcG proteins assemble into two major complexes, Polycomb Repressive Complex 1 (PRC1) and PRC2, which function cooperatively to mediate transcriptional repression 3,4. In mammals, PRC1 contains the E3 ubiquitin ligases RING1A and RING1B, which catalyze monoubiquitination of histone H2A at lysine 119 (H2AK119ub1) 5,6. The PRC2 comprises the methyltransferases EZH1 and EZH2, which catalyze mono-, di-, and tri-methylation of histone H3 at lysine 27 (H3K27me1/2/3) 7-9. These complexes are typically recruited to promoters of key developmental regulators where they deposit H2AK119ub1 and H3K27me3, hallmarks of facultative heterochromatin 10,11. Collectively, PRC1 and PRC2 mediate epigenetic silencing by promoting chromatin compaction 12 and counteracting transcription 13.

Oogenesis provides a unique in vivo model to investigate chromatin-mediated gene regulation, as postnatal oocytes grow for extended periods of time without undergoing cell division. Shortly after birth, oocytes are enclosed in primordial follicles and arrested at the diplotene stage of meiotic prophase I, known as the germinal vesicle (GV) stage. After primordial follicle activation, they enter growth phase to progress from primary follicles to pre-antral follicles, and pre-ovulatory follicles. During this growth phase (~3-weeks in mouse), GV oocytes undergo significant cytoplasmic and nuclear enlargement without cell cycle progression and ultimately mature into fully grown GV oocytes (FGOs). During this time, oocytes accumulate maternal RNAs and proteins essential for meiotic resumption and early embryonic development 14.

In parallel with transcriptional and translational changes, oocytes acquire a distinct epigenome, including the formation of broad, non-canonical domains of H2AK119ub1 and H3K27me3 15-19. Notably, PRC1 and PRC2 have different functions during postnatal oocyte development. Loss of PRC1 in oocytes leads to widespread gene de-repression and disrupts the organization of Polycomb-associated three-dimensional chromatin domains 20,21. As a result, PRC1-null oocytes fail to support embryonic development beyond the two-cell stage 20. In contrast, maternal loss of PRC2 has only a modest effect on oogenesis and primarily impacts non-canonical genomic imprinting dependent on maternal H3K27me3, with developmental consequences emerging after implantation 22-28. Despite these insights, how broad Polycomb domains are established and maintained during oocyte postnatal growth remains unclear. In particular, the mechanisms that restrict the spread of facultative heterochromatin into euchromatic regions during this period are poorly understood.

In addition to Polycomb domains, FGOs exhibit pervasive acetylation of histone H3 at lysine 27 (H3K27ac), a marker of active euchromatin 29,30. This non-canonical, widespread H3K27ac is mutually exclusive with H3K27me3 domains, spatially segregating euchromatin and facultative heterochromatin in oocytes 31. The broad H3K27ac feature is also found in early-stage human preimplantation embryos 32. Remarkably, many FGO-specific putative enhancers, marked by distal H3K27ac, reside in gene-poor regions, and are linked to oocyte-specific gene activation 29. However, the mechanisms governing the formation and function of these oocyte-specific broad H3K27ac domains remain largely unknown. These observations highlight the current knowledge gaps of how chromatin regulators coordinate the formation of spatially distinct active and repressive domains during oocyte growth.

The ubiquitin carboxy-terminal hydrolase BAP1 is the catalytic subunit of the Polycomb Repressive-Deubiquitinase (PR-DUB) complex, which removes monoubiquitin from H2AK119ub1 33-35. In Drosophila embryos, loss of BAP1 leads to elevated H2AK119ub1 levels at Polycomb target genes and disrupts gene repression by impairing PRC1-mediated chromatin compaction 36. In mouse embryonic stem cells (mESCs), PR-DUB broadly promotes gene activation by limiting pervasive deposition of H2AK119ub1 across the genome, yet it is also responsible for Polycomb repression via a mechanism distinct from that in Drosophila 37-40. By contrast, studies in human cell lines have revealed that BAP1 functions as a transcriptional activator, acting to restrict PRC1-mediated H2AK119ub1 accumulation at gene regulatory elements, including enhancers 41,42. These findings underscore the context-dependent role of BAP1 in gene regulation, suggesting that it may act to support both Polycomb repression and transcriptional activation, depending on cellular and developmental context. Thus, the precise mechanisms by which BAP1 regulates transcription remain enigmatic, and it is essential to dissect its in vivo function in balancing Polycomb-mediated silencing and transcriptional activation.

In this study, we aimed to determine the role of PR-DUB in regulating H2AK119ub1 in oocytes and early embryos, and to dissect how its activity contributes to the formation of facultative heterochromatin and euchromatin during oogenesis. We reveal that PR-DUB safeguards the oocyte epigenome and female fertility by protecting euchromatin from ectopic Polycomb activity, rather than by enforcing transcriptional repression.

Results
Identification of candidate H2AK119ub1 deubiquitinases in early embryos
To identify candidate DUBs responsible for H2AK119ub1 removal in vivo, we first analyzed the expression dynamics of known H2A DUBs during oogenesis and early embryonic development using publicly available RNA-seq and ribosome profiling datasets 43,44. Based on high expression levels, ribosome association in oocytes and preimplantation embryos, and lack of activity toward monoubiquitinated histone H2B, we selected five H2A DUBs for further analysis: USP16 45, USP28 46, USP21 47, BAP1 33, and MYSM1 48(Supplementary Fig. 1 a-d). To assess their functional activity toward H2AK119ub1, we microinjected zygotes with Flag-tagged mRNAs encoding each candidate immediately after fertilization and evaluated H2AK119ub1 levels by immunostaining in late zygotes (~6 hours post-injection) (Fig. 1a). Subcellular localization analyses revealed that USP16 and USP21 were primarily cytoplasmic, whereas BAP1, USP28, and MYSM1 exhibited no strong preference between nucleus and cytoplasm (Fig. 1b).

Among these candidates, USP21 overexpression led to a robust depletion of H2AK119ub1 within 6 hours (Fig. 1b, c). In contrast, BAP1 alone did not affect H2AK 119ub1 levels, but co-expression with ASXL1 significantly depleted the H2AK119ub1 signal. This finding aligns with previous studies showing that ASXL1 is required to activate BAP1’s H2A DUB function 49. Overexpression of USP16, USP28, or MYSM1 had a mild impact, if any, on H2AK119ub1 levels. Together, these data suggest that USP21 and BAP1 potentially regulate H2AK119ub1 during early development. Since Usp21 null mice are viable and fertile 50,51, we selected BAP1 for detailed loss-of-function analysis in this study. It should be noted that we cannot exclude the possibility that USP16 (discussed further below), USP28, and MYSM1 may function as H2A DUBs at other developmental stages or require additional cofactors in zygotes. Future loss-of-function studies will be necessary to determine their contributions to H2AK119ub1 regulation during oogenesis and early embryonic development.

BAP1 limits genome wide pervasive H2AK119ub1 accumulation in oocytes
Having identified BAP1 as a potential key H2A DUB in zygotes, we next examined the expression dynamics of other PR-DUB subunits during oogenesis and early embryonic development. Reanalysis of publicly available RNA-seq and ribosome profiling datasets 43,44 revealed that key PR-DUB components, including Bap1, Asxl1, Foxk2, and Ogt, are highly expressed and associated with ribosomes throughout oocyte maturation and preimplantation stages (Fig. 1d, Supplementary Fig. 1e). Consistent with these findings, immunoblot analysis confirmed the presence of BAP1 protein in FGOs and early embryos (Fig. 1e).

To investigate the function of PR-DUB during oogenesis and early development, we generated an oocyte-specific conditional knockout (CKO) of Bap1 by crossing a floxed Bap1 line (see Methods), in which exons 6-12 are flanked by loxP sites, with a Gdf9-iCre transgenic line 52 (Supplementary Fig. 2a). Gdf9-iCre is specifically expressed in oocytes of primordial follicles by postnatal day 3 52, enabling targeted deletion of Bap1 in early-stage oocytes. Cre-mediated recombination is predicted to produce a truncated BAP1 protein lacking catalytic activity and the ability to interact with ASXL partners 49 (Supplementary Fig. 2b). Throughout this study, Bap1fl/fl mice were used as controls (CTR), and Gdf9-iCre; Bap1fl/fl mice were used as CKO. At 6-9 weeks of age, comparable number of FGOs were recovered from CTR and CKO female mice (Supplementary Fig. 2c-d). RNA-seq and immunoblot analyses confirmed effective depletion of Bap1 at both the transcript and protein levels in CKO FGOs (Fig. 1f, Supplementary Fig. 2e).

Immunofluorescence analyses revealed that H2AK119ub1 levels in CKO FGOs were significantly elevated, approximately 2- to 3-fold, compared to those in CTR oocytes (Fig. 1g, h). To determine the genomic regions where H2AK119ub1 increases, we performed spike-in normalized Cleavage Under Targets and Release Using Nuclease (CUT&RUN) (see Methods; Supplementary Fig. 2f). Remarkably, this analysis uncovered a pervasive accumulation of H2AK119ub1 across the genome (Fig. 1i, j; Supplementary Fig. 2g). The extent of H2AK119ub1 elevation in BAP1-deficient oocytes appeared more pronounced than that in mESCs 37 (Fig. 1k). Notably, putative enhancers, defined by distal H3K27ac peaks 29, exhibited a particularly strong increase in H2AK119ub1 (Fig. 1k), suggesting a role of BAP1 in regulating enhancer activities. Together, these findings indicate that BAP1 functions to restrain widespread H2AK119ub1 accumulation in oocytes.

BAP1 primarily activates gene expression in oocytes
We next investigated transcriptomic changes resulting from Bap1 deletion in FGOs. Comparative analysis identified 101 upregulated and 827 downregulated genes in CKO FGOs (Fig. 1l, Supplementary Data 1). The predominance of downregulated genes suggests that BAP1 primarily acts as a gene activator in oocytes, likely by counteracting H2AK119ub1, a key player in PRC1-mediated gene silencing 53,54. Downregulated genes are enriched for gene ontology (GO) terms implicated in oogenesis, such as “cell adhesion molecule binding” (Cdh2, Dsc2, Cdh18) and “growth factor binding” (Fgf10, Egfr, Foxp2) (Supplementary Fig. 2h). To link the observed transcriptional dysregulation to changes in H2AK119ub1, we examined H2AK119ub1 levels at these genes. While downregulated genes had similar baseline H2AK119ub1 enrichment in CTR oocytes, they acquired significantly higher levels of H2AK119ub1 in CKO FGOs compared to non-differentially expressed genes (DEGs) (Fig. 1m, n). Notably, this increase occurred across both genic and intergenic regions.

It has been previously reported that the methyl-binding protein MBD6 recruits PR-DUB to retrotransposons by recognizing m5C-modified chromatin-associated RNAs, a process critical for retrotransposon activation 55. However, we found that BAP1 deficiency in oocytes had only a modest effect on the expression of repeat elements (Supplementary Fig. 2i, Supplementary Data 1). This suggests a limited role for MBD6-mediated PR-DUB recruitment in retrotransposon regulation during oogenesis, consistent with the low RNA-seq and Ribo-seq expression levels of Mbd6 in oocytes (Fig. 1d, Supplementary Fig. 1e). In sum, these findings indicate that the widespread accumulation of H2AK119ub1 following BAP1 loss primarily leads to gene repression, with a subset of genes being more susceptible than others (further discussed below).

BAP1 preserves H3K27ac by preventing ectopic H3K27me3 deposition in oocytes.
Previous studies have shown that H2AK119ub1 guides H3K27me3 deposition 56-59, and that H3K27me3 and H3K27ac are mutually exclusive. We therefore examined how BAP1 loss affects the H3K27me3 and H3K27ac landscapes in oocytes. Unlike the dramatic increase in H2AK119ub1, immunofluorescence analyses revealed that global levels of both H3K27me3 and H3K27ac remained largely unchanged in CKO FGOs (Fig. 2a, b). This observation was further supported by spike-in normalized CUT&RUN analyses, which confirmed that most broad H3K27me3 and H3K27ac domains, known to be oocyte-specific 15,29, were retained in CKO FGOs (Fig. 2c, Supplementary Fig. 3a).

However, closer inspection of genome browser tracks revealed specific loci where H3K27ac was reduced and H3K27me3 correspondingly increased in CKO FGOs (Fig. 2d, Supplementary Fig. 3b). To systematically characterize these changes, we identified 9,043 H3K27ac-lost regions (ranging from 10 to 375 kb) in CKO oocytes (see Methods, Supplementary Data 2). Loss of H3K27ac in Bap1 CKO FGOs is globally associated with a corresponding gain of H3K27me3 (Fig. 2e). In addition, the H3K27ac-lost regions already displayed relatively lower H3K27ac and higher H3K27me3 in CTR oocytes compared to regions with unchanged H3K27ac (Supplementary Fig. 3c). H2AK119ub1 levels increased similarly in both H3K27ac-lost and H3K27ac-unchanged regions in CKO FGOs (Fig. 2e and Supplementary Fig. 3c), suggesting that the increase in H2AK119ub1 is not the sole determining factor that dictates which regions lose H3K27ac and gain H3K27me3. Most H3K27ac-lost regions (69%) were in intergenic regions and gene bodies, suggesting potential enhancer activity (Fig. 2f). Genes associated with H3K27ac loss, either at promoters or within gene bodies, were preferentially downregulated in CKO FGOs (Fig. 2g). Together, these results indicate that BAP1 deficiency causes H3K27ac loss and H3K27me3 gain at selective loci, contributing to gene repression in oocytes.

