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• W2088910050 abstract "•SCML2 regulates global silencing of somatic/progenitor genes in the male germline•SCML2 is a germline-specific subunit of Polycomb repressive complex 1•SCML2 establishes H2A ubiquitination to silence somatic/progenitor genes on autosomes•SCML2 prevents H2A ubiquitination on meiotic sex chromosomes Gametogenesis is dependent on the expression of germline-specific genes. However, it remains unknown how the germline epigenome is distinctly established from that of somatic lineages. Here we show that genes commonly expressed in somatic lineages and spermatogenesis-progenitor cells undergo repression in a genome-wide manner in late stages of the male germline and identify underlying mechanisms. SCML2, a germline-specific subunit of a Polycomb repressive complex 1 (PRC1), establishes the unique epigenome of the male germline through two distinct antithetical mechanisms. SCML2 works with PRC1 and promotes RNF2-dependent ubiquitination of H2A, thereby marking somatic/progenitor genes on autosomes for repression. Paradoxically, SCML2 also prevents RNF2-dependent ubiquitination of H2A on sex chromosomes during meiosis, thereby enabling unique epigenetic programming of sex chromosomes for male reproduction. Our results reveal divergent mechanisms involving a shared regulator by which the male germline epigenome is distinguished from that of the soma and progenitor cells. Gametogenesis is dependent on the expression of germline-specific genes. However, it remains unknown how the germline epigenome is distinctly established from that of somatic lineages. Here we show that genes commonly expressed in somatic lineages and spermatogenesis-progenitor cells undergo repression in a genome-wide manner in late stages of the male germline and identify underlying mechanisms. SCML2, a germline-specific subunit of a Polycomb repressive complex 1 (PRC1), establishes the unique epigenome of the male germline through two distinct antithetical mechanisms. SCML2 works with PRC1 and promotes RNF2-dependent ubiquitination of H2A, thereby marking somatic/progenitor genes on autosomes for repression. Paradoxically, SCML2 also prevents RNF2-dependent ubiquitination of H2A on sex chromosomes during meiosis, thereby enabling unique epigenetic programming of sex chromosomes for male reproduction. Our results reveal divergent mechanisms involving a shared regulator by which the male germline epigenome is distinguished from that of the soma and progenitor cells. The germline is the only heritable lineage across generations, and it ensures continuity of life. Although biological strategies to specify the germline are diverse among species, suppression of somatic transcriptional programs is a common feature in the germline (Nakamura et al., 2010Nakamura A. Shirae-Kurabayashi M. Hanyu-Nakamura K. Repression of early zygotic transcription in the germline.Curr. Opin. Cell Biol. 2010; 22: 709-714Crossref PubMed Scopus (23) Google Scholar). In mammals, primordial germ cells are specified during early embryonic development after gastrulation and actively migrate into developing gonads, where they differentiate into spermatozoa or oocytes according to the presence or absence of Y chromosomes (Svingen and Koopman, 2013Svingen T. Koopman P. Building the mammalian testis: origins, differentiation, and assembly of the component cell populations.Genes Dev. 2013; 27: 2409-2426Crossref PubMed Scopus (252) Google Scholar). During this developmental period, the germline undergoes unique epigenetic programing distinct from that in somatic lineages and is naturally reprogrammed in the next generation (Gill et al., 2012Gill M.E. Erkek S. Peters A.H. Parental epigenetic control of embryogenesis: a balance between inheritance and reprogramming?.Curr. Opin. Cell Biol. 2012; 24: 387-396Crossref PubMed Scopus (35) Google Scholar, Kota and Feil, 2010Kota S.K. Feil R. Epigenetic transitions in germ cell development and meiosis.Dev. Cell. 2010; 19: 675-686Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar, Saitou et al., 2012Saitou M. Kagiwada S. Kurimoto K. Epigenetic reprogramming in mouse pre-implantation development and primordial germ cells.Development. 