The Histone Modification Reader ZCWPW1 Links Histone Methylation To PRDM9-induced Double-strand Break Repair

Meiosis is a unique,specialized cellular division process fundamental to the production of viable gametes in all sexually reproducing organisms.During meiosis,one round of DNA replication followed by two rounds of cell divisions results in generation of haploid gametes(sperm and eggs in mammals).During the prophase I of meiosis,homologous(parental and maternal)chromosomes must find each other,pair,recombine and then become tethered to each other until separate.The normal progress of these events ensures the stable transmission of genetic material between biological generations,and thus produces genetic diversity.The number and distribution of meiotic recombination on chromosomes are tightly regulated.The abnormal number and location of chromosomal recombination that occur during human meiosis lead to the inability to produce gametes or poor quality of gametes,which eventually leads to infertility,spontaneous abortion or birth defects.The in-depth understanding of the regulation mechanism of meiosis will ultimately enable us to diagnose various causes of infertility based on meiotic abnormalities,and to more effectively apply assisted reproductive technology to obtain functional gametes.At the molecular level,meiotic homologous recombination is initiated by the induction of programmed double strand break(DSB),followed by homologous recombination for DSB repair,leading to the formation of crossover and the conversion of genetic material.DSB induction is a complex process,and DSB locations are known to be marked at the very earliest stages of meiosis by trimethylation of histone H3 on lysine 4(H3K4me3),which are thought to mark the future sites of DSB formation.In mammals,this histone modification,which represents the formation site of DSB,is catalyzed by PRDM9(PR/SET domain-containing protein 9),which is mainly expressed in the leptonema and zygonema of the meiosis prophase I.PRDM9 contains an N-terminal KRAB domain involved in protein-protein interactions,an SSXRD nuclear localization signal,a SET domain with histone methyltransferase activity,and a carboxyl end with a long and highly variable in the genetic structure of zinc finger domains,which determines its specificity of the combination of the genome(which is used to define the recombination hotspots,also the DSB formation location).In yeast,the histone modification reader SPP1(Suppressor of PRP Protein 1)recognizes H3K4me3 near the promoter through its PHD(Plant homeodomain)domain and mediates the formation of DSB.In mice,although multiple studies have shown that PRDM9-mediated modifications of H3K4me3 and H3K36me3 control the formation sites of DSB,little is known about any protein that might read these epigenetic markers and thus help to promote the meiotic recombination process.ZCWPW1 is one of the members of the protein family including zinc finger CW(zf-CW)domain and PWWP domain.Its zf-CW domain has three conserved tryptophan residues and four conserved cysteine residues.Structural analysis shows that the zf-CW domain of human ZCWPW1 is a histone modification H3K4me3 reader,chromatin co-precipitation analysis also confirmed that the zf-CW domain of ZCWPW1 can recognize the H3K4me3 marker.Studies have confirmed that the zf-CW domain and the PHD domain are highly homologous in recognizing the histone modification H3K4me3.The PWWP domain is another "reader" that has been shown to specifically recognize H3K36me3.Based on the above research background,we raise the following scientific questions:The histone modification"reader" ZCWPW1 may be involved in the process of meiosis by identifying PRDM9-mediated histone modifications(H3K4me3/H3K36me3).ZCWPW1 is specifically and highly expressed in germ cells.Western blotting and immunofluorescence have confirmed that ZCWPW1 was mainly expressed in the nucleus of spermatocytes and oocytes in meiosis prophase I.In different embryonic stages(E12:leptonema,E14:zygonema,E16:pachynema)ovaries,the expression of Zcwpw1 was significantly up-regulated in zygonema,while in Stra8 and Dazl knockout mice(The initiation of meiosis is abnormal,without zygonema spermatocytes in the testis),the expression of Zcwpw1 is significantly down-regulated.All the above results suggest that ZCWPW1 may play a role in the prophase I of meiosis.In order to investigate whether ZCWPW 1 is involved in the regulation of meiosis,we constructed Zcwpw1 gene knockout mice.The testes of male mice became smaller,no spermatocytes in the late stage of meiosis were observed in the seminiferous tubules,and no mature sperm cells were observed in the epididymis,indicating complete sterility.Pathological analysis found that spermatocyte meiosis arrest,apoptotic cells increased and empty seminiferous tubules appeared in Zcwpw1 knockout mice.The staining of chromosome spreading revealed that spermatocytes of Zcwpw1 knockout mice arrested at zygotene stage,and there were no spermatids in pachytene or later stage.There was an average of 7-8 pairs of homologous chromosomes that can initiate or complete the synapsis,and the high-resolution structured illumination microscopy(SIM)results show that the structure of the synaptonemal complex is normal.We have confirmed that the spermatocytes in Zcwpw1 knockout mouse were arrested in the zygotene stage by a series of pathological analysis.In meiosis prophase I,along with the homologous chromosome synapsis,there will be SPO11 and other molecular-mediated programmed DNA double-strand breaks and homologous recombination processes.In order to further investigate the reason of Zcwpw1 knockout mice spermatocytes arrest in the zygonema,we analyzed the production,repair and homologous recombination of DSB during meiosis prophase I.By co-staining spermatocyte chromosomes with yH2AX/SYCP3,we found that the Zcwpw1 knockout mice spermatocytes arrested at the pachytene-like stage,and there was still a large number of yH2AX signals on the un-synapsed chromosomes,and there was no XY body formation.These results