To better understand the mechanisms underlying locus specific H3K27ac loss in CKO FGOs, we examined the genomic features of the affected regions. A striking characteristic of the H3K27ac-lost regions is their preferential localization within gene-poor regions, as opposed to gene-rich regions (Fig. 2d, 2h, Supplementary Fig. 3b; see Methods). Previous studies have shown that H3K27ac peaks in gene-poor regions are more prevalent in FGOs compared to growing oocytes 29. Consistently, we found that these H3K27ac-lost regions exhibit low H3K27ac levels in non-grown oocytes at postnatal day 1 (P1) and growing oocytes at P7 and P10 but gain high levels of H3K27ac during the final stage of oocyte growth, as seen in FGOs (Fig. 2i, 2j, Supplementary Fig. 3d). In contrast, regions that unaffected by Bap1 deletion acquire H3K27ac even before Gdf9-iCre activation on P3 (Fig. 2j, Supplementary Fig. 3d). These findings suggest that BAP1 is critical for the establishment of new H3K27ac marks during oocyte growth, rather than for maintaining pre-existing H3K27ac marks. Supporting this idea, only 9.9-13.4% of H3K27ac-lost regions overlapped with H3K27ac peaks in P7/P10 growing oocytes, whereas this overlap increased to 49-57.9% for H3K27ac-unchanged regions (Supplementary Fig. 3e). Further supporting this model, genes unaffected by Bap1 deletion are already highly expressed early during oocyte growth, whereas genes downregulated in CKO FGOs reach peak expression only at the fully grown stage and show lower expression levels than the other group (Fig. 2k, Supplementary Fig. 3f). Together, these data suggest that the timing of H3K27ac establishment during oocyte development underlies the locus specific H3K27ac loss observed in Bap1 CKO FGOs.

BAP1 plays a limited role in Polycomb-mediated silencing in oocytes
Having established that BAP1 preserves oocyte-specific broad H3K27ac domains by preventing ectopic H3K27me3 deposition, we next examined its potential role in Polycomb-mediated gene silencing. In mESCs, it has been proposed that widespread accumulation of H2AK119ub1 in PR-DUB mutants may sequester PRC2, thereby diminishing H3K27me3 deposition at canonical Polycomb group (PcG) target loci 37-39. To evaluate this model in oocytes, we assessed H2AK119ub1, H3K27me3, and H3K27ac profiles at the promoters of oocyte PcG target genes as defined previously 60. This analysis revealed a marked increase in H2AK119ub1 at PcG target promoters in Bap1 CKO FGOs, accompanied by a relatively modest decrease in H3K27me3 and a corresponding gain in H3K27ac (Fig. 3a, b).

To assess whether the observed chromatin alterations could lead to transcriptional de-repression of PcG targets, we re-analyzed gene expression in Bap1 CKO FGOs. For comparison, we examined publicly available RNA-seq datasets from PRC1 and PRC2 CKO oocytes 21. Consistent with the established importance of PRC1 in postnatal oocyte development 20, PRC1 loss led to a greater number of DEGs (2,417 upregulated and 700 downregulated) compared to PRC2 loss (414 upregulated and 67 downregulated) (Supplementary Fig. 4a-b). Among these, 479 and 83 PcG targets were de-repressed in PRC1 and PRC2 CKO oocytes, respectively (Fig. 3c). By contrast, only 7 PcG targets were upregulated in Bap1 CKO FGOs, and the extent of their upregulation was substantially lower than that observed in PRC1/2 mutants (Fig. 3d). Analyses of PcG targets previously defined in mESCs 61 also led to a similar conclusion (Supplementary Fig. 4c-d). These findings suggest that, although the global accumulation of H2AK119ub1 in BAP1-deficient oocytes may partially impair PRC2 recruitment and reduce H3K27me3 deposition at PcG targets, the overall integrity of Polycomb-mediated silencing is largely preserved. Taken together, these results indicate that BAP1 plays a limited role in the regulation of Polycomb-dependent gene silencing in oocytes.

Loss of maternal BAP1 severely impairs preimplantation development
Having established the critical role of BAP1 in safeguarding the oocyte epigenome and transcriptome, we next assessed how maternal BAP1 deficiency may affect female fertility. To this end, CTR and CKO females were co-caged with wild-type males for six months. Bap1 CKO females exhibited a significant reduction in fertility that was attributable to both fewer litters and smaller litter sizes (Fig. 4a-c). Ovarian morphologies of Bap1 CKO females appear largely normal (Supplementary Fig. 5a), which is consistent with the fact that comparable numbers of FGOs are retrieved from control and CKO females at 6-9 weeks of age (Supplementary Fig. 2c, d), However, in vitro meiotic maturation analysis revealed a mild reduction in the metaphase II rate in CKO oocytes (Supplementary Fig. 5b, c). Additionally, the number of embryos recovered at embryonic day 3.5 (E3.5) was slightly smaller in the CKO group (Fig. 4d), suggesting that ovulation is moderately impaired. Importantly, the embryos from Bap1 CKO females showed defective preimplantation development, with most (~90%) delayed at the 8-16-cell or morula stage at E3.5, whereas control embryos (~96%) progressed to the blastocyst stage by this time point (Fig. 4e, f). Although extended ex vivo culture for an additional 24 hours allowed most mutant embryos to form blastocysts, these were of poor quality, exhibiting reduced total cell numbers and abnormal expression of lineage markers, including NANOG (epiblast), GATA4 (primitive endoderm), and CDX2 (trophectoderm) (Fig. 4g, h).

To further evaluate developmental competence of the blastocysts, we dissected embryos at E6.5 and found that the average number of decidua (5.2 ± 3.3) and recovered embryos (2.2 ± 3.2) in the matKO group was lower than those in controls (10.0 ± 1.2, 9.2 ± 1.8)(Fig. 4i, j). The matKO E6.5 embryos were overall smaller than CTR embryos (Fig. 4k). These data are consistent with the compromised blastocyst development of matKO embryos. We next performed cesarean section (C-section) at E18.5 and found that only 3.9 ± 2.0 matKO embryos reached term, representing just 41.4% of the number observed in CTR females (9.7 ± 1.5) (Fig. 4l, m, Supplementary Fig. 5d), indicating a high rate of post-implantation embryonic loss likely attributable to compromised blastocyst quality. Notably, both placentae and fetuses from the maternal knockout (matKO) group were slightly heavier than those of controls (Fig. 4n, o), possibly due to reduced litter size. Thus, these data indicate that BAP1-deficient oocytes could not support normal preimplantation development to give rise to competent blastocysts.

We next sought to determine when developmental delays first arise in matKO embryos during preimplantation development. By monitoring embryos derived through in vitro fertilization (IVF), we observed that developmental delays began as early as the 2- to 4-cell transition (Fig. 4p-r). The delay became more pronounced by 72 hours post-IVF (hpi), at which point only ~30% of CKO embryos had reached the morula stage. Ultimately, only ~30% of CKO embryos developed into blastocysts, in contrast to the ~85% blastocyst rate observed in the control group. The more pronounced developmental arrest observed in IVF-derived matKO embryos likely reflects the increased sensitivity of mutant embryos to suboptimal ex vivo culture conditions relative to the in vivo environment.

Because the paternal Bap1 allele remains intact in matKO embryos (Fig. 4p), it is possible that Bap1 expression from the paternal genome during zygotic genome activation (ZGA) at the 2-cell stage partially compensates for the maternal loss. To test this hypothesis, we generated Prm-Cre; Bap1fl/fl male mice, in which Bap1 is deleted during the late haploid spermatid stage. When Bap1 CKO oocytes were fertilized with sperm from these males, the resulting embryos, referred to as maternal-zygotic knockout (mzKO), displayed markedly more severe developmental defects (Fig. 4q-r). Few mzKO embryos progressed beyond the 2- to 4-cell stages. The observed phenotype was not due to intrinsic defects in Prm-Cre; Bap1fl/fl sperm, as their fertilization of control oocytes resulted in normal progression to the blastocyst stage (Fig. 4q-r). Together, these findings demonstrate that maternal BAP1 is essential for preimplantation development, and loss of both maternal and zygotic Bap1 further exacerbates the phenotype.

Loss of maternal BAP1 causes maternal-to-zygotic transition (MZT) defects
Having established that maternal BAP1 is essential for preimplantation development, we next sought to investigate the underlying mechanisms responsible for the developmental arrest and/or delay observed in Bap1 matKO embryos. The MZT is a critical developmental window during which, maternal RNAs and proteins are degraded, and embryonic development becomes increasingly dependent on zygotic gene expression initiated during ZGA 62. Minor ZGA occurs at late 1-cell (L1C) and early 2-cell (E2C) stages with the activation of hundreds of genes, whereas major ZGA takes place at late 2-cell (L2C) stage, during which thousands of genes are transcriptionally activated 63,64.

Given that the developmental defect in Bap1 matKO embryos emerges as early as the 2- to 4-cell transition, we hypothesized that loss of maternal BAP1 disrupts the MZT. To test this hypothesis, we performed total RNA sequencing of control and Bap1 mutant oocytes/embryos at the MII stage, L1C, E2C, and L2C stages (Fig. 5a, Supplementary Fig. 6a). Comparative transcriptome analyses identified 886, 869, 1096, and 2740 DEGs between CTR and mutant groups at the MII stage, L1C, E2C, and L2C stages, respectively (Fig. 5b, Supplementary Data 1). The number of DEGs was comparable across the GV, MII, and L1C stages, with a predominant trend of gene downregulation in Bap1 mutants (Fig. 1i and 5b). Notably, ~67% (511 genes) of L1C-downregulated genes were already repressed in mutant oocytes (Fig. 5c), and these genes were enriched for GO terms related to cell adhesion (e.g., Cdh2, Cdh18, Cdh20, Dsc2) and growth factor binding (e.g., Gata3, Fgf10, Egfr, Foxp2) (Fig. Supplementary Fig. 6b). Thus, these data suggest the aberrant transcriptomes in Bap1 CKO oocytes are inherited into 1-cell embryos.

We next analyzed compositions of DEGs at E2C and L2C stages. Downregulated genes at these stages significantly overlapped with those classified as “minor ZGA,” “major ZGA,” and “2-cell transient” in the DBTMEE v2 database 65, while upregulated genes were enriched for transcripts categorized as “maternal RNA” (Fig. 5d), implicating defects in both zygotic activation and maternal RNA decay. To further assess the impact of maternal BAP1 loss on ZGA, we compared DEGs in Bap1 mutants with previously defined zygote and E2C minor ZGA gene sets 30. Among 23 zygote minor ZGA genes detected in our dataset, three (Sox6, Stk39, Lcp2) were significantly downregulated in Bap1 matKO embryos at L1C (Fig. 5e), with additional minor ZGA genes such as Dux and Usp17la/c/d showing a trend of downregulation (Supplementary Data 1). Furthermore, 22% of minor ZGA genes and 32% of major ZGA genes were downregulated at the E2C and L2C stages, respectively (Fig. 5e, f), including key regulators of early development such as Zscan4, Cdk2ap1, Nr5a2, and Obox3/6 (Fig. 5f)66-73. Downregulated genes at L2C were also significantly enriched for GO terms related to ribosome biogenesis and rRNA processing (Fig. 5g), potentially accounting for the developmental defects at the 2- to 4-cell transition. In addition, MT2/MERVL retrotransposons, known to be essential for early development 74-76, were markedly downregulated in Bap1 mutants at the E2C stage (Supplementary Fig. 6c). Interestingly, a subset of E2C minor ZGA genes showed aberrant upregulation at L2C, possibly reflecting delayed downregulation (Fig. 5f). Lastly, 13.5% of maternal transcripts were upregulated at the L2C stage, indicating defective maternal RNA degradation (Fig. 5e). Collectively, these findings demonstrate that loss of maternal BAP1 causes defects in both ZGA and maternal transcript clearance during MZT.

Ubiquitin-specific peptidase 16 (USP16) has previously been implicated in H2A deubiquitination during the GV-to-MII transition and is essential for mouse ZGA 77. To evaluate whether USP16 and BAP1 act through similar or distinct mechanisms during oogenesis and early embryogenesis, we reanalyzed the published transcriptomic data. To our surprise, there was minimal overlap between DEGs in Usp16 and Bap1 mutants at both L1C and L2C stages (Fig. 5h, Supplementary Fig. 6d), suggesting that maternal USP16 and BAP1 function via distinct regulatory pathways. Consistent with this notion, loss of BAP1, but not USP16, causes increase in H2AK119ub1 in FGOs (Fig. 1)77. These findings underscore the mechanistic divergence between USP16 and BAP1 in regulating H2A deubiquitination during development and highlight the need for further investigation into their individual and potentially complementary roles.