2012; 139: 15-31Crossref PubMed Scopus (298) Google Scholar, Sasaki and Matsui, 2008Sasaki H. Matsui Y. Epigenetic events in mammalian germ-cell development: reprogramming and beyond.Nat. Rev. Genet. 2008; 9: 129-140Crossref PubMed Scopus (637) Google Scholar). However, it remains elusive how the germline epigenome is distinctly established from that of somatic lineages. A potential candidate is a Polycomb group (PcG)-based mechanism, which regulates epigenetic gene repression and is responsible for stem cell renewal, differentiation, and development (Aloia et al., 2013Aloia L. Di Stefano B. Di Croce L. Polycomb complexes in stem cells and embryonic development.Development. 2013; 140: 2525-2534Crossref PubMed Scopus (225) Google Scholar, Simon and Kingston, 2013Simon J.A. Kingston R.E. Occupying chromatin: Polycomb mechanisms for getting to genomic targets, stopping transcriptional traffic, and staying put.Mol. Cell. 2013; 49: 808-824Abstract Full Text Full Text PDF PubMed Scopus (534) Google Scholar). Recent evidence has demonstrated that multiple Polycomb subunits are exchanged to acquire specific functions for different biological contexts (Gao et al., 2012Gao Z. Zhang J. Bonasio R. Strino F. Sawai A. Parisi F. Kluger Y. Reinberg D. PCGF homologs, CBX proteins, and RYBP define functionally distinct PRC1 family complexes.Mol. Cell. 2012; 45: 344-356Abstract Full Text Full Text PDF PubMed Scopus (589) Google Scholar, Tavares et al., 2012Tavares L. Dimitrova E. Oxley D. Webster J. Poot R. Demmers J. Bezstarosti K. Taylor S. Ura H. Koide H. et al.RYBP-PRC1 complexes mediate H2A ubiquitylation at polycomb target sites independently of PRC2 and H3K27me3.Cell. 2012; 148: 664-678Abstract Full Text Full Text PDF PubMed Scopus (435) Google Scholar). Therefore, if a germline-specific PcG protein exists, it could mediate germline-specific functions. In this context, it should be noted that germline genes are repressed by a PcG protein, L(3)mbt, in somatic cells of the fly. Importantly, tumors with a deletion of L(3)mbt exhibit soma-to-germline transformation (Janic et al., 2010Janic A. Mendizabal L. Llamazares S. Rossell D. Gonzalez C. Ectopic expression of germline genes drives malignant brain tumor growth in Drosophila.Science. 2010; 330: 1824-1827Crossref PubMed Scopus (202) Google Scholar). This study suggests that suppression of germline genes is important for tumor suppression in somatic cells, as also proposed in humans (Simpson et al., 2005Simpson A.J. Caballero O.L. Jungbluth A. Chen Y.T. Old L.J. Cancer/testis antigens, gametogenesis and cancer.Nat. Rev. Cancer. 2005; 5: 615-625Crossref PubMed Scopus (1259) Google Scholar). It further suggests that a PcG-based mechanism is a critical determinant between soma versus germline transcriptomes. However, it is unknown whether a germline-specific epigenetic silencer exists that suppresses the common features of the somatic program to define the unique epigenome of the germline. One of the PcG complexes in mammals, Polycomb repressive complex 1 (PRC1), plays a central role during development by suppressing large numbers of genes through mono-ubiquitination of H2A at lysine 119 (H2AK119ub). This is mediated by a PRC1 core subunit, RNF2 (also known as RING1B) (Wang et al., 2004Wang H. Wang L. Erdjument-Bromage H. Vidal M. Tempst P. Jones R.S. Zhang Y. Role of histone H2A ubiquitination in Polycomb silencing.Nature. 2004; 431: 873-878Crossref PubMed Scopus (1288) Google Scholar). In female primordial germ cells, PRC1 prevents precocious entry into meiosis and coordinates the timing of sex differentiation (Yokobayashi et al., 2013Yokobayashi S. Liang C.Y. Kohler H. Nestorov P. Liu Z. Vidal M. van Lohuizen M. Roloff T.C. Peters A.H. PRC1 coordinates timing of sexual differentiation of female primordial germ cells.Nature. 2013; 495: 236-240Crossref PubMed Scopus (86) Google Scholar). It is unknown whether any specific PcG subunits have critical roles during spermatogenesis, the process in which spermatogonia undergo self-renewal, enter meiosis, and differentiate into sperm. In this study, our unbiased proteomics screen unexpectedly identified Sex comb on midleg-like 2 (SCML2), a homolog of the Drosophila PRC1 subunit Sex comb on midleg (Scm). We demonstrate that SCML2 is a specific and essential epigenetic modifier in the male germline. SCML2 is one of the malignant brain tumor (MBT) domain-containing proteins (Montini et al., 1999Montini E. Buchner G. Spalluto C. Andolfi G. Caruso A. den Dunnen J.T. Trump D. Rocchi M. Ballabio A. Franco B. Identification of SCML2, a second human gene homologous to the Drosophila sex comb on midleg (Scm): a new gene cluster on Xp22.Genomics. 