SUMMARY 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.

Sex chromosomes are uniquely subject to chromosome-wide silencing during male meiosis, and silencing persists into post-meiotic spermatids. Against this background, a select set of sex chromosome-linked genes escapes silencing and is activated in post-meiotic spermatids. Here, we identify a novel mechanism that regulates escape gene activation in an environment of chromosome-wide silencing in murine germ cells. We show that RNF8-dependent ubiquitination of histone H2A during meiosis establishes active epigenetic modifications, including dimethylation of H3K4 on the sex chromosomes. RNF8-dependent active epigenetic memory, defined by dimethylation of H3K4, persists throughout meiotic division. Various active epigenetic modifications are subsequently established on the sex chromosomes in post-meiotic spermatids. These RNF8-dependent modifications include trimethylation of H3K4, histone lysine crotonylation (Kcr), and incorporation of the histone variant H2AFZ. RNF8-dependent epigenetic programming regulates escape gene activation from inactive sex chromosomes in post-meiotic spermatids. Kcr accumulates at transcriptional start sites of sex-linked genes activated in an RNF8-dependent manner, and a chromatin conformational change is associated with RNF8-dependent epigenetic programming. Furthermore, we demonstrate that this RNF8-dependent pathway is distinct from that which recognizes DNA double-strand breaks. Our results establish a novel connection between a DNA damage response factor (RNF8) and epigenetic programming, specifically in establishing active epigenetic modifications and gene activation.

Repressive H3K27me3 and active H3K4me2/3 together form bivalent chromatin domains, molecular hallmarks of developmental potential. In the male germline, these domains are thought to persist into sperm to establish totipotency in the next generation. However, it remains unknown how H3K27me3 is established on specific targets in the male germline. Here, we demonstrate that a germline-specific Polycomb protein, SCML2, binds to H3K4me2/3-rich hypomethylated promoters in undifferentiated spermatogonia to facilitate H3K27me3. Thus, SCML2 establishes bivalent domains in the male germline of mice. SCML2 regulates two major classes of bivalent domains: Class I domains are established on developmental regulator genes that are silent throughout spermatogenesis, while class II domains are established on somatic genes silenced during late spermatogenesis. We propose that SCML2-dependent H3K27me3 in the male germline prepares the expression of developmental regulator and somatic genes in embryonic development.

SUMMARY Precise epigenetic regulation of the sex chromosomes is vital for the male germline. Here, we analyze meiosis in eight mouse models deficient for various DNA damage response (DDR) factors, including Fanconi anemia (FA) proteins. We reveal a network of FA and DDR proteins in which FA core factors FANCA, FANCB, and FANCC are essential for FANCD2 foci formation, whereas BRCA1 (FANCS), MDC1, and RNF8 are required for BRCA2 (FANCD1) and SLX4 (FANCP) accumulation on the sex chromosomes during meiosis. In addition, FA proteins modulate distinct histone marks on the sex chromosomes: FA core proteins and FANCD2 regulate H3K9 methylation, while FANCD2 and RNF8 function together to regulate H3K4 methylation independently of FA core proteins. Our data suggest that RNF8 integrates the FA-BRCA pathway. Taken together, our study reveals distinct functions for FA proteins and illuminates the male sex chromosomes as a model to dissect the function of the FA-BRCA pathway.

HERV-M (human endogenous retrovirus M), related to the super family of HERV-K, has a methionine (M) tRNA primer-binding site, and is located within the periphilin gene on human chromosome 12q12. HERV-M has been integrated into the periphilin gene as the truncated form, 5'LTR-gag-pol-3'LTR. Polymerase chain reaction (PCR) and reverse transcription-polymerase chain reaction (RT-PCR) approaches were conducted to investigate its evolutionary origins. Interestingly, the insertion of retroelements in a common ancestor genome can make different transcript variants in different species. In the case of the periphilin gene, human (10 variants) and mouse (2 variants) lineages show different transcript variants. Insertion of HERV-M (variant 1-3) could affect the protein-coding region. Also, Alusq/x (variant 4-9) and L1ME4a (mammalian-wide subfamilies of LINE-1) (variant 10) in humans and SINE (short interspersed repetitive element) and RLTR15 (the mouse putative long terminal repeat) (variant 2) in mice could be driving forces in transcript diversification of the periphilin gene during mammalian evolution. The HERV-M derived transcripts (variant 1-3) were expressed in different human tissues, whereas they were not detected in crab-eating monkey and squirrel monkey tissues by RT-PCR amplification. Taken together, HERV-M seems to have been integrated into our common ancestor genome after the divergence of simians and prosimians, and then was actively expressed during hominoid evolution.