Maternal BAP1 regulates embryonic enhancer activity during ZGA
Having demonstrated MZT defects in Bap1 matKO embryos, we next investigated how the loss of maternal BAP1 affects chromatin dynamics during early embryogenesis. To this end, we performed immunostaining for H2AK119ub1, H3K27me3, and H3K27ac in CTR and Bap1 matKO embryos at the 1-cell (6 and 10 hpi), L2C (28 hpi), and 4-cell (48 hpi) stages. Since developmental arrest in Bap1 matKO embryos becomes evident as early as the 2- to 4-cell transition, only morphologically normal 4-cell embryos were included in the analysis. Similar to FGOs, H2AK119ub1 levels were significantly elevated in Bap1 matKO embryos compared to CTR at all stages examined (Fig. 6a, Supplementary Fig. 7). In contrast, H3K27me3 immunostaining levels were largely comparable between the two groups. Interestingly, H3K27ac signals were markedly reduced at the 2-cell and 4-cell stages in the absence of maternal BAP1 (Fig. 6a, Supplementary Fig. 7), suggesting impaired formation of active euchromatin that likely occurs independently of H3K27me3.

To further explore this, we performed CUT&RUN profiling of H3K27ac and H3K27me3 at the L2C stage, when major ZGA occurs. Consistent with the immunofluorescence data, the global H3K27me3 landscape was largely unchanged between control and matKO embryos, whereas H3K27ac signals were moderately decreased (Supplementary Fig. 8a, b). Notably, putative L2C enhancers, defined by distal H3K27ac peaks (Liu et al., 2024), were reduced in matKO embryos (Fig. 6b, Supplementary Fig. 8c). In contrast, H3K27ac levels at promoter regions of both maternal and major ZGA genes remained largely unaffected by the loss of maternal BAP1 (Fig. 6b). These findings suggest that impaired activation of L2C enhancers contributes to defective ZGA in maternal BAP1-deficient embryos. Moreover, H3K27ac levels at the embryonic enhancers were low in FGOs and showed no difference between control and mutant oocytes (Supplementary Fig. 8d), indicating that this defect arises post-fertilization. Furthermore, H3K27me3 levels at maternal genes, major ZGA loci, and L2C enhancers were comparable between control and matKO embryos (Fig. 6c), excluding aberrant H3K27me3 deposition as a contributing factor. This also suggests that the observed upregulation of maternal transcripts (Fig. 5e-f) likely reflects impaired maternal mRNA decay rather than transcriptional derepression resulting from altered H3K27me3. Collectively, these results indicate that maternal BAP1 is required for proper embryonic enhancer activities during ZGA, independent of H3K27me3 regulation.

Aberrant H3K27me3 landscape, but not H3K27ac changes, in BAP1-null oocytes persists into early embryos
Given that H3K27me3-deficient states in Eed- or Pcgf1/6-null oocytes are irreversibly transmitted to early embryos 17,23, we next examined whether the aberrant H3K27me3 landscape observed in Bap1-null FGOs (Fig. 2-3) similarly persists post-fertilization. Leveraging the fact that our L2C embryos are derived from B6/129 × PWK F1 hybrids, we performed allele-specific analysis of H3K27me3 distribution. We first focused on genomic regions, primarily located in gene deserts, that acquire ectopic H3K27me3 and corresponding H3K27ac loss in Bap1-deficient oocytes (Fig. 2). In control L2C embryos, these regions displayed paternally biased H3K27me3 enrichment (Fig. 6d, e and Supplementary Fig. 8e), consistent with prior reports that paternal H3K27me3 in preimplantation embryos predominantly localizes to gene-poor regions 15. Remarkably, these loci acquired maternal-specific H3K27me3 enrichment in Bap1 matKO embryos, and this ectopic gain was already evident in CKO FGOs (Fig. 6d, e and Supplementary Fig. 8e), suggesting inheritance of the abnormal chromatin state from the oocyte. In contrast, H3K27ac levels at these loci declined sharply from FGO to L2C stages in both control and mutant embryos, with the CTR maternal allele showing only a slightly higher H3K27ac than that in mutants (Fig. 6d, e and Supplementary Fig. 8e). This suggests that this H3K27ac reduction reflects a normal developmental progression and is not specific to BAP1 loss. Notably, the inherited ectopic H3K27me3 may not initiate gene repression in embryos, as the downregulated genes within these domains were already transcriptionally reduced at earlier stages (Supplementary Fig. 8f, g), implying that these transcriptional defects arise during oogenesis rather than being induced post-fertilization.

We next assessed H3K27me3 and H3K27ac profiles at the typical Polycomb targets in control and Bap1 matKO L2C embryos. Previous studies have shown that H3K27me3 is largely lost from promoter regions of Polycomb targets following fertilization, with only a subset retaining residual H3K27me3 during preimplantation development 15,61. Analysis of the Polycomb targets that retain H3K27me3 at the L2C stage (n = 719; see Methods) revealed a maternal bias in H3K27me3 enrichment in control embryos (Fig. 6f, g). In contrast, this maternal H3K27me3 signal was either completely lost or markedly reduced in Bap1 matKO embryos (Fig. 6f, g). Notably, the reduction observed in mutant L2C embryos was more pronounced than that in Bap1 CKO oocytes, suggesting progressive loss post-fertilization. By contrast, the modest H3K27ac gain seen at Polycomb targets in CKO FGOs was not sustained in early embryos (Supplementary Fig. 8h, i). Together, these results demonstrate that the aberrant H3K27me3 landscape, but not H3K27ac changes, established in BAP1-null oocytes is inherited into early embryonic stages.

The ectopic H3K27me3 domains in Bap1 matKO embryos are resolved by early post-implantation stages
The intergenerational inheritance of aberrant H3K27me3 domains in Bap1 matKO embryos prompted us to determine how long these domains persist beyond the two-cell stage. To this end, we performed H3K27me3 CUT&RUN in morulae, the developmental stage immediately preceding the first lineage differentiation. We also dissected post-implantation E6.5 embryos into epiblast (EPI), visceral endoderm (VE), and extra-embryonic ectoderm (EXE), followed by H3K27me3 CUT&RUN profiling of each lineage. The CUT&RUN biological replicates were highly reproducible (Supplementary Fig. 9a), and hierarchical clustering revealed clear separation between morulae and post-implantation lineages (Supplementary Fig. 9b). As validation of data quality, non-canonical imprinted genes (e.g., Gab1, Jade1/Phf17, and Sfmbt2) exhibited the expected maternally biased H3K27me3 enrichment at the morula stage (Supplementary Fig. 9c). Furthermore, lineage marker genes, Pou5f1 in EPI, Gata6 in VE, and Gata3 in EXE, displayed lineage-specific deletion of H3K27me3 in their respective tissues, further validating data quality (Supplementary Fig. 9d-f).

Having established data quality, we next examined the developmental dynamics of the aberrant H3K27me3 domains induced by loss of BAP1. We found that ectopic H3K27me3 gains in Bap1-null oocytes persist through the morula stage but are resolved in both embryonic and extraembryonic lineages by E6.5 (Fig. 7a-b). This observation resonates with our previous findings on non-canonical genomic imprinting 26, in which the maternally inherited H3K27me3 domains are maintained during preimplantation development but are subsequently lost following implantation.

A recent study reported that knock out of Ezhip, a negatively regulator of PRC2 78,79, causes excessive genome-wide H3K27me3 accumulation in early embryos and paradoxically disrupts non-canonical genomic imprinting 60. Prompted by this observation, we asked whether pervasive increase of H2AK119ub1 observed in Bap1-null oocytes might similarly affect allelic gene expression in later development. Consistent with the preserved allelic H3K27me3 enrichment at the non-canonical imprinted loci (Supplementary Fig. 9c), imprinted expression of these genes was largely maintained in the VE and EXE lineages (Fig. 7c, bolded genes). Similarly, canonical imprinting was minimally affected by maternal BAP1 loss (Fig. 7c). These data demonstrate that maternal BAP1 is dispensable for both canonical and non-canonical genomic imprinting.

Methods
Mouse strains and breeding
For DUB overexpression experiments and for western blot analyses of BAP1 protein in oocytes and early embryos, we used B6D2F1/J (BDF1; JAX strain #100006) male and female mice for IVF. C57BL/6J males (JAX strain #000664) were used for Bap1 CTR and CKO breeding trials and for embryonic development analyses at E3.5 and E18.5. To enable allelic resolution in chromatin profiling and RNA sequencing, PWK/PhJ males (JAX strain #003715) were used to generate late 2-cell, morula, and E6.5 embryos.

To generate oocyte specific Bap1 KO, Bap1flox animals (Jax strain #: 031874, B6 background) were crossed with Gdf9-iCre transgenic mice (Jax strain #: 011062, B6/129 mixed background) to produce Gdf9-iCre, Bap1 flox/+ males and Bap1 flox/+ females. These mice were intercrossed to obtain Bap1 flox/flox and Gdf9-iCre, Bap1 flox/flox females, which were used as CTR and CKO, respectively. To generate sperm specific Bap1 KO, Bap1flox animals were crossed with Prm-Cre mice (Jax strain #: 007252, B6/129 mixed background). The resulting Prm-Cre, Bap1 flox/+ females were subsequently bred with Bap1 flox/+ males to obtain Prm-Cre, Bap1 flox/flox males for downstream experiments. Genotypes were confirmed by PCR using genomic DNA extracted from tail biopsies. Genotyping primers are listed in Supplementary Data 3. Mice were euthanized in CO chamber followed by cervical dislocation.

Mice were housed under a 12:12 h light:dark cycle with controlled ambient temperature and humidity. All animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC) at Cincinnati Children’s Hospital Medical Center (protocol IACUC2025-0089).

Mouse superovulation, IVF, embryo culture, and fertility assessment
To induce superovulation, 8-12-week-old female mice were intraperitoneally injected with either 7.5 IU Pregnant Mare Serum Gonadotropin (PMSG; BioVendor) or ~0.15 mL CARD HyperOva (Cosmo Bio). After 44-48 hours, 7.5 IU Human Chorionic Gonadotropin (hCG; Sigma) was administered via intraperitoneal injection.

Approximately 16-18 hours post-hCG injection, cumulus-oocyte complexes (COCs) were collected from the oviductal ampullae. IVF was performed using sperm isolated from the cauda epididymis of 9-15-week-old male mice. For collection of Bap1 CTR and matKO embryos used in immunostaining, RNA-seq, and developmental rate analyses, sperm were obtained from wild-type C57BL/6J (Jax strain #: 000664) or Prm-Cre, Bap1flox/flox males and capacitated in Human Tubal Fluid (HTF; Millipore Sigma) medium for ~1 hour at 37 °C in a CO₂ incubator with ambient air. For CUT&RUN experiments, sperm were isolated from wild-type PWK/PhJ mice (Jax strain #: 003715) and capacitated in CARD FEARTIUP Preincubation Medium (Cosmo Bio).

The time of sperm addition to COCs was defined as 0 hours post-insemination (hpi). At ~6 hpi, zygotes exhibiting two pronuclei were selected, washed in M2 medium (Millipore), and cultured in KSOM medium (Millipore) at 37 °C in a CO₂ incubator under ambient air conditions. Brightfield images of oocytes and embryos were acquired using EVOSII cell imaging system (Thermo Fisher Scientific).

For fertility assessment and embryo collection at E3.5 and E18.5, Bap1 CTR and CKO females were co-caged with wild-type C57BL/6J males (Jax strain #: 000664). The presence of a vaginal plug the following morning was designated as E0.5. For E3.5 collections, blastocysts were flushed from the uterus using M2 medium (Millipore). For E18.5 collections, pregnant females were euthanized, and the number and weight of fetuses and placentas were assessed.

Dissection of E6.5 embryos into epiblast (EPI), visceral endoderm (VE), and extra-embryonic ectoderm (EXE) was described previously 90. Specifically, after removing Reichert’s membrane and ectoplacental cone, embryos were incubated on ice for 5 min in a disassociation solution containing 2.5% pancreatin (Sigma), 0.25% trypsin (Fisher Scientific), and 0.5% polyvinylpyrrolidone (Irvine Scientific). The digestion was stopped by adding an equal volume of 10% fetal bovine serum (FBS, HyClone) in PBS (Gibco). The VE layer was separated by gentle pipetting through a fine glass needle. EPI and EXE were then separated by cutting at the boundary between embryonic and extraembryonic regions using 27-gauge needles. The isolated EPI, VE, and EXE were washed three times in 0.2% BSA/PBS and stored in 0.2 ml tubes at -80°C.

Whole-mount immunostaining and histology analyses
For immunostaining, FGOs, zygotes, two-cell embryos, and blastocysts were fixed and permeabilized in 3.7% paraformaldehyde (Sigma) and 0.2% Triton X-100 (Sigma) in PBS (Gibco) at room temperature for 20 min. After four washes in 1% BSA/PBS, oocytes/embryos were incubated with primary antibodies at 4°C overnight. The following day, samples were washed four times in 1% BSA/PBS before incubated with secondary antibody at room temperature for 1 hr. Both primary and secondary antibodies were diluted in 1% BSA/PBS. Oocyte/embryos were then mounted using VECTASHIELD Antifade Mounting Medium with DAPI. Fluorescence was detected under a laser-scanning confocal microscope (Nikon A1R inverted), and images were analyzed using ImageJ (NIH).