1999; 58: 65-72Crossref PubMed Scopus (32) Google Scholar), which often function together with PRC1. Two other MBT domain-containing proteins, SCMH1 and SFMBT1, have been suggested to be involved in spermatogenesis (Takada et al., 2007Takada Y. Isono K. Shinga J. Turner J.M. Kitamura H. Ohara O. Watanabe G. Singh P.B. Kamijo T. Jenuwein T. et al.Mammalian Polycomb Scmh1 mediates exclusion of Polycomb complexes from the XY body in the pachytene spermatocytes.Development. 2007; 134: 579-590Crossref PubMed Scopus (46) Google Scholar, Zhang et al., 2013Zhang J. Bonasio R. Strino F. Kluger Y. Holloway J.K. Modzelewski A.J. Cohen P.E. Reinberg D. SFMBT1 functions with LSD1 to regulate expression of canonical histone genes and chromatin-related factors.Genes Dev. 2013; 27: 749-766Crossref PubMed Scopus (59) Google Scholar). However, Sfmbt1 knockout mice are fertile (Qin et al., 2012Qin J. Whyte W.A. Anderssen E. Apostolou E. Chen H.H. Akbarian S. Bronson R.T. Hochedlinger K. Ramaswamy S. Young R.A. Hock H. The Polycomb group protein L3mbtl2 assembles an atypical PRC1-family complex that is essential in pluripotent stem cells and early development.Cell Stem Cell. 2012; 11: 319-332Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar), and subfertility of Scmh1 knockout mice is completely rescued by the additional deletion of another PRC1 subunit, PHC2 (Takada et al., 2007Takada Y. Isono K. Shinga J. Turner J.M. Kitamura H. Ohara O. Watanabe G. Singh P.B. Kamijo T. Jenuwein T. et al.Mammalian Polycomb Scmh1 mediates exclusion of Polycomb complexes from the XY body in the pachytene spermatocytes.Development. 2007; 134: 579-590Crossref PubMed Scopus (46) Google Scholar). In addition, the deletion of another MBT domain protein, L3MBTL1, does not affect fertility in mice (Qin et al., 2010Qin J. Van Buren D. Huang H.S. Zhong L. Mostoslavsky R. Akbarian S. Hock H. Chromatin protein L3MBTL1 is dispensable for development and tumor suppression in mice.J. Biol. Chem. 2010; 285: 27767-27775Crossref PubMed Scopus (22) Google Scholar). These results indicate that SFMBT1, SCMH1, PHC2, and L3MBTL1 are not essential in the male germline. Unlike these proteins, here we show that SCML2 is essential for spermatogenesis and that SCML2 suppresses a large group of genes that are commonly expressed in somatic lineages and in spermatogenesis progenitor cells. Additionally, we demonstrate that, in meiosis, SCML2 is essential for epigenetic programming of sex chromosomes, where unsynapsed sex chromosomes are epigenetically silenced by the action of DNA damage response (DDR) proteins through a process known as meiotic sex chromosome inactivation (MSCI) (Ichijima et al., 2012Ichijima Y. Sin H.S. Namekawa S.H. Sex chromosome inactivation in germ cells: emerging roles of DNA damage response pathways.Cell. Mol. Life Sci. 2012; 69: 2559-2572Crossref PubMed Scopus (61) Google Scholar, Turner, 2007Turner J.M. Meiotic sex chromosome inactivation.Development. 2007; 134: 1823-1831Crossref PubMed Scopus (478) Google Scholar). Based on these two specific functions, SCML2 uniquely defines the specific epigenome of the male germline. To elucidate unique features of the male germline, we compared the transcriptomes of the spermatogenic cells at different stages and somatic lineages in mice. Specifically, we compared RNA sequencing (RNA-seq) data of germline stem (GS) cells that represent mitotically active undifferentiated spermatogonia, the THY1+ undifferentiated spermatogonia that enrich the stem cell phase, the meiotic spermatocyte at the pachytene stage (pachytene spermatocytes [PS]), and post-meiotic round spermatids (RS) (described in Figure 1A) with those of other cell types by generating a heatmap that included all of the expressed RefSeq genes in the genome of these samples (16,475 genes) (Figure 1B). Importantly, we found that whole-genome transcriptomes in PS and RS were largely different from those of somatic lineages, embryonic stem (ES) cells, and the mitotic phase of germ cells including GS cells and THY1+ cells (Figure 1B). A large group of genes