Role For Histone Methyltransferase SETDB1 On The Proliferation And Differentiation Of Spermatogonial Stem Cells And Its Mechanism

Spermatogenesis is the basis for the continuous production of sperm in males,which mainly includes the self-renewal and differentiation of spermatogonial stem cells(SSCs),meiosis,and the transformation of haploid sperm cells.Spermatogenesis is delicately regulated by dynamic gene expression.The conditional knockout of H3K9me2demethylatase gene Jmjd1a and Jmjd1b in embryonic germ cells results in disrupted gene expression and impairs spermiogenesis.Thus,exploration of the regulatory mechanisms for histone methylation modification in SSCs has guiding significance in diagnosis and treatment of male infertility.SETDB1 has been reported to catalyze methylation of lysine 9on histone H3(H3K9),and play a crucial role in pluripotency maintenance of stem cells and neurogenesis.However,the role of SETDB1 in proliferation and differentiation of SSCs remains elusive.Therefore,immunofluorescence,chromatin immunoprecipitation,RNA-seq,ATAC-seq and Ch IP-seq were used to determine the molecular mechanisms of SETDB1 in proliferation and differentiation of SSCs.The main results were as follows:(1)Ed U and flow cytometry analysis showed that Setdb1-KD impaired proliferation and resulted in cell cycle arrest in the S phase in SSCs.Immunofluorescence and TUNEL assay showed that knockdown of Setdb1 triggered DNA double-strand breaks and apoptosis.These data suggest that Setdb1-KD impairs SSC proliferation and induces apoptosis.(2)The expression of NOX4 and P22PHOX(NOX4 regulatory subunits)and the level of ROS are increased in SSCs induced by Setdb1-KD.The rescue study revealed that compared with the Setdb1-KD group,the level of ROS was decreased and the phenotype of apoptosis and decreased proliferation were partially rescued in Setdb1-KD and Nox4-KD group.In addition,melatonin is a biological free radical scavenger and antioxidant that not only removed ROS directly,but also inhibited apoptosis that had been induced by Setdb1-KD.These results showed that NOX4 meditated ROS increase which was involved in Setdb1-KD-induced apoptosis and in proliferation decrease.(3)Knockdown of Setdb1 resulted in increase of p-P38 and p-JNK,and activation of FOXO4.These results suggested that Setdb1-KD activated the p38/JNK-FOXO4 signal pathway downstream of ROS.(4)According to the results of SETDB1-ChIP-qPCR and H3K9me3-Ch IP-q PCR,there was no SETDB1 and H3K9me3 recruitment in the promoter of Nox4,suggesting that the regulation of Nox4 is not directly exerted by SETDB1.It is possible that Setdb1-KD induces the activation of the transposon flanking Nox4,thereby increasing the expression of NOX4.(5)In mouse spermatogonia,Ythdf2-KO resulted in a decrease of the SETDB1 and H3K9me3 modification level.The results of Co-IP revealed that SETDB1 interacted with?H2AX in spermatogonia.When there was DNA damage,Setdb1-KD reduced the ability of spermatogonia to form cell clones,and increased apoptosis and the number of?H2AX-positive cells.We also found that Ythdf2-KO spermatogonia showed a phenotype similar to Setdb1-KD.When treated with etoposide,a DNA damage induction agent,the average number of?H2AX-and c PARP-positive cells in each seminiferous tubule in Ythdf2 c KO mice(Stra8-Cre,Ythdf2^loxp/loxp)was higher than that in the control group.These results suggest that YTHDF2 and SETDB1 are involved in DNA damage response,and YTHDF2 could regulate the expression of Setdb1 in mouse spermatogonia.(6)The rescue study revealed that when DNA damage occurs,SETDB1overexpression in Ythdf2-KO spermatogonia could partly rescue the colony number of spermatogonia which was decreased by Ythdf2-KO.Moreover,RNA-seq analysis indicated that SETDB1-OE reduced the transcription of pro-apoptotic genes in Ythdf2-KO spermatogonia under the circumstance of DNA damage.These results suggested that the increased sensitivity of spermatogonia to DNA damage which was induced by Ythdf2-KO may be due to the down-regulation of Setdb1.(7)We isolated porcine SSCs and differentiating spermatogonia by FACS and STA-PUT,respectively.Then,we performed ATAC-seq,Ch IP-seq and RNA-seq to describe the dynamics of SETDB1 and H3K9me3 modification and the chromatin accessibility.RNA-seq analysis showed that 684 genes were up-regulated and 702 genes were down-regulated during the differentiation of SSCs.Moreover,the enrichment levels of SETDB1 and H3K9me3 modification were decreased and the chromatin accessibility was increased at the promoter of c-Kit during the differentiation process.Therefore,we speculate that SETDB1 may directly regulate the expression of promoter differentiation-related genes through its H3K9me3 modification function.In conclusion,this study reported that SETDB1 regulated the survival of mouse SSCs by regulating the expression of NOX4 and the content of ROS.In mouse spermatogonia,Ythdf2-KO-induced downregulation of SETDB1 led to increased sensitivity of spermatogonia to DNA damage.During porcine SSC differentiation,SETDB1 regulated differentiation-related gene expression changes by regulating chromatin accessibility and catalyzing H3K9me3 modification,thus regulating the differentiation of porcine SSCs.Taken together,this study provides a theoretical reference for the regulatory mechanisms for epigenetic modification in the development of male germ cells.