Primary antibodies were rabbit anti-H3K27me3 (1:500, Cell Signaling, 9733), rabbit anti-H2AK119ub1 (1:2,000, Cell Signaling, 8240), rabbit anti-H3K27ac (1:500, Millipore Sigma, 07360), mouse anti-Flag (1:200, Sigma, F1804), rabbit anti-NANOG (1:200, Cosmo Bio, ATL-HPA072117-25), Goat anti-CDX2 (1:200, R&D, AF3665), or Mouse anti-GATA4 (1:200, R&D MAB2606). Secondary antibodies were Alexa Fluor 488 donkey anti-mouse IgG (1:200), Alexa Fluor 568 donkey anti-rabbit IgG (1:200), or Alexa Fluor 647 donkey anti-goat IgG (1:200) (Life Technologies). For histology analyses, paraffin-embedded ovaries were sectioned at 5 μm, and stained with hematoxylin and eosin.

Plasmid construction and mRNA preparation
Coding sequences for mouse Usp16 (NM_024258), Usp21 (NM_013919), Usp28 (NM_175482), Mysm1 (NM_177239), Bap1 (NM_027088), and the N-terminal region (amino acids 1–476) of Asxl1 (NM_015338) were synthesized by Twist Bioscience. Each sequence was cloned into the pcDNA3.1-Flag-poly(A)83 vector 91, inserted between an N-terminal Flag tag and an 83-nucleotide polyadenylation sequence. Plasmids were linearized by restriction enzyme digestion and used as templates for in vitro transcription with the mMESSAGE mMACHINE® T7 Ultra Kit (Life Technologies), following the manufacturer’s instructions. The resulting mRNA was purified by LiCl precipitation and quantified using a NanoDrop spectrophotometer.

Micro-injection of H2A DUB mRNAs
IVF experiments using BDF1 (Jax strain #: 100006) MII oocytes and sperm were the same as in previous sections. At 2 hpi, fertilized zygotes with the presence of 2nd polar body or fertilization cone were transferred into M2 medium (Sigma), and mRNA was injected into the cytoplasm of zygotes (~2–4 hpi) using a Piezo impact-driven micromanipulator (Eppendorf). Following injection, embryos were cultured in HTF (Millipore) medium for an additional 4 h before being transferred to KSOM (Millipore). The mRNA concentrations for each H2A DUB mRNA were 2 µg µl−1 except that 1 µg µl−1 were used for Bap1 and Asxl1 mRNAs when they were co-injected.

Western blotting
Oocytes and embryos collection and immunoblot analysis were as previously described 92. Specifically, oocytes and embryos were collected, and the zona pellucida were removed by a brief incubation in acid Tyrode’s solution (Sigma). After three washes with prewarmed 1% polyvinyl alcohol (PVA; Sigma) in PBS, samples in 5 µl of 1%PVA/PBS were transferred using a glass pipette into 15 µl of loading buffer containing 5µl of 4 × LDS sample buffer (Thermo Fisher Scientific) and 10 µl of water. Samples were snap-frozen in dry ice for 5 min, and the freeze-thaw cycle was repeated once before storage at -80°C. Samples were heated at 90°C for 5 min and loaded onto a 4-12% Bis-Tris Protein gel (Thermo Fisher Scientific). Proteins were transferred to a PVDF membrane using the Trans-Blot Turbo Transfer System (Bio-Rad). Membranes were blocked in 3% BSA/PBS for 1 h at room temperature and incubated with primary antibodies overnight at 4 °C. The following day, membranes were washed and incubated with HRP-conjugated secondary antibodies for 1 h at room temperature. Chemiluminescence was detected using an Amersham Imager 680. Primary antibodies against BAP1 (1/1000 dilution; CST 13271), Tubulin (1/1000 dilution; CST 2144A), Beta-actin (1/1000, sc-47778) were used in this study. Secondary antibodies were HRP-linked anti-rabbit IgG (CST 7074P2; 1:1000) and anti-mouse IgG (CST 7076P2; 1:1000).

RNA-seq and CUT&RUN library preparation and sequencing
For oocytes and preimplantation embryos, RNA-seq libraries were generated using the SMART-Seq Stranded Kit (Takara) according to the manufacturer’s instructions. Briefly, oocytes and embryos were incubated in acid Tyrode’s solution to remove zona pellucida and then washed three times in 0.2% BSA/PBS. Samples were transferred to 0.2-ml PCR tubes using a glass needle with minimal carryover (~0.5-1 µl). After adding 7 µl of RNase inhibitor water and 6 µl of shearing mix, samples were lysed at 85 °C to fragment RNA. First-strand cDNA synthesis was performed using random primers, followed by an initial PCR amplification (10 cycles). The resulting cDNA was purified, and rRNA-derived sequences were removed by probe-mediated cleavage. Final RNA-seq libraries were prepared with an additional PCR amplification (10 cycles).

For E6.5 embryos, RNA-seq libraries were generated using SMART-Seq mRNA (Takara) in combination with the Nextera XT Sample Preparation Kit (Illumina). Reverse transcription was performed using oligo-dT according to the manufacturer’s instructions, and cDNA was subsequently amplified by PCR (13 cycles). For each sample, 400 pg of amplified cDNA was used for library preparation. The Nextera XT kit was then used for cDNA fragmentation, adaptor ligation, and library amplification, following the manufacturer’s protocols.

CUT&RUN library preparation was performed as previously described 93 with minor modifications. For FGOs, the zona pellucida was removed by brief incubation in acid Tyrode’s solution. For late 2-cell embryos, zona-free embryos were further incubated in TrypLE (Thermo Fisher Scientific) supplemented with 0.5% PVP (Irvine Scientific) to remove polar bodies. Oocytes and embryos were transferred to 0.2 ml CUT&RUN 8-strip tubes (EpiCypher) containing 60 µl wash buffer (20 mM HEPES-NaOH pH 7.5, 150 mM NaCl, 0.5 mM spermidine, and 1× Roche protease inhibitor) and 10 µl Concanavalin A-coated beads (Polysciences) pre-activated in bead binding buffer (20 mM HEPES-KOH pH 7.9, 10 mM KCl, 1 mM CaCl₂, 1 mM MnCl₂). For some CUT&RUN reactions, Drosophila cell nuclei (Active motif) were used for spike-in normalization. The number of Drosophila cell nuclei used for each experiment are included in Supplementary Data 3. Beads and cells were mixed by gentle tapping and rotated on a nutator for 10-15 min at room temperature. Tubes were briefly spun down and placed on a magnetic stand to remove supernatant without disturbing the bead-cell complexes. Samples were incubated with primary antibodies diluted in antibody incubation buffer (1× wash buffer, 0.02% digitonin, 2 mM EDTA) at 4 °C overnight on a thermomixer (300 rpm). The following day, samples were washed once with 200 µl Dig-wash buffer (1× wash buffer, 0.02% digitonin) and once with 100 µl Dig-wash buffer, with each wash performed for 30 s on a magnetic stand. Beads/cells were then incubated with 5.6 ng/ml pAG-MNase (homemade) at 4 °C on a thermomixer (300 rpm) for 2–3 h. Afterward, samples were washed twice with 200 µl Dig-wash buffer and incubated with 100 µl Dig-wash buffer on ice for 3 min. To initiate pAG-MNase digestion, 2 µl of 100 mM CaCl₂ was added and mixed by gentle tapping. Digestion proceeded on ice for 30 min and was stopped by adding 100 µl 2× STOP buffer (340 mM NaCl, 20 mM EDTA, 4 mM EGTA, 50 µg/ml RNase A, 100 µg/ml glycogen, 0.02% digitonin). Samples were incubated at 37 °C for 30 min to release short DNA fragments, after which the supernatant was supplemented with 40 ng carrier RNA and purified by phenol-chloroform extraction. CUT&RUN libraries were prepared using the NEBNext Ultra II DNA Library Prep Kit (NEB) according to the manufacturer’s instructions.

For morula-stage CUT&RUN, a modified protocol was used in which primary antibody binding and pAG-MNase binding were performed prior to binding cells to Concanavalin A beads 94. Postnatal day 1 oocyte collection and H3K27ac CUT&TAG was as previously described 95.

All libraries were sequenced on the Illumina NovaSeq X Plus platform at Novogene (2 ✕ 150bp). The number of oocytes and embryos used for RNA-seq/CUT&RUN, sequencing depth, and reads alignment are included in Supplementary Data 3.

RNA-seq data processing
Adaptor trimming, read alignment, and read count generation for both genes and repeat subfamilies were performed as previously described 74. Differential gene expression analysis was carried out using DESeq2 (v1.38.3)96, applying the criteria: adjusted p-value < 0.05 and fold change ≥ 2. For a gene to be considered differentially expressed, the higher-expression group was additionally required to have FPKM > 0.5. Gene Ontology (GO) term enrichment among differentially expressed genes (DEGs) was assessed using the enrichGO function in the clusterProfiler R package (v4.6.2)97. BigWig files for visualization were generated using deepTools (v2.0.0)98 with the following parameters: --binSize 25 --normalizeUsing RPKM --scaleFactor 1. Gene sets corresponding to zygote minor ZGA genes, early 2-cell minor ZGA genes, late 2-cell major ZGA genes, and maternal transcripts were obtained from 30. Enrichment of DEGs across gene categories defined in the DBTMEE_v2 database 65 was analyzed using the enrichr function in clusterProfiler (v4.6.2)97. For E6.5 stage RNA-seq data, to avoid alignment bias toward the reference genome (C57BL/6), reads were aligned to a custom genome where single-nucleotide polymorphisms (SNPs) between PWK/PhJ and B6/129 strains were masked with "N". SNPsplit (v0.3.2)(Krueger and Andrews, 2016) was used to determine parental origin of read pairs. Imprinted gene list at E6.5 were obtained from 24.

CUT&RUN data processing
Raw paired-end reads from CUT&RUN experiments were trimmed using TrimGalore (v0.6.6) (https://github.com/FelixKrueger/TrimGalore) with the --paired option. For FGOs, trimmed reads were aligned to the mm10 mouse reference genome using Bowtie2 (v2.4.2)99 with the parameters: --no-unal --no-mixed --no-discordant -I 0 -X 1000. Uniquely mapped reads were filtered using Sambamba (v0.6.8) 100 with the criteria: -F "mapping_quality >= 30 and not (unmapped or mate_is_unmapped)". PCR duplicates were removed using Picard (v2.18.22) (http://broadinstitute.github.io/picard/). BigWig coverage files were generated using deepTools (v2.0.0)98 with the options: --binSize 25 --normalizeUsing RPKM --scaleFactor 1.

For late 2-cell (L2C), morula, and E6.5 stage CUT&RUN data, to avoid alignment bias toward the reference genome (C57BL/6), reads were aligned to a custom genome where SNPs between PWK/PhJ and B6/129 strains were masked with "N". SNPsplit (v0.3.2)101 was used to determine parental origin of read pairs. For H2AK119ub1, H3K27me3, and H3K27ac oocyte CUT&RUN datasets, spike-in normalization was performed by aligning reads to the Drosophila melanogaster reference genome (fly46), and scaling factors were calculated as described by 102(see Supplementary Data 3). Since oocyte chromatin exhibits non-canonical, broad enrichment of H2AK119ub1, H3K27me3, and H3K27ac, a hidden Markov model (HMM)-based domain caller 18 was used to determine broad domain boundaries for these histone modifications.

Reproducibility between biological replicates was assessed by calculating the Pearson correlation coefficient of FPKM values computed in non-overlapping 5-kb genomic bins. For downstream analyses, biological replicates were pooled using the merge command from Sambamba (v0.6.8)100. The heatmaps of CUT&RUN profiles were generated using deepTools (v2.0.0)98 computeMatrix and plotHeatmap.

Identification of H3K27ac-lost regions in Bap1 CKO FGOs
FPKM values were computed in non-overlapping 5-kb genomic bins across the genome. Bins with FPKM ≥ 2 and fold change ≥ 1.5 between conditions were classified as differentially enriched bins. To define broader enriched regions, adjacent differentially enriched 5-kb bins were merged using BEDTools (v2.30.0) 103. Only merged regions larger than 10 kb were retained and defined as differentially enriched regions. The regions were annotated using ‘‘makeTxDbFromGFF’’ and ‘‘annotatePeak’’ function from the ‘‘ChIPpeakAnno (v4.4.1)’’, ‘‘GenomicFeatures (v4.4.1)’’, and ‘‘ChIPseeker (v4.4.2)’’ Bioconductor R packages 104-106. Note that multiple H3K27ac-lost regions could be identified from a single H3K27ac domain defined by the HMM-based method.