was expressed in somatic lineages, ES cells, GS cells, and the THY1+ fraction, but this group was specifically repressed in PS and RS. We defined this group of genes as somatic/progenitor genes because this group is commonly active in somatic lineages and mitotic phases of spermatogenesis-progenitor cells. In contrast, a large group of late spermatogenesis genes that are associated with male reproduction was specifically expressed in PS and RS. In accordance with this finding, previous reports have demonstrated that germ cells undergo a dynamic transcriptome change during the late stages of spermatogenesis (Chalmel et al., 2007Chalmel F. Rolland A.D. Niederhauser-Wiederkehr C. Chung S.S. Demougin P. Gattiker A. Moore J. Patard J.J. Wolgemuth D.J. Jégou B. Primig M. The conserved transcriptome in human and rodent male gametogenesis.Proc. Natl. Acad. Sci. U S A. 2007; 104: 8346-8351Crossref PubMed Scopus (207) Google Scholar, Khil et al., 2004Khil P.P. Smirnova N.A. Romanienko P.J. Camerini-Otero R.D. The mouse X chromosome is enriched for sex-biased genes not subject to selection by meiotic sex chromosome inactivation.Nat. Genet. 2004; 36: 642-646Crossref PubMed Scopus (247) Google Scholar, Namekawa et al., 2006Namekawa S.H. Park P.J. Zhang L.F. Shima J.E. McCarrey J.R. Griswold M.D. Lee J.T. Postmeiotic sex chromatin in the male germline of mice.Curr. Biol. 2006; 16: 660-667Abstract Full Text Full Text PDF PubMed Scopus (314) Google Scholar, Shima et al., 2004Shima J.E. McLean D.J. McCarrey J.R. Griswold M.D. The murine testicular transcriptome: characterizing gene expression in the testis during the progression of spermatogenesis.Biol. Reprod. 2004; 71: 319-330Crossref PubMed Scopus (412) Google Scholar). Our present study demonstrates that suppression of the somatic/progenitor program is a unique feature of the late stages of the male germline and also that a specific set of late spermatogenesis genes is activated at these stages. The next key question is whether there are any factors involved in establishing the unique transcriptomes of the late stages of the male germline. Interestingly, a factor we independently identified as a component of meiotic sex chromosomes has a role in shaping transcriptomes in the male germline (described below). In our attempt to identify a germline-specific epigenetic modifier, we focused on MSCI, a unique germline-specific event that is essential to male meiosis, which involves activation of the DDR pathway and deposition of γH2AX on sex chromosomes. Although several DDR proteins are required for MSCI, we reasoned that a germline-specific protein could regulate this process. To test this possibility, we performed immunoprecipitation of nucleosomes containing γH2AX, an essential modification of silent sex chromosomes, combined with mass spectrometry and identified an X chromosome linked protein, SCML2 (Figures 1C and 1D). Consistent with our previous study indicating the interaction of γH2AX and MDC1 on meiotic sex chromosomes (Ichijima et al., 2011Ichijima Y. Ichijima M. Lou Z. Nussenzweig A. Camerini-Otero R.D. Chen J. Andreassen P.R. Namekawa S.H. MDC1 directs chromosome-wide silencing of the sex chromosomes in male germ cells.Genes Dev. 2011; 25: 959-971Crossref PubMed Scopus (127) Google Scholar), the mass spectrometry score for MDC1 was the highest. The score for SCML2 was the second highest. Subsequent immunoprecipitation, combined with western blotting, confirmed that SCML2 interacts with γH2AX in testicular extracts (Figure 1E). Immunostaining revealed that the expression of SCML2 commenced in embryonic germ cells after entering the gonad in both males and females (Figures 1F and S1). In adult testes, SCML2 highly accumulated in ZBTB16 (also known as PLZF)-positive undifferentiated spermatogonia, which include the stem cell population (Figure 1G). Later during meiosis, SCML2 slightly accumulated on entire nuclei from the leptotene to the early pachytene stage (Figure S1) and highly accumulated on the sex chromosomes from the early to mid-pachytene stage to the diplotene stage (Figure 1H), consistent with the fact that γH2AX has a role in transcriptionally silencing sex chromosomes and interacts with SCML2. Similar to SCML2 protein localization, Scml2 transcripts were specifically detected in the germline (Figure 1I). Scml2 expression was especially high in GS cells that represent undifferentiated spermatogonia. Paradoxically, SCML2 is transcriptionally silenced by MSCI because of its X linkage (Mueller et al., 2008Mueller J.L. Mahadevaiah S.K. Park P.J. Warburton P.E. Page D.C. Turner J.M. The mouse X chromosome is enriched for multicopy testis genes showing postmeiotic expression.Nat. Genet. 