indicated that DSB could be produced in the spermatocytes of Zcwpw1 knockout mice,but could not be completely repaired.The early recombinant molecules RPA2/SYCP3,RAD51/SYCP3 and DMC1/SYCP3 were co-stained on the chromosome spreads of Zcwpw1 knockout mice spermatocytes.A large number of RPA2/RAD51/DMC1 foci remained on the un-synapsed chromosome axis of pachytene-like spermatocytes in the knockout group.The recombinant node MLH1/SYCP3 was co-stained with the spermatocytes chromosome spread,and no MLH1 signal was found in the Zcwpw1 knockout spermatocytes.These results suggested that Zcwpw1 knockout resulted in abnormal homologous recombination of spermatocytes and failure to form recombinant nodes.When genomic DNA is damaged,it will activate the DNA damage response.During meiosis,DDR related proteins promote the process of homologous recombination and the synapsis of homologous chromosomes.In mammals,DDR-related proteins can recognize and mediate the transcriptional silencing of unsynapsed chromatin(Meiotic silencing of unsynapsed chromatin,MSUC),which is restricted to the X and Y chromosomes that are not fully synapsed during male meiosis,a process also known as Meiotic sex chromosome inactivation(MSCI).The chromosome spread of spermatocytes was co-stained with the MSUC initiation factor BRCAl/SYCP3.In the Zcwpw1 knockout spermatocytes,the BRCA1 signal was abundantly enriched on the un-synapsed chromosome,suggesting the existence of MSUC abnormal in the Zcwpw1 knockout spermatocytes.Transcriptome sequencing was performed on the testicular tissue of mice at 14 days after birth(meiosis progressed to the zygotene stage,and there are a large number of un-synapsed chromosomes in spermatocytes of both genotypes),and it was found that compared with the control group,81.8%(464/567)of the differentially expressed genes were down-regulated in the testes of Zcwpw1 knockout mice,suggesting that Zcwpw1 knockout resulted in abnormal MSUC.Preliminary phenotypic analysis found that Zcwpw1 knockout spermatocytes arrested at the zygotene stage,unable to form the XY body.In order to study whether Zcwpw1 knockout would cause abnormalities in MSCI,transcriptome at 17 days after birth(meiosis progressed to the pachytene stage,and MSCI appear)found that the sex chromosome-related gene expression was significantly up-regulated in the testes of the Zcwpw1 knockout mice compared with the control group,indicating that Zcwpw1 knockout lead to abnormal MSCI.Proteomic analysis of testicular tissue of 14-day-old mice showed that Zcwpw1 knockout resulted in dysregulation of chromatin remodeling related genes,suggesting that ZCWPW1 may participate in the regulation of meiosis by affecting chromatin remodeling.The meiosis of spermatocytes and oocytes has a significant difference in time(meiosis in male mice starts at 8 days after birth,meiosis in female mice starts on embryonic day 13.5).The expression and localization of ZCWPW1 in spermatocyte and oocyte also exist certain differences(ZCWPW1 locates on XY body in pachytene spermatocyte,and diffusely distributed throughout the nucleus in pachytene oocytes).Zcwpw1 Knockout male mice are azoospermia,while Zcwpw1 knockout female mice show premature ovarian failure.Fertility is normal until three months,but significantly reduced at five months,and premature ovarian failure occurs at eight months.Further analysis revealed that the ovarian reserve of Zcwpw1 knockout mice was normal on embryonic day 13.5 at the beginning of meiosis,and the ovarian reserve decreased significantly at 1 day after birth,and by 8 days after birth,knockout mice ovarian reserve is only about 15%of the control mice.Chromosome spread analysis showed that the delay of meiosis during embryonic stage resulted in the massive loss of oocytes after birth.Phenotypic analysis of Zcwpw1 knockout mice confirmed that ZCWPW1 was involved in the regulation of meiosis.To clarify whether ZCWPW1 is involved in meiosis regulation by recognizing PRDM9-mediated histone modifications,we used ChIP-Seq assay in a series of animal models and found that ZCWPW1 is specifically enriched in the histone modified H3K4me3 and H3K36me3 regions near the recombinant hotspots.Analysis of mice with point mutation of ZCWPW1 zf-CW domain confirmed that ZCWPW1's chromatin recognition is dependent on its zf-CW domain.Analysis of Prdm9 knockout mice confirmed that the localization of ZCWPW1 to the chromatin histone modified H3K4me3 and H3K36me3 regions depend on the catalytic activity of PRDM9.In conclusion,our study found that the H3K4me3 reader ZCWPW1 is specifically required for meiosis prophase I progression in male but not in female germ cells in mice.Loss of Zcwpw1 in male mice caused a complete failure of synapsis,resulting in meiotic arrest at the zygotene to pachytene stage,accompanied by incomplete DNA double-strand break repair and lack of crossover formation,leading to male infertility.In oocytes,deletion of Zcwpw1 only somewhat slowed down meiosis prophase I progression;Zcwpw1 knockout oocytes were able to complete meiosis,and Zcwpw1 knockout female mice had normal fertility until mid-adulthood.We conclude that the H3K4me3 reader ZCWPW1 is indispensable for meiosis synapsis in males but is dispensable for females.It has been shown that the induction and repair of programmed DSB at the early stage of the meiosis prophase I is the molecular basis of chromosomal behavior in meiosis.PRDM9-mediated H3K4me3 and H3K36me3 modification plays an important role in the formation of programmed DSB sites.We have demonstrated for the first time that ZCWPW1 specifically recognizes H3K4me3 and H3K36me3 modifications near the recombination hotspot mediated by PRDM9,and participate in the repair of meiotic DNA double-strand breaks.Our series of studies provide evidence to elucidate the role of epigenetic regulation in meiosis and the differences between male and female in the process of meiosis,and hopefully provide candidate target molecules for the diagnosis and treatment of male infertility and female POI,as well as the development of novel drugs.