Analyses at PcG targets
The list of oocyte and ESC PcG target genes was obtained from 60 and 61, respectively. The oocyte Polycomb targets were identified based on the H3K27me3 enrichment in fully grown oocytes 60. To identify PcG targets that retain H3K27me3 at the late 2-cell stage, FPKM values at promoter regions (±2.5 kb around the transcription start site) were calculated. Genes with FPKM ≥ 2 were considered as retaining H3K27me3. This cutoff was determined based on visual inspection of representative loci, and the resulting gene list was found to be comparable to 61.

Annotations and genomic intervals
The gene annotation file (GFF3 format) was downloaded from GENCODE (release M25) (https://www.gencodegenes.org/). Gene promoters were defined as ±2.5 kb surrounding the transcription start sites (TSS). Gene bodies were defined from the TSS to the annotated transcription end site (TES). Intergenic regions were defined as genomic regions that do not overlap with annotated promoters or gene bodies. Putative enhancers specific to FGOs and late 2-cell embryos were obtained from 29. Gene-rich and gene-poor genomic regions were defined as previously described 107. All mm9-based genomic coordinates were converted to the mm10 assembly using the liftOver function from the rtracklayer R package (v1.58.0)108.

Visualization and statistical analyses
All statistical analyses were performed using R (v4.2.2). Statistical tests and sample sizes are indicated in the figure legends. All genomic browser tracks were visualized using the UCSC genome browser 109. Figure 8 was generated using BioRender.

Reanalysis of Public datasets
All public ChIP-seq, CUT&RUN, and RNA-seq datasets were processed using pipelines and parameters similar to those used for in-house data.