2008; 40: 794-799Crossref PubMed Scopus (223) Google Scholar), despite being localized on the sex chromosomes during meiosis. To better understand the function of SCML2, we generated Scml2-knockout (Scml2-KO) mice using zinc finger nuclease technology (Cui et al., 2011Cui X. Ji D. Fisher D.A. Wu Y. Briner D.M. Weinstein E.J. Targeted integration in rat and mouse embryos with zinc-finger nucleases.Nat. Biotechnol. 2011; 29: 64-67Crossref PubMed Scopus (269) Google Scholar). We established two independent females harboring a heterozygous deletion at the X-linked Scml2 locus (Figure 2A). In male progeny possessing either of the mutations, SCML2 was not detectable by western blotting or immunostaining (Figures 2B and 2C), indicating that the mutations nullified SCML2 expression. Thus, we refer hereafter to both mutant lines as Scml2-KO mice. SCML2 was detected at 140 kDa in testicular lysate, although SCML2 is subject to protein degradation if a protease inhibitor is not added (Figure 2B). Scml2-KO males were born at Mendelian ratios and looked healthy (data not shown). However, their testes were smaller than littermate control testes, and Scml2-KO males were not able to impregnate female mice (Figures 2D–2F). Therefore, we conclude that Scml2-KO males are infertile and that SCML2 is essential in spermatogenesis. To elucidate gene expression changes in meiotic and post-meiotic stages resulting from SCML2 deletion, we performed RNA-seq using purified THY1+ fraction, PS, and RS from Scml2-KO mice. Because there was no germ cell arrest at a particular stage in Scml2-KO mice despite the occurrence of progressive apoptosis during differentiation in spermatogenesis (as shown below), purified germ cells enabled us to evaluate the gene expression change caused by SCML2 depletion without the secondary effect of developmental delay. Remarkably, the expression patterns of PS and RS were globally changed compared with those of wild-type mice; notably, somatic/progenitor genes were largely derepressed in Scml2-KO PS and RS, although gene expression in the THY1+ fraction was not largely altered (Figure 1B). Hereafter the autosomes and sex chromosomes were analyzed separately because the sex chromosomes are subject to MSCI. To identify specific targets regulated by SCML2, we applied stringent criteria to our RNA-seq data set (padj < 0.05, >2-fold difference, reads per kilobase per million (RPKM) ≥ 5 for higher-expression genes). We identified 853 and 613 autosomal genes that were significantly up- and downregulated, respectively, in Scml2-KO PS (Figure 2G; gene lists are included in Table S1). In RS, 213 and 214 autosomal genes were significantly up- and downregulated, respectively. On the other hand, only 17 and 28 autosomal genes were up- and downregulated in Scml2-KO THY1+ cells. The genes upregulated in Scml2-KO PS and RS were mainly expressed in somatic lineages and spermatogenesis progenitor cells and strongly suppressed in wild-type PS and RS (Figures 2H and S2). By contrast, downregulated genes in Scml2-KO PS and RS were specifically expressed in PS and RS (Figures 2I and S2). Gene ontology (GO) enrichment analysis further confirmed that the set of genes downregulated in Scml2-KO cells showed enrichment for genes involved in the late stages of spermatogenesis (Figure S2). Taken together, SCML2 is essential for the suppression of somatic/progenitor genes and the activation of late-spermatogenesis-specific genes in PS and RS. To further investigate the role of SCML2, we performed histological analyses. In Scml2-KO testes, massive loss of differentiated germ cells occurred (Figure 3A) and polynucleated cells were observed (Figure 3B). Additionally, we found that elongated spermatids in the Scml2-KO failed to condense (Figures 3C and 3D), and spermatozoa were rarely seen in epididymides (Figure 3E). Next, to investigate which cell types are affected by SCML2 deficiency, stage-specific spermatogenic cells were