Statistics and Reproducibility
Statistical analyses were performed with R (https://www.r-project.org/) and GraphPad Prism 9.5 software. All statistical methods, sample sizes, and p-values are indicated in Figures or Figure legends. Reproducibility between RNA-seq and CUT&RUN biological replicates were evaluated by calculating Pearson’s r coefficient. No statistical method was used to predetermine sample size. The experiments were not randomized. The investigators were not blinded to allocation during experiments and outcome assessments.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In this paper, the authors analyzed the role of BAP1 in the formation of specific chromatin domains in oocytes and demonstrated that BAP1 prevents the accumulation of pervasive H2AK119ub1, protects oocyte-specific H3K27ac, and prevents the establishment of H3K27me3. Experimental results showed that loss of internal BAP1 leads to abnormalities in oocyte developmental potential and fertility. These results suggest that PR-DUB plays a role in establishing the oocyte epigenome and protecting its reproductive potential. While the discovery that BAP1 regulates H2AK119ub1 as a PR-DUB component is not surprising, given previous reports in mouse ES cells and other studies, their experiments using oocytes support the authors' conclusions and provide a logical explanation for the role of BAP1 in properly establishing the oocyte epigenome. We believe this paper is worthy of publication in Nature Communications. However, to improve the manuscript, we offer several comments below.\n1. The experiments in Fig. 1a-c raised questions about how PR-DUB activity is regulated. BAP1 alone has no effect on H2AK119ub1, but co-expression with ASXL1 reduces H2AK119ub1. ASXL1 appears to be expressed at endogenous levels at this stage, so dependence on ASXL1 is unclear. Furthermore, the significance of the experiments in Fig. 1a-c is unclear. Given their overexpression, can we discuss the importance of DUBs in the zygote? Rather, it is more important to examine the effect of loss of function of DUBs on H2AK119ub1. It is known that BAP1 is involved in the de-ubiquitination of H2AK119ub1, and the authors demonstrate this in Fig. 1g-h. What happens if other DUBs are knocked out? Furthermore, NAR (https://doi.org/10.1093/nar/gkac468) reports that USP16 is involved in the de-ubiquitination of H2AK119ub1 at this stage. What is the reason for this contradiction?\n2. The authors claim in Fig. 5 that maternal BAP1 KO results in abnormal maternal RNA decay, but it is unclear how BAP1 is involved in RNA decay. Is it possible that BAP1 KO simply delays the development, or is BAP1 involved in repression of the RNAs? Are there any changes in the levels of H2AK119ub1 and H3K27me3 at genes whose expression is upregulated in BAP1 KO?\n3. In Fig. 2, the relationship between changes in H2AK119ub1 and H3K27ac/me3 is unclear. Direct inspection of those changes will be more informative.", "reviewer_2": "Kang et al, examine the importance of the erasers of histone modifications in oocytes and female fertility. In particular, they focus on the histone modification H2AK119ub1. They began the study with an intriguing overexpression screen of all reported deubiquitinases for this histone modification- this is a fantastic experiment as no previous study has directly compared these erasers in the same assay. This revealed that USP21 and the PR-DUB complex are the key erasers for H2AK119ub1- certainly in terms of global levels of the modification.\nThey then established a BAP1 conditional knockout system to remove this enzymatic subunit of the PR-DUB complex. They showed that this impacted the epigenome by inducing pervasive accumulation of H2AK119ub1 nearly genome-wide, in keeping with previous studies in drosophila and embryonic stem cells. Unlike these previous studies, that reported a dual role for BAP1 in repressing transcription of Polycomb target genes and activating other genes, in oocytes the team discovered that BAP1 primarily serves as an activator and has limited impact on maintaining Polycomb target gene repression.\nInstead, they found that the ectopic deposition of H2AK119ub1 led to changes in active enhancer maintenance ultimately causing their invasion by the repressive modification H3K27me3. Most strikingly, they went on to show that BAP1 plays a critical role in fertility, as its loss had a massive impact on number of litters and pups per litter. Indeed, a further model system that removed residual paternal BAP1 from sperm in addition to maternal BAP1 loss displayed an even more striking phenotype. Together the data show a critical role for BAP1 in reproduction and indeed in remodelling the epigenome following fertilisation- as aberrant H3K27me3 patterns from oocytes were shown to persist in the early embryo. This coincided with major defects in gene expression, particularly in maternal-to-zygotic transition in this BAP1 conditional knockout system.\nOverall, the authors have made some critical discoveries that will be very important to the chromatin, epigenetics and developmental biology fields. This essential role for an eraser of Polycomb-mediated modifications in fertility is extremely striking and the mechanistic work builds upon previous mechanisms to further unravel cell-type specific functions of BAP1. The work itself is of very high quality, with the majority of experiments being technically and statistically sound. In particular, the authors did an excellent job managing interpretations of their work and clearly putting it in the context of other work in the field. There are some points below which could strengthen the manuscript.\nMajor point\n• Spike-in normalisation has become the standard for chromatin mapping assays including ChIP-seq and CUT&RUN. The authors utilized this for CUT&RUN of H2AK119ub1 in figure 1 however, they did not use spike-in normalisation for H3K27ac and H3K27me3 CUT&RUN in figure 2. This normalisation is particularly critical for histone modifications that are abundant at gene poor regions. To interpret the three histone modifications CUT&RUNs using the same analysis but with only one including the correct spike-in normalisation is not prudent. This could have a major impact on the interpretations of figure 2 and therefore the importance of enhancer activation in the proposed BAP1 mechanism. I would suggest repeating these CUT&RUN with the spike-in normalisation procedure they used for H2AK119ub1 to ensure correct interpretation of the results.\n• In figure 2 the relationship between changes in H3K27ac and H3K27me3 is generally examined on an individual locus basis at specific exemplar loci but never genome-wide or at multiple domains/peaks. The authors should generate heatmaps using the same domains/peaks for both histone modifications in CTR and CKO conditions to examine if H3K27ac is lost where H3K27me3 is gained. An alternative approach would be to generate XY scatterplots of the fold change in each histone modification abundance at genomic regions. This would allow a direct assessment of the relationship between H3K27ac and H3K27me3 on a genome-wide scale rather than relying on individual example loci. These additional analyses may help support the figure 2 title statement in particular. The relationship between lost H3K27ac and gained H2AK119ub1 is also not examined, this could provide a more direct mechanistic link between the observed changes in H3K27ac and BAP1 function.\n• The authors state that they identified 10,363 H3K27ac domains in CTR (control) cells (figure 2) and find that 9,043 H3K27ac domains are lost (figure 6). This doesn’t seem to match well with the scale of difference between CTR and BAP1 CKO cells shown in figure 2C. The ‘domain’ system that is being used is seemingly at odds with typical peak based definition of sites enriched for histone modifications – through programs such as MACS or SEACR which are more commonly found in chromatin mapping studies.\n• A major finding of this study compared to previous research on BAP1 is that there are few PRC1 or PRC2 target genes activated in response to BAP1 loss. The authors state that this may be a cell-type specific feature of oocytes. In figure 3, the genes that are defined as Polycomb targets are based on a mESCs dataset. Polycomb complexes are highly mobile in different cell types and stages of development. Therefore, representative PRC1 and PRC2 complex member CUT&RUNs are required to define their specific targets in Oocytes to ensure the authors interpretation that there is minimal effect on Polycomb silencing in BAP1 CKO is correct.\n• An additional point regarding this distinctive transcriptional phenotype is that while its in oocytes, late 1-cell and early 2-cell stages there are very few upregulated genes compared to downregulated (figure 5B). However, at the late 2-cell stage there are >1000 upregulated genes in BAP1 mutant cells. It would be very interesting to compare the overlap of the upregulated genes across these different cell stages to assess at which point PRC1 and PRC2 target genes begin to be upregulated, as they are in ESCs.\nMinor point\n• Citing previously described methods, instead of reiterating the method is poor practice and can lead to reproducibility issues as small details can be lost in translation. Please include full methods descriptions, particularly for CUT&RUN, but most method sections seem to be overly brief.\n• The authors should be clearer on what regions/ peaks are being examined in the heatmaps in figure 1J. The legends says these are H2AK119ub1 domains but the main text says actively transcribed regions. They appear largely devoid of H2AK119ub1 in control cells so defining the sites clearly is key to interpreting the data. This should also be made clear in the figure 2C legend for H3K27ac and H3K27me3 domains heatmaps. The legends direct the reader to the methods but these simply reference a previous HMM model and do not inform the reader on what these domains actually are (intergenic, active promoter, enhancer etc).\n• Figure 2E shows genomic localisation of ‘H3K27ac lost’ domains, and appears to be making the point that enhancers are specifically effected by BAP1 loss. However, it doesn’t show how this differs from H3K27ac retained domains, or all H3K27ac domains. Specificity cannot be inferred without including this information.\n• ‘Polycom’ -> ‘Polycomb’ typo in the figure 3 title.", "reviewer_3": "This manuscript by Kang et al. investigates the role of the Polycomb Repressive-Deubiquitinase (PR-DUB) complex, and specifically its catalytic subunit BAP1, during mouse oogenesis and early embryogenesis. Using conditional knockout models and multi-omics profiling, the authors show that BAP1 removes H2AK119ub1 to restrain Polycomb activity, prevent ectopic H3K27me3 deposition, and maintain broad oocyte-specific H3K27ac domains. Loss of maternal BAP1 leads to transcriptional repression of oocyte genes, defective zygotic genome activation, and female subfertility. Importantly, aberrant H3K27me3 domains established in BAP1-deficient oocytes persist into early embryos, suggesting an intergenerational transmission of repressive chromatin states.\nThe study is technically strong and extends well-established Polycomb principles to a physiologically relevant setting. However, the mechanistic insight is limited, as the findings largely confirm the known model in which BAP1 counterbalances PRC1-mediated H2AK119ub1 to restrict PRC2 spreading. The main novelty lies in demonstrating this mechanism in the oocyte context and connecting it to fertility and developmental competence. The assertion that BAP1 protects H3K27ac is intriguing but remains correlative; direct evidence that increased H2AK119ub1 leads to loss of H3K27ac is lacking.\nThe most original and conceptually important aspect of this work concerns the intergenerational inheritance of abnormal H3K27me3 domains. This observation resonates with prior findings across species. In mice, Inoue and colleagues showed that maternal loss of H3K27me3 can persist after fertilization and influence imprinting; in Drosophila, Zenk and collaborators demonstrated germline transmission of Polycomb-associated chromatin; and in C. elegans, Gaydos and co-workers revealed PRC2-dependent transgenerational inheritance of H3K27me3. Kang et al. now describe the converse phenomenon: the persistence of maternal gain of H3K27me3 in embryos. This represents an important addition to the field of epigenetic inheritance. However, this intergenerational aspect is not sufficiently emphasized in the current manuscript. It should be more thoroughly developed, both in the results and in the discussion, as it constitutes the most significant conceptual contribution of the study. The above studies should be cited.\nOverall, this is a high-quality descriptive study with clear developmental and reproductive relevance, though its mechanistic novelty remains modest. The authors should strengthen causality and clarify whether BAP1 directly maintains H3K27ac or acts indirectly by limiting PRC2 activity. It would also greatly enhance the paper to deepen the discussion and analysis of the intergenerational dimension, which links this work to a broader literature on chromatin-based inheritance across species.\nTo reinforce their conclusions, the authors should conduct targeted experiments that test causality between H2AK119ub1 accumulation and H3K27ac loss. A mechanistic rescue or inhibition approach, such as PRC2 inhibition using GSK126 or EED226, or USP21/BAP1+ASXL1 overexpression, could determine whether blocking PRC2 spreading restores H3K27ac at affected loci. They should also examine whether enhancer machinery components such as p300/CBP, BRD4, or MLL3/4 change occupancy at the regions that lose H3K27ac, which would clarify whether BAP1 directly supports enhancer activation. Temporal analyses or acute depletion experiments could further demonstrate whether H2AK119ub1 elevation precedes H3K27me3 gain and H3K27ac loss, establishing the directionality of events. Finally, the authors should extend their intergenerational analysis by quantifying how long the ectopic H3K27me3 domains persist beyond the two-cell stage and by comparing this to the inheritance mechanisms described by Inoue, Zenk, and Gaydos.\nMinor issues include clarifying the terminology of PR-DUB (whether it refers exclusively to the BAP1–ASXL complex), providing full information on biological replicates and statistical details, improving figure clarity by using distinct color scales for H3K27ac and H3K27me3.\nIn summary, this paper offers a valuable dataset and an important extension of Polycomb biology into mammalian oogenesis and inheritance. The work convincingly demonstrates that BAP1 is essential for safeguarding the oocyte epigenome and fertility, but its broader impact will depend on strengthening mechanistic causality and expanding the intergenerational perspective, which represents the most compelling aspect of this study. With these revisions, the manuscript could meet the conceptual standards of Nature Communications.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_5": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "Responses to the Reviewers’ comments\nWe thank the reviewers for their time and for providing constructive feedback on our manuscript.\nBelow, we provide point-by-point responses to each comment. In this letter, we use “Fig. #” to\nrefer to figures in the manuscript and “Fig. R#” to refer to figures included in this response.\nReviewer #1 (Remarks to the Author)\nIn this paper, the authors analyzed the role of BAP1 in the formation of specific chromatin domains\nin oocytes and demonstrated that BAP1 prevents the accumulation of pervasive H2AK119ub1,\nprotects oocyte-specific H3K27ac, and prevents the establishment of H3K27me3. Experimental\nresults showed that loss of internal BAP1 leads to abnormalities in oocyte developmental potential\nand fertility. These results suggest that PR-DUB plays a role in establishing the oocyte epigenome\nand protecting its reproductive potential. While the discovery that BAP1 regulates H2AK119ub1\nas a PR-DUB component is not surprising, given previous reports in mouse ES cells and other\nstudies, their experiments using oocytes support the authors' conclusions and provide a logical\nexplanation for the role of BAP1 in properly establishing the oocyte epigenome. We believe this\npaper is worthy of publication in Nature Communications. However, to improve the manuscript,\nwe offer several comments below.\nResponse: We appreciate the reviewer’s positive summary and support for the publication of our\nwork.\nComment 1. The experiments in Fig. 1a-c raised questions about how PR-DUB activity is\nregulated. BAP1 alone has no effect on H2AK119ub1, but co-expression with ASXL1 reduces\nH2AK119ub1. ASXL1 appears to be expressed at endogenous levels at this stage, so\ndependence on ASXL1 is unclear.\nResponse: We were also surprised that overexpression of BAP1 alone did not alter H2AK119ub1\nlevels in zygotes, despite the presence of endogenous ASXL1. It is likely that the overexpressed\nBAP1, when introduced alone, exceeds the available pool of ASXL binding partners. Since ASXL\nproteins are essential cofactors for BAP1’s catalytic activity, the unbound (“free”) BAP1 would\nthus remain catalytically inactive and unable to globally remove H2AK119ub1.\nComment 2. Furthermore, the significance of the experiments in Fig. 1a-c is unclear. Given their\noverexpression, can we discuss the importance of DUBs in the zygote? Rather, it is more\nimportant to examine the effect of loss of function of DUBs on H2AK119ub1. It is known that BAP1\nis involved in the de-ubiquitination of H2AK119ub1, and the authors demonstrate this in Fig. 1g-\nh. What happens if other DUBs are knocked out?\nResponse: We agree with the reviewer that a gain-of-function approach is less definitive than a\nloss-of-function strategy for elucidating gene function, and we have been cautious in interpreting\nour results. The experiments in Fig. 1a-c were intended primarily as a preliminary screen to\nidentify candidate DUBs that may function as key erasers of H2AK119ub1 at this stage, providing\na basis for subsequent, more rigorous loss-of-function analyses. Since other DUBs are highly\nexpressed in oocytes, their efficient depletion would require ZP3Cre- or Gdf9Cre-mediated\nconditional knockouts. Such experiments are beyond the scope of the current study but represent\nan important direction for future work.\nWe have included following texts related to the above response in lines 105-109: “It should be\nnoted that we cannot exclude the possibility that USP16 (discussed further below), USP28, and\nMYSM1 may function as H2A DUBs at other developmental stages or require additional cofactors\nin zygotes. Future loss-of-function studies will be necessary to determine their contributions to\nH2AK119ub1 regulation during oogenesis and early embryonic development.”\nComment 3. Furthermore, NAR (https://doi.org/10.1093/nar/gkac468) reports that USP16 is\ninvolved in the de-ubiquitination of H2AK119ub1 at this stage. What is the reason for this\ncontradiction?\nResponse: We do not think that these two studies contradict each other. Rong et al did not directly\nexamine whether overexpression of USP16 affects H2AK119ub1 levels in zygotes as we\ndescribed in our manuscript. Instead, they showed that loss of USP16 causes increase of\nH2AK119ub1 specifically during GV-to-MII transition, but not in GV oocytes nor zygotes (Rong et\nal., Fig. 4A-B and Fig. S6A-B). Future studies will be needed to perform side-by-side\ncomparisons to determine how the loss of different DUBs affects H2AK119ub1 and oocyte\ndevelopmental competence.\nComment 4. The authors claim in Fig. 5 that maternal BAP1 KO results in abnormal maternal\nRNA decay, but it is unclear how BAP1 is involved in RNA decay. Is it possible that BAP1 KO\nsimply delays the development, or is BAP1 involved in repression of the RNAs? Are there any\nchanges in the levels of H2AK119ub1 and H3K27me3 at genes whose expression is upregulated\nin BAP1 KO?\nResponse: As shown in Fig. 6C, there is minimal enrichment of H3K27me3 at maternal genes in\nBap1 control and maternal KO (matKO) embryos. Therefore, H3K27me3 is unlikely to be directly\ninvolved in the repression of maternal transcripts in late 2-cell embryos. Given that ~800 genes\nwere already downregulated in Bap1 CKO MII oocytes, the subsequent maternal-to-zygotic\ndefects, including abnormal maternal RNA decay, may represent secondary effects and/or\nembryonic delay resulting from the reduced developmental competence of Bap1 null oocytes.\nWe have included following texts related to the above response in lines 330-331: “This also\nsuggests that the observed upregulation of maternal transcripts (Fig. 5e-f) likely reflects impaired\nmaternal mRNA decay rather than transcriptional derepression resulting from altered H3K27me3.”\nand lines 443-445: “Failure to activate these genes compromises the developmental competence\nof Bap1-null oocytes, and the matKO embryos subsequently fail to properly complete the MZT,\nresulting in defective preimplantation development.”\nComment 5. In Fig. 2, the relationship between changes in H2AK119ub1 and H3K27ac/me3 is\nunclear. Direct inspection of those changes will be more informative.\nResponse: We thank the reviewer for raising this important point. As suggested, we examined\nthe enrichment of histone modifications at H3K27ac-lost and H3K27ac-unchanged regions in both\nBap1 control (CTR) and conditional knockout (CKO) FGOs. As shown in Fig. R1, loss of H3K27ac\nin Bap1 CKO FGOs is globally associated with a corresponding gain of H3K27me3. In contrast,\nH2AK119ub1 levels increased similarly in both H3K27ac-lost and H3K27ac-unchanged regions.\nThis heatmap is also consistent with the boxplot analyses (Supplementary Fig. 3c). These\nresults suggest that the increase in H2AK119ub1 is not the sole determining factor that dictates\nwhich regions lose H3K27ac and gain H3K27me3.\nWe have included Fig. R1 in the revised manuscript as new Fig. 2e. Related text updates are in\nlines 184-188.\nFig. R1. Heatmap illustrating changes of\nhistone modifications at the H3K27ac-\nlost (n = 9,043) and H3K27ac-no change\n(n = 27,287) regions in Bap1 CTR and\nCKO fully grown oocytes (FGOs).\nReviewer #2 (Remarks to the Author)\nKang et al, examine the importance of the erasers of histone modifications in oocytes and female\nfertility. In particular, they focus on the histone modification H2AK119ub1. They began the study\nwith an intriguing overexpression screen of all reported deubiquitinases for this histone\nmodification- this is a fantastic experiment as no previous study has directly compared these\nerasers in the same assay. This revealed that USP21 and the PR-DUB complex are the key\nerasers for H2AK119ub1- certainly in terms of global levels of the modification.\nThey then established a BAP1 conditional knockout system to remove this enzymatic subunit of\nthe PR-DUB complex. They showed that this impacted the epigenome by inducing pervasive\naccumulation of H2AK119ub1 nearly genome-wide, in keeping with previous studies in drosophila\nand embryonic stem cells. Unlike these previous studies, that reported a dual role for BAP1 in\nrepressing transcription of Polycomb target genes and activating other genes, in oocytes the team\ndiscovered that BAP1 primarily serves as an activator and has limited impact on maintaining\nPolycomb target gene repression.\nInstead, they found that the ectopic deposition of H2AK119ub1 led to changes in active enhancer\nmaintenance ultimately causing their invasion by the repressive modification H3K27me3. Most\nstrikingly, they went on to show that BAP1 plays a critical role in fertility, as its loss had a massive\nimpact on number of litters and pups per litter. Indeed, a further model system that removed\nresidual paternal BAP1 from sperm in addition to maternal BAP1 loss displayed an even more\nstriking phenotype. Together the data show a critical role for BAP1 in reproduction and indeed in\nremodelling the epigenome following fertilisation- as aberrant H3K27me3 patterns from oocytes\nwere shown to persist in the early embryo. This coincided with major defects in gene expression,\nparticularly in maternal-to-zygotic transition in this BAP1 conditional knockout system.\nOverall, the authors have made some critical discoveries that will be very important to the\nchromatin, epigenetics and developmental biology fields. This essential role for an eraser of\nPolycomb-mediated modifications in fertility is extremely striking and the mechanistic work builds\nupon previous mechanisms to further unravel cell-type specific functions of BAP1. The work itself\nis of very high quality, with the majority of experiments being technically and statistically sound.\nIn particular, the authors did an excellent job managing interpretations of their work and clearly\nputting it in the context of other work in the field. There are some points below which could\nstrengthen the manuscript.\nResponse: We thank the reviewer’s detailed summary and the enthusiasm on our work.\nMajor point\nComment 1. • Spike-in normalisation has become the standard for chromatin mapping assays\nincluding ChIP-seq and CUT&RUN. The authors utilized this for CUT&RUN of H2AK119ub1 in\nfigure 1 however, they did not use spike-in normalisation for H3K27ac and H3K27me3 CUT&RUN\nin figure 2. This normalisation is particularly critical for histone modifications that are abundant at\ngene poor regions. To interpret the three histone modifications CUT&RUNs using the same\nanalysis but with only one including the correct spike-in normalisation is not prudent. This could\nhave a major impact on the interpretations of figure 2 and therefore the importance of enhancer\nactivation in the proposed BAP1 mechanism. I would suggest repeating these CUT&RUN with\nthe spike-in normalisation procedure they used for H2AK119ub1 to ensure correct interpretation\nof the results.\nResponse: We agree with the reviewer’s point. As the reviewer suggested, we performed\nH3K27me3 and H3K27ac CUT&RUN with fly nuclei spike-in normalization. The scale factors for\nH3K27me3 and H3K27ac range from 1.0-1.1 (Supplementary Table S3), making the spike-in\nnormalized data very similar to the standard FPKM normalized data. As a result, the use of spike-\nin normalized data does not change our interpretations of original Fig. 2 and Fig. 3.\nWe have updated following figure panels using the spike-in normalized data: Fig. 2c-e, 2j, 3a-b,\nand Supplementary Fig. 3a-d.\nComment 2. • In figure 2 the relationship between changes in H3K27ac and H3K27me3 is\ngenerally examined on an individual locus basis at specific exemplar loci but never genome-wide\nor at multiple domains/peaks. The authors should generate heatmaps using the same\ndomains/peaks for both histone modifications in CTR and CKO conditions to examine if H3K27ac\nis lost where H3K27me3 is gained. An alternative approach would be to generate XY scatterplots\nof the fold change in each histone modification abundance at genomic regions. This would allow\na direct assessment of the relationship between H3K27ac and H3K27me3 on a genome-wide\nscale rather than relying on individual example loci. These additional analyses may help support\nthe figure 2 title statement in particular. The relationship between lost H3K27ac and gained\nH2AK119ub1 is also not examined, this could provide a more direct mechanistic link between the\nobserved changes in H3K27ac and BAP1 function.\nResponse: We thank the reviewer for raising this important point. As suggested, we examined\nthe enrichment of histone modifications at H3K27ac-lost and H3K27ac-unchanged regions in both\nBap1 control (CTR) and conditional knockout (CKO) FGOs. As shown in Fig. R1, loss of H3K27ac\nin Bap1 CKO FGOs is globally associated with a corresponding gain of H3K27me3. In contrast,\nH2AK119ub1 levels increased similarly in both H3K27ac-lost and H3K27ac-unchanged regions.\nThis heatmap is also consistent with the boxplot analyses (Supplementary Fig. 3c). These\nresults suggest that the increase in H2AK119ub1 is not the sole determining factor that dictates\nwhich regions lose H3K27ac and gain H3K27me3.\nWe have included Fig. R1 in the revised manuscript as new Fig. 2e. Related text updates are in\nlines 184-188.\nComment 3. • The authors state that they identified 10,363 H3K27ac domains in CTR (control)\ncells (figure 2) and find that 9,043 H3K27ac domains are lost (figure 6). This doesn’t seem to\nmatch well with the scale of difference between CTR and BAP1 CKO cells shown in figure 2C.\nThe ‘domain’ system that is being used is seemingly at odds with typical peak based definition of\nsites enriched for histone modifications – through programs such as MACS or SEACR which are\nmore commonly found in chromatin mapping studies.\nResponse: The oocyte chromatin exhibits non-canonical, broad enrichment of several histone\nmodifications, including H2AK119ub1, H3K27me3, and H3K27ac. For this reason, we employed\nthe HMM-based method previously described in Chen et al., 2021 (PMID: 33821005), as it more\naccurately captures these widespread chromatin domains. In contrast, SEACR produces less\ncoherent results, yielding fragmented peaks with mixed widths (Fig. R2). Furthermore, multiple\nsubregions within a single control H3K27ac domain can display reduced H3K27ac in CKO\noocytes (Fig. R3), which may account for the seemingly odd domain numbers noted by the\nreviewer.\nWe have included more clarifications in Methods: lines 639-641, 654-655 and Fig. 1 and 2 legends.\nFig. R2. Genome browser view of H3K27me3 and H3K27ac at the Xist locus in fully grown oocytes.\nFig. R3. Several regions showing loss of H3K27ac in a large H3K27ac domain in CKO oocytes.\nComment 4. • A major finding of this study compared to previous research on BAP1 is that there\nare few PRC1 or PRC2 target genes activated in response to BAP1 loss. The authors state that\nthis may be a cell-type specific feature of oocytes. In figure 3, the genes that are defined as\nPolycomb targets are based on a mESCs dataset. Polycomb complexes are highly mobile in\ndifferent cell types and stages of development. Therefore, representative PRC1 and PRC2\ncomplex member CUT&RUNs are required to define their specific targets in Oocytes to ensure\nthe authors interpretation that there is minimal effect on Polycomb silencing in BAP1 CKO is\ncorrect.\nResponse: We agree with the reviewer’s point. As suggested, we reanalyzed Fig. 3 using the\n1700 oocyte Polycomb targets recently reported by Wei Xie’s lab (Zeng et al., 2025 PMID:\n41118764). These genes are enriched for EZH2, EED, H3K27me3, and H2AK119ub1 in oocytes\n(Fig. R4)(Zeng et al., 2025). The change of gene list does not alter our conclusion that few\nPolycomb targets are derepressed in oocytes upon BAP1 loss.\nWe have included Fig. R4 as new Fig. 3. We thought analyses of mESC PcG targets are\ninformative and included the results in new Supplementary Fig. 4. We have also modified\nrelevant texts in lines 201-202 and lines 209-213.\nFig. R4. BAP1 plays a limited role in Polycomb-mediated gene silencing in oocytes.\na) Metaplots displaying signal intensities of H2AK119ub1, H3K27me3, and H3K27ac at 1700 oocyte\nPolycomb group (PcG) target genes in Bap1 control (CTR) and conditional knockout (CKO) fully\ngrown oocytes (FGOs).\nb) Genome browser views of EED, EZH2, H2AK119ub1, H3K27me3, and H3K27ac signals at the\nPcG target loci Plxnd1 and Gata5. Public EED and EZH2 CUT&RUN data are included for\ncomparison (Zeng et al., 2025).\nc) Venn diagram showing the overlap of oocyte PcG target genes that are de-repressed in PRC1,\nPRC2, and Bap1 CKO FGOs. PRC1/2 RNA-seq are from public datasets (Du et al., 2020).\nd) Heatmap depicting expression levels of oocyte PcG targets that are de-repressed in Bap1 CKO\nFGOs, compared across PRC1 and PRC2 CKO oocytes.\nComment 5. • An additional point regarding this distinctive transcriptional phenotype is that\nwhile its in oocytes, late 1-cell and early 2-cell stages there are very few upregulated genes\ncompared to downregulated (figure 5B). However, at the late 2-cell stage there are >1000\nupregulated genes in BAP1 mutant cells. It would be very interesting to compare the overlap of\nthe upregulated genes across these different cell stages to assess at which point PRC1 and\nPRC2 target genes begin to be upregulated, as they are in ESCs.\nResponse: We thank the reviewer for raising this interesting point. We don’t think that the\nupregulated genes at the late 2-cell stage are due to derepression of PRC1/2 targets for following\nreasons. First, the upregulated genes are enriched for maternal genes (Fig. 5b, 5d-f), reflecting\nmaternal decay defects. The maternal decay defects are likely a result of secondary effects and/or\nembryonic delay caused by reduced developmental competence of Bap1 CKO oocytes. Second,\nthe maternal genes are minimally enriched for H3K27me3 in both CTR and mutant late 2-cell\nembryos (Fig. 6b), suggesting that the observed upregulations of maternal transcripts are not due\nto altered H3K27me3.\nWe have included following texts related to the above response in lines 330-331: “This also\nsuggests that the observed upregulation of maternal transcripts (Fig. 5e-f) likely reflects impaired\nmaternal mRNA decay rather than transcriptional derepression resulting from altered H3K27me3.”\nand lines 443-445: “Failure to activate these genes compromises the developmental competence\nof Bap1-null oocytes, and the matKO embryos subsequently fail to properly complete the MZT,\nresulting in defective preimplantation development.”\nMinor point\nMinor Comment 1. • Citing previously described methods, instead of reiterating the method is\npoor practice and can lead to reproducibility issues as small details can be lost in translation.\nPlease include full methods descriptions, particularly for CUT&RUN, but most method sections\nseem to be overly brief.\nResponse: We thank the reviewer for this suggestion. We have rewritten most of our method\nsection. We provided detailed description of the CUT&RUN procedures.\nMinor Comment 2. • The authors should be clearer on what regions/ peaks are being examined\nin the heatmaps in figure 1J. The legends says these are H2AK119ub1 domains but the main text\nsays actively transcribed regions. They appear largely devoid of H2AK119ub1 in control cells so\ndefining the sites clearly is key to interpreting the data. This should also be made clear in the\nfigure 2C legend for H3K27ac and H3K27me3 domains heatmaps. The legends direct the reader\nto the methods but these simply reference a previous HMM model and do not inform the reader\non what these domains actually are (intergenic, active promoter, enhancer etc).\nResponse: The previously published HMM model-based method is more suitable than SEACR\nfor identifying non-canonical and broad H2AK119ub1, H3K27me3, and H3K27ac domains in\nmouse oocytes. These HMM-defined domains are the regions used for the heatmaps in Fig. 1J\nand Fig. 2C. The reason why these domains appear weakly enriched for H2AK119ub1 in the\ncontrol group is due to the overall increased H2AK119ub1 level in the CKO oocytes. To improve\nclarity, we have included these HMM domain tracks in Fig. 1i and Supplementary Fig. 2g. To\nminimize confusion, we have also removed “a relatively modest increase at actively transcribed\ngenes” in line 132 and clarified the Figure 1 and 2 legends.\nMinor Comment 3. • Figure 2E shows genomic localisation of ‘H3K27ac lost’ domains, and\nappears to be making the point that enhancers are specifically effected by BAP1 loss. However,\nit doesn’t show how this differs from H3K27ac retained domains, or all H3K27ac domains.\nSpecificity cannot be inferred without including this information.\nResponse: We agree with the reviewer’s point. We have now analyzed genomic localization of\nthe “H3K27ac unchanged” domains as suggested (new Fig. 2f). The “H3K27ac unchanged”\ndomains show greater promoter overlap, approximately 22% higher, than the “H3K27ac lost”\ndomains. This comparison clarifies that the domains losing H3K27ac upon BAP1 depletion are\nmore likely to be enriched at putative enhancer regions.\nMinor Comment 4. • ‘Polycom’ -> ‘Polycomb’ typo in the figure 3 title.\nResponse: We have corrected the typo and thank you for catching this.