counted. The number of undifferentiated spermatogonia was unchanged (Figures 3F and 3G), suggesting that SCML2 is apparently not involved in the maintenance of spermatogonial stem cells, consistent with minor gene expression change in this population. In contrast, the number of STRA8-positive cells, which includes differentiating spermatogonia and pre-leptotene spermatocytes, as well as SYCP3-positive meiotic spermatocytes, was significantly decreased in the Scml2-KO (Figures 3H–3K). An increase of spermatocytes and spermatids undergoing apoptosis in Scml2-KO testes suggest that the reduction of differentiating cells is caused by apoptosis (Figures 3L–3N). In Scml2-KO testes, apoptosis occurred progressively in each stage following spermatogonia: H1T-negative spermatocytes (before the mid-pachytene stage), H1T-positive spermatocytes (during and after the mid-pachytene stage), and RS (Figure 3N). Although the decrease of SYCP3-positive meiotic spermatocytes could be due in part to the decrease of differentiated spermatogonia, apoptosis mainly occurred in pre-leptotene spermatocytes (the majority of STRA8-positve cells) and in later stages. Therefore, consistent with the global disruption of transcriptomes of Scml2-KO PS and RS, we conclude that SCML2 regulates the differentiation of spermatogenic cells. To elucidate the molecular function of SCML2, we investigated whether SCML2 is a part of PRC1 in spermatogenic cells because a previous proteomics study identified SCML2 as a PRC1 component (Gao et al., 2012Gao Z. Zhang J. Bonasio R. Strino F. Sawai A. Parisi F. Kluger Y. Reinberg D. PCGF homologs, CBX proteins, and RYBP define functionally distinct PRC1 family complexes.Mol. Cell. 2012; 45: 344-356Abstract Full Text Full Text PDF PubMed Scopus (589) Google Scholar). RNF2, a catalytic core component of PRC1, and RNF2-mediated H2AK119ub, which was detected by a rabbit monoclonal antibody (D27C4), were both detected in cells progressing from undifferentiated spermatogonia to spermatocytes (Figure S3). Consistent with the histological data, co-immunoprecipitation experiments revealed that SCML2 interacts with RNF2 in testicular extracts (Figure 4A). Furthermore, chromatin immunoprecipitation (ChIP) sequencing (ChIP-seq) using GS cells, where SCML2 is abundantly expressed, demonstrated that SCML2 frequently bound to PRC1 target genes, such as the Hoxd cluster (Woo et al., 2010Woo C.J. Kharchenko P.V. Daheron L. Park P.J. Kingston R.E. A region of the human HOXD cluster that confers Polycomb-group responsiveness.Cell. 2010; 140: 99-110Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar) and Htra1 locus, and shared peaks with other PRC1 subunits, RNF2 and BMI1 (Figures 4B and S3). More than three-quarters (75.4% [n = 2,808]) of all SCML2-positive transcription start sites (TSSs; n = 3,723) overlapped with those of RNF2 (Figures 4C and S3), and 44.2% (n = 3,237) of all SCML2-positive islands (n = 7,326) were associated with RNF2-positive islands and enrichment of H2AK119ub throughout the entire genome (Figure 4D). Moreover, similar to other PcG proteins, SCML2 was also enriched at CpG islands, where gene-regulatory elements including promoters and enhancers reside (Ku et al., 2008Ku M. Koche R.P. Rheinbay E. Mendenhall E.M. Endoh M. Mikkelsen T.S. Presser A. Nusbaum C. Xie X. Chi A.S. et al.Genomewide analysis of PRC1 and PRC2 occupancy identifies two classes of bivalent domains.PLoS Genet. 2008; 4: e1000242Crossref PubMed Scopus (789) Google Scholar) (Figure 4D). The ChIP-seq data were reproducible between native ChIP and cross-linking ChIP (Figure S4). Taken together, these results suggest that SCML2 forms a complex with PRC1 and binds to gene-regulatory regions in GS cells. Conversely, 13,771 RNF2-positive islands did not overlap with SCML2-positive islands (such as Tbx5 locus; Figure S3), suggesting that other PRC1 complexes, which lack SCML2, regulate these islands. Because SCML2 forms a complex with RNF2 (Figure 4), we reasoned that SCML2 regulates target genes through H2AK119ub. To test this possibility, we first examined whether accumulation of H2AK119ub is associated with SCML2 in undifferentiated spermatogonia. Because we were not able to establish Scml2-KO GS cells, RNA-seq data from a THY1+ fraction of wild-type and Scml2-KO mice were used for this analysis. SCML2, RNF2, and