\nReviewer #3 (Remarks to the Author):\nThis manuscript by Kang et al. investigates the role of the Polycomb Repressive-Deubiquitinase\n(PR-DUB) complex, and specifically its catalytic subunit BAP1, during mouse oogenesis and\nearly embryogenesis. Using conditional knockout models and multi-omics profiling, the authors\nshow that BAP1 removes H2AK119ub1 to restrain Polycomb activity, prevent ectopic\nH3K27me3 deposition, and maintain broad oocyte-specific H3K27ac domains. Loss of maternal\nBAP1 leads to transcriptional repression of oocyte genes, defective zygotic genome activation,\nand female subfertility. Importantly, aberrant H3K27me3 domains established in BAP1-deficient\noocytes persist into early embryos, suggesting an intergenerational transmission of repressive\nchromatin states.\nResponse: We appreciate the reviewer’s accurate summary of this work.\nComment #1: The study is technically strong and extends well-established Polycomb principles\nto a physiologically relevant setting. However, the mechanistic insight is limited, as the findings\nlargely confirm the known model in which BAP1 counterbalances PRC1-mediated H2AK119ub1\nto restrict PRC2 spreading. The main novelty lies in demonstrating this mechanism in the\noocyte context and connecting it to fertility and developmental competence. The assertion that\nBAP1 protects H3K27ac is intriguing but remains correlative; direct evidence that increased\nH2AK119ub1 leads to loss of H3K27ac is lacking.\nResponse: We thank the reviewer for the positive comment that “this study is technically strong”,\nand for highlighting the novel link of BAP1 to fertility and oocyte developmental competence.\nAlthough we agree with the reviewer that some aspects of our findings are consistent with\nestablished PR-DUB functions, our study reveals important in vivo roles and extends existing\nmodels by providing new mechanistic insight into how PR-DUB acts as a critical determinant of\nthe oocyte epigenome, with consequences for early embryogenesis. While BAP1’s role in\ncounteracting widespread H2AK119ub1 accumulation in oocytes (Fig. 1) is consistent with\nobservations in other systems, our study is, to our knowledge, the first to demonstrate that BAP1\nregulates the non-canonical H3K27me3 and H3K27ac domains unique to mouse oocytes (Fig.\n2). These chromatin features are absent in mESCs and somatic cells, and therefore our study\nrevealed oocyte-specific regulatory mechanisms in which BAP1 operates. Furthermore, our data\nshow that BAP1 functions primarily as a transcriptional activator in oocytes and plays a limited\nrole in Polycomb-mediated repression (Fig. 3). This contrasts with previous findings in mESCs,\nwhere BAP1 exhibits a dual role, both repressing canonical Polycomb targets and activating other\ngene sets. Thus, our results uncover a previously unrecognized and oocyte-specific regulatory\nmechanism that diverges from established models.\nTo provide further insights regarding how BAP1 regulates H3K27ac in oocytes, we have\nperformed additional experiments and analyses.\n1) Increase in H2AK119ub1 is not the sole determining factor that dictates which regions lose\nH3K27ac and gain H3K27me3 in Bap1 CKO oocytes. We examined the enrichment of histone\nmodifications at H3K27ac-lost and H3K27ac-unchanged regions in both Bap1 control (CTR) and\nconditional knockout (CKO) fully grown oocytes (FGOs). As shown in Fig. R1, loss of H3K27ac\nin Bap1 CKO FGOs is globally associated with a corresponding gain of H3K27me3. In contrast,\nH2AK119ub1 levels increased similarly in both H3K27ac-lost and H3K27ac-unchanged regions.\nThese results suggest that the increase in H2AK119ub1 is not the sole determining factor that\ndictates which regions lose H3K27ac and gain H3K27me3.\nFig. R1. Heatmap illustrating changes of\nhistone modifications at the H3K27ac-\nlost (n = 9043) and H3K27ac-no change\n(n = 27287) domains in Bap1 CTR and\nCKO fully grown oocytes (FGOs).\n2) BAP1 is important for the establishment of H3K27ac during postnatal oocyte growth but is\ndispensable for maintenance of pre-existing H3K27ac marks. To better understand the\nmechanisms underlying locus specific H3K27ac loss in Bap1 CKO FGOs, we included new spike-\nin H3K27c CUT&TAG data in postnatal day 1 (P1) non-growing oocyte (NGOs), when Gdf9-\nCre\nhas not yet expressed, in collaboration with the Namekawa lab at UC Davis. Regions that are\nsensitive to BAP1 loss gain H3K27ac during the final stage of oocyte growth, as seen in FGOs\n(pink areas in Fig. R5a). In contrast, regions unaffected by Bap1 deletion already acquire\nH3K27ac before Bap1 deletion in P1 NGOs (Fig. R5a). These findings suggest that PR-DUB is\nrequired for the establishment of new H3K27ac marks during oocyte growth, rather than for\nmaintaining pre-existing H3K27ac marks.\nWe have included Fig. R1 in the revised manuscript as new Fig. 2e, and updated Fig. 2i, 2j,\nSupplementary Fig. 3d. Related text updates are in lines 169-175.\nFIGURE REDACTED\nFig. R5. a) Genome browser view of H3K27ac, H3K27me3, and RNA-seq profiles at the Fgf10\nlocus. b) Heatmap showing H3K27ac signal during oocyte growth across H3K27ac-lost domains.\nThe H3K27ac data for postnatal day 7 (P7), P10 growing oocytes and FGOs are from public\ndatasets (Liu et al., 2024).\nComment #2: The most original and conceptually important aspect of this work concerns the\nintergenerational inheritance of abnormal H3K27me3 domains. This observation resonates with\nprior findings across species. In mice, Inoue and colleagues showed that maternal loss of\nH3K27me3 can persist after fertilization and influence imprinting; in Drosophila, Zenk and\ncollaborators demonstrated germline transmission of Polycomb-associated chromatin; and in C.\nelegans, Gaydos and co-workers revealed PRC2-dependent transgenerational inheritance of\nH3K27me3. Kang et al. now describe the converse phenomenon: the persistence of maternal\ngain of H3K27me3 in embryos. This represents an important addition to the field of epigenetic\ninheritance. However, this intergenerational aspect is not sufficiently emphasized in the current\nmanuscript. It should be more thoroughly developed, both in the results and in the discussion, as\nit constitutes the most significant conceptual contribution of the study. The above studies should\nbe cited.\nResponse: We appreciate the reviewer for highlighting the significant conceptual advance of the\nstudy. As suggested, we extended our intergenerational analysis by quantifying how long the\nectopic H3K27me3 domains persist beyond the two-cell stage. To this end, we performed\nH3K27me3 CUT&RUN in morulae, the developmental stage immediately preceding the first\nlineage differentiation. We also dissected post-implantation E6.5 embryos into epiblast (EPI),\nFig. R6. Quality control of H3K27me3 CUT&RUN data in morulae and E6.5 embryos. a) Scatter\nplots showing reproducibility of H3K27me3 CUT&RUN profiles between biological replicates in morulae\nand E6.5 embryos. Correlation between Bap1 control (CTR) extra-embryonic ectoderm (EXE) and a\npublished dataset (Chen et al., 2019) is shown. Pearson correlation coefficients are indicated. b)\nHierarchical clustering of H3K27me3 between different samples. c) Genome browser view of allelic\nH3K27me3 at non-canonical imprinted gene loci in morulae. d) Genome browser view of H3K27me3\nand RNA-seq profiles at the lineage marker gene loci. e) Expression of lineage markers in CTR and\nmatKO samples. f) Correlation heatmap between biological replicates of RNA-seq samples.\nvisceral endoderm (VE), and extra-embryonic ectoderm (EXE), followed by H3K27me3\nCUT&RUN profiling of each lineage. The CUT&RUN biological replicates were highly reproducible\n(Fig. R6a), and hierarchical clustering revealed clear separation between morulae and post-\nimplantation lineages (Fig. R6b). As validation of data quality, non-canonical imprinted genes\n(e.g., Gab1, Jade1/Phf17, and Sfmbt2) exhibited the expected maternally biased H3K27me3\nenrichment at the morula stage (Rig. R6c). Furthermore, lineage marker genes, Pou5f1 in EPI,\nGata6 in VE, and Gata3 in EXE, displayed lineage-specific deletion of H3K27me3 in their\nrespective tissues, further validating data quality (Fig. R6d-f).\nHaving verified data quality, we next examined the developmental dynamics of the ectopic\nH3K27me3 domains induced by BAP1 loss. We found that ectopic H3K27me3 gain in Bap1-null\noocytes persists through the morula stage but is erased in all three lineages by E6.5 (Fig. R7a–\nb). This resonates with our previous findings on non-canonical imprinting (Chen et al., 2019),\nin\nwhich maternally inherited H3K27me3 domains are maintained during preimplantation\ndevelopment but are subsequently lost following implantation.\nFig. R7. Ectopic H3K27me3 in Bap1-null oocytes persist through the morula stage but are lost in\npost-implantation embryos. a) Genome browser view of allelic H3K27me3 in late 2-cell (L2C),\nmorula, and E6.5 control (CTR) and maternal KO (matKO) embryos. b) Metaplots showing allelic\nH3K27me3 enrichment at the ectopic H3K27me3 domains across different developmental stages. c)\nHeatmaps showing allelic expression bias of imprinted genes. MEG, maternally expressed genes.\nPEGs, paternally expressed genes. Pat: paternal. Mat: Maternal. SNP: single nucleotide polymorphism.\nA recent study reported that knock out of Ezhip, a negatively regulator of PRC2 (Jain et al., 2019;\nRagazzini et al., 2019), causes excessive genome-wide H3K27me3 accumulation in early\nembryos and paradoxically disrupts non-canonical genomic imprinting (Zeng et al., 2025).\nPrompted by this observation, we asked whether pervasive increase of H2AK119ub1 observed\nin Bap1-null oocytes might similarly affect allelic gene expression in later development. Consistent\nwith the preserved allelic H3K27me3 enrichment at the non-canonical imprinted loci (Fig. R6c),\nimprinted expression of these genes was largely maintained in the VE and EXE lineages (Fig.\nR7c, bolded genes). Similarly, canonical imprinting was minimally affected by maternal BAP1 loss\n(Fig. R7c). These data demonstrate that loss of maternal BAP1 is dispensable for both canonical\nand non-canonical genomic imprinting.\nWe have included Fig. R6 as new Supplementary Fig. 9 and Fig. R7 as new Fig. 7. We have\nalso included related texts in Result and Discussion sections (line 364-389, 454-470) including\ncitations to the suggested references.\nComment #3: Overall, is this a high-quality descriptive study with clear developmental and\nreproductive relevance, though its mechanistic novelty remains modest. The authors should\nstrengthen causality and clarify whether BAP1 directly maintains H3K27ac or acts indirectly by\nlimiting PRC2 activity. It would also greatly enhance the paper to deepen the discussion and\nanalysis of the intergenerational dimension, which links this work to a broader literature on\nchromatin-based inheritance across species.\nResponse: We thank the reviewer for this point. We have extended our intergenerational\nanalysis by quantifying how long the ectopic H3K27me3 domains persist beyond the two-cell\nstage. Please find the related response to Comment #2. Regarding whether BAP1 directly\nmaintains H3K27ac or acts indirectly by limiting PRC2 activity. Please find our responses to the\nfollowing Comment #4 and #5 and discussions from line 401-413.\nComment #4: To reinforce their conclusions, the authors should conduct targeted experiments\nthat test causality between H2AK119ub1 accumulation and H3K27ac loss. A mechanistic\nrescue or inhibition approach, such as PRC2 inhibition using GSK126 or EED226, or\nUSP21/BAP1+ASXL1 overexpression, could determine whether blocking PRC2 spreading\nrestores H3K27ac at affected loci.\nResponse: We thank the reviewer for this constructive comment. We performed immunostaining\nanalyses in postnatal day 7 (P7) oocytes, ~4 days after Gdf9-Cre activation. Compared to control,\nP7 CKO oocytes already exhibit a significant increase in H2AK119ub1 and a modest increase in\nFig. R8. Immunostaining analyses of postnatal day 7 (P7) growing oocytes. Left panel\nshows the representative images of oocyte nuclei, and right panel shows the quantification\nof relative immunostaining intensities. Each dot represents one oocyte analyzed. p-value:\ntwo-sided t-test. Scale bar, 20 μm.\nH3K27me3, while H3K27ac level remains unchanged at this stage (Fig. R8). These data suggest\nthat gain of H2AK119ub1 and H3K27me3 precedes the reduction of H3K27ac loss in Bap1 CKO\noocytes. Although these data do not directly demonstrate causality between H2AK119ub1\naccumulation and H3K27ac loss, they suggest that H3K27ac is likely regulated downstream of\nPolycomb-associated marks.\nGiven these observations, we agree with the reviewer that it would be very interesting to test\nwhether blocking PRC2 activity restores H3K27ac at the affected loci in Bap1 CKO oocytes.\nHowever, it is technically challenging to achieve chemical inhibition of PRC2 specifically in\ngrowing oocytes from P3 to ~7-8-weeks of age without affecting somatic cells. A more feasible\nstrategy would be to generate Gdf9-Cre, Bap1fl/fl, Eed fl/fl double conditional KO mice. However,\nbreeding mice carrying three genetically engineered alleles is time-consuming and could take at\nleast ~1 year. Furthermore, comprehensive reproductive phenotyping and multi-omic profiling of\nthe single and double CKO mice would likely require an additional year. Therefore, we feel that\nthese experiments are beyond the scope of the current study but represent an exciting avenue\nfor future investigation.\nComment #5: They should also examine whether enhancer machinery components such as\np300/CBP, BRD4, or MLL3/4 change occupancy at the regions that lose H3K27ac, which would\nclarify whether BAP1 directly supports enhancer activation.\nResponse: This is a constructive comment given the known link between BAP1 and the\nCOMPASS complex. However, it remains technically challenging to profile non-histone\nmodifications with scares materials such as mouse oocytes. Typically, it requires substantial effort\nto identify high-quality antibodies that work reliably with ~200-500 cells. As noted in a recent\nNSMB paper on H2A.Z from Azusa Inoue’s lab: “In the future, it will be informative to map the\ndistribution of MLL2-COMPASS and H2A.Z chaperones in oocytes, warranting further\nimprovement in low-input chromatin analysis technologies.” This underscores the current\ntechnical limitations of such experiments.\nComment #6: Temporal analyses or acute depletion experiments could further demonstrate\nwhether H2AK119ub1 elevation precedes H3K27me3 gain and H3K27ac loss, establishing the\ndirectionality of events.\nResponse: Based our immunostaining results shown in Fig. R8, H2AK119ub1 elevation\nprecedes H3K27me3 and H3K27ac changes upon Bap1 deletion. This suggests that H3K27ac\nis likely regulated downstream of Polycomb-associated marks.\nComment #7: Finally, the authors should extend their intergenerational analysis by quantifying\nhow long the ectopic H3K27me3 domains persist beyond the two-cell stage and by comparing\nthis to the inheritance mechanisms described by Inoue, Zenk, and Gaydos.\nResponse: Please find our response for Comment #2. We have also cited the work by Inoue,\nZenk, and Gaydos.\nMinor comment #1: Minor issues include clarifying the terminology of PR-DUB (whether it\nrefers exclusively to the BAP1–ASXL complex),\nResponse: According to Scheuermann et al., 2010 (PMID 20436459), PR-DUB refers to the\nBAP1-ASXL in mammals or Calypso-ASX in fly. We made this statement in the Introduction “The\nubiquitin carboxy-terminal hydrolase BAP1 is the catalytic subunit of the Polycomb Repressive-\nDeubiquitinase (PR-DUB) complex, which removes monoubiquitin from H2AK119ub1 (Ge et al.,\n2023; Scheuermann et al., 2010; Thomas et al., 2023).”\nMinor comment #2: providing full information on biological replicates and statistical details,\nResponse: We have included biological replicates and statistical details in figure legends and\nSupplemental tables.\nMinor comment #3: improving figure clarity by using distinct color scales for H3K27ac and\nH3K27me3.\nResponse: As suggested, we have used different colors for H3K27ac and H3K27me3\nheatmaps and genome browser tracks.\nIn summary, this paper offers a valuable dataset and an important extension of Polycomb\nbiology into mammalian oogenesis and inheritance. The work convincingly demonstrates that\nBAP1 is essential for safeguarding the oocyte epigenome and fertility, but its broader impact will\ndepend on strengthening mechanistic causality and expanding the intergenerational\nperspective, which represents the most compelling aspect of this study. With these revisions,\nthe manuscript could meet the conceptual standards of Nature Communications.\nResponse: We thank the reviewer for constructive comments, which have allowed us to\nsubstantially improve the manuscript, and for pointing potentially exciting directions for future\nstudies.\nReviewer #4 (Remarks to the Author)\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is\npart of the Nature Communications initiative to facilitate training in peer review and to provide\nappropriate recognition for Early Career Researchers who co-review manuscripts.\nReviewer #5 (Remarks to the Author)\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is\npart of the Nature Communications initiative to facilitate training in peer review and to provide\nappropriate recognition for Early Career Researchers who co-review manuscripts."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have sincerely addressed the concerns raised by us and the other reviewers, and we find the revision to be convincing.", "reviewer_2": "The authors have comprehensively addressed all of my technical and conceptual questions. This has led to the inclusions of new data and text to explain certain technical aspects. I very enthusiastically support the publication of this work in Nature Communications.", "reviewer_3": "The authors have clearly and thoroughly addressed all reviewer concerns. At this point, I believe the manuscript is ready for publication.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_5": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": ""}]

Decision letter