H2AK119ub highly accumulated on the upregulated genes in Scml2-KO THY1+ cells compared with those downregulated (Figure 5A), suggesting that SCML2 regulates establishment of H2AK119ub for gene repression in undifferentiated spermatogonia. Next, we sought to determine whether suppression of somatic/progenitor genes that occurs in PS and RS are also mediated through the regulation of H2AK119ub. Consistent with the active transcription of somatic/progenitor genes in the THY1+ fraction, SCML2-regulated genes in PS and RS are largely distinct from those in the THY1+ fraction (Figure 5B). To examine the enrichment of H2AK119ub, we performed ChIP-seq of H2AK119ub in purified PS from the wild-type and the Scml2-KO (Figure 5C). Focusing on somatic/progenitor genes that are upregulated in Scml2-KO PS, H2AK119ub was relatively high around TSSs of such genes in the wild-type, compared with the Scml2-KO (Figure 5C). In contrast, when focusing on genes found downregulated in the Scml2-KO, accumulation of H2AK119ub was not observed around TSSs in either the wild-type or the Scml2-KO (Figure 5C). This result suggests that SCML2 promotes establishment of H2AK119ub for repression of somatic/progenitor genes in late spermatogenesis. Because SCML2 was highly accumulated on entire nuclei in undifferentiated spermatogonia, but was not on the autosome regions during meiosis (Figures 1G, 1H, and S1), we hypothesized that SCML2 establishes H2AK119ub prior to spermatogenic differentiation. Consistent with our cytological data, in PS, SCML2 is no longer associated with TSSs of somatic/progenitor genes found upregulated in Scml2-KO PS (Figure 5D). Moreover, in GS cells, SCML2, RNF2, and H2AK119ub were relatively high at TSSs of somatic/progenitor genes that were found to be repressed by SCML2 in the later stages (genes upregulated in the Scml2-KO PS and RS: Figures 5E and S4). This tendency is evident in genes upregulated in the Scml2-KO RS. These results suggest that, prior to spermatogenic differentiation, SCML2 functions with RNF2 and regulates establishment of H2AK119ub, and that H2AK119ub persists into later stages for repression of somatic/progenitor genes after the removal of SCML2 from their TSSs. Because SCML2 was not especially high around TSSs of genes that were found downregulated in the Scml2-KO, activation of late spermatogenesis genes may be indirectly mediated through SCML2. Because SCML2 was identified through its interaction with γH2AX and intensely localized on meiotic sex chromosomes (Figure 1), we next examined the function of SCML2 in the regulation of the sex chromosomes during meiosis. Surprisingly, H2AK119ub was decreased on the area of sex chromosomes in wild-type cells, but intensely accumulated there in Scml2-KO cells (Figures 6A and S5). Consistent with this, ChIP-seq data confirmed the enrichment of SCML2 on the X chromosomes in wild-type PS compared with that on autosomes (Figure 6B) and higher accumulation of H2AK119ub on the X chromosomes in Scml2-KO PS compared with that on the X chromosome in wild-type PS (Figure 6C). Importantly, this staining pattern was distinct from that of ubiquitination mediated by RNF8, which establishes polyubiquitination of H2A in the context of the DDR in somatic cells (Feng and Chen, 2012Feng L. Chen J. The E3 ligase RNF8 regulates KU80 removal and NHEJ repair.Nat. Struct. Mol. Biol. 2012; 19: 201-206Crossref PubMed Scopus (160) Google Scholar, Huen et al., 2007Huen M.S. Grant R. Manke I. Minn K. Yu X. Yaffe M.B. Chen J. RNF8 transduces the DNA-damage signal via histone ubiquitylation and checkpoint protein assembly.Cell. 2007; 131: 901-914Abstract Full Text Full Text PDF PubMed Scopus (814) Google Scholar, Kolas et al., 2007Kolas N.K. Chapman J.R. Nakada S. Ylanko J. Chahwan R. Sweeney F.D. Panier S. Mendez M. Wildenhain J. Thomson T.M. et al.Orchestration of the DNA-damage response by the RNF8 ub" @default.
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• W2088910050 creator A5022212436 @default.
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• W2088910050 date "2015-03-01" @default.
• W2088910050 modified "2023-10-06" @default.
• W2088910050 title "SCML2 Establishes the Male Germline Epigenome through Regulation of Histone H2A Ubiquitination" @default.
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