Molecular Mechanism Of How Histone Methytransferase Setdb1 Regulate The Survival Of Male Germ Stem Cells

Both gonocytes and spermatogonia with stem cell characteristicsare known as male germ stem cells,which can self-renew and maintain the stem cell pool and differentiate into sperm.Spermatogonial stem cells(SSCs)are the only adult stem cells that can transfer their genetic information to their offspring.SSCs can differentiate into spermatogonia and initiate spermatogenesis,spermatogenesis requires a precisely ordered regulation of gene expression.Gene expression is regulated by histone modifications.Three distinct methylation states,which are mono-(me1),di-(me2),and tri-(me3)methylation that occur at lysine(K)of histone H3 and H4(such as K4,K9,and K27 of histone H3 and K20 of histone H4),are present in mammalian cells.Generally,H3K4 and H3K36 are related to transcriptional activation,whereas H3K9,H3K27,and H4K20 are associated with transcriptional repression.These histone methylations are modulated by methyltransferases and demethylases.Histone methyltransferase SETDB1(SET domain,bifurcated 1),also known as ESET,suppresses gene expression and modulates heterochromatin formation through H3K9me2/3.SETDB1 plays an important role in the maintenance of embryonic stem cells and the survival of neural cells.This study revealed the function and the molecular mechanism of SETDB1 in regulating the survival of mouse spermatogonial stem cell,and the regulation of SETDB1 in maintaining the survival of gonocytes in pigs using protein coimmunoprecipitation,chromatin immunoprecipitation and immunofluorescence techniques.The main results are as follows:(1)Setdb1 knockdown induced the mitochondrial membrane potential of C18-4 cells,valgus fracture membrane phosphatidylserine,and DNA breakage,andSetdb1-KD induced up-regulation of apoptosis associated genes Bax,Apaf1,p53,Caspase 9,decrease of anti-apoptosis gene XIAP expression.These results suggest that Setdb1 interference can regulate the expression of apoptosis-related genes to induce apoptosis.(2)Setdb1-KD activates PTEN,which inhibits the phosphorylation of AKT and inhibits its activity,leading to the activation of FOXO1.Pten-KD reversed the change elicited by Setdb1-KD,in terms of upregulation of the pro-apoptosis gene BAX,Caspase9,Apaf1,Puma and Bim.Importantly,Pten-KD inverted the reduction of phospho-AKT,phospho-FOXO1 and BCL2,meanwhile decreased the level of cleaved-Caspase3 compared with the solely knockdown of Setdb1,suggesting that Pten-KD rescues the Setdb1-KD induced phenotype.(3)SETDB1 can interact with AKT,and SETDB1 coordinated with AKT to enhance the inhibitory effect of AKT on FOXO1 activity,which can induce the FOXO1 to be located in the cytoplasm and inhibit its own transcriptional activity.Setdb1-KD promoted cytoplasmic-to-nuclear translocation of FOXO1 and activate the expression of Bim and Puma.These results suggest that SETDB1 can coordinate with AKT to regulate FOXO1 activity and expression of the downstream target genes.(4)The results of ChIP showed that H3K9me3 levels on Pten,Foxo3 and Bim were decreased by Setdb1 deficiency,whereas Setdb1 deficiency did not cause noticeable change of H3K9me3 on the promoter regions of Foxo1,Puma,Bax and Bcl2.In addition,SETDB1 directly bound to the promoter regions of Pten,BimPuma and Bax Collectively,these data suggest that SETDB1 directly binds to Pten and Bimand represses theirexpression via elevated H3K9me3 level to regulate the survival of mouse spermatogonial stem cells.(5)In the process of pig testis development,the expression of SETDB1 at the transcriptional and translational level was gradually increasing.It achieved the highest level at adult stage.However,H3K9me3 level was contrary to the SETDB1 expression.The abundance of H3K9me3 was the highest in the neonatal piglet testis tissue,and then decreased.In 7d and 2m testis,SETDB1 exhibited significant cytoplasmic localization and weakly in nucleus in gonocytes/undifferentiated spermatogonia,H3K9me3 showed extensive perinuclear distribution in gonocytes/undifferentiated spermatogonia.SETDB1 was expressed in the nuclear of undifferentiated spermatogonia and differentiated spermatogonia in adult testes,H3K9me3 distributed in perinuclear of SSCs,while displayed intranuclear distribution in differentiated spermatogonia.(6)In order to study the function of SETDB1 in gonocytes,the purity of enriched gonocytes reached 76.5% ± 3.9% by differential adherent enrichment.Western blot and RT-PCR results showed that SETDB1 was mainly expressed in gonocytes.These results suggest that the expression pattern of SETDB1 may have a specific role for the survival of the gonocytes.(7)SETDB1-KD induced apoptosis of gonocytes,and apoptosis was not dependent on H3K9me3.In addition,the results revealed that SETDB1 can interacted with EZH2,which catalyzes H3K27 methylation,and SETDB1 knockdown decreased the levels of H3K27me3,indicating knockdown of SETDB1 in gonocytes induces apoptosis via regulating H3K27me3 rather than H3K9me3.In summary,in mouse studies,Setdb1-KD induced spermatogonial stem cell apoptosis is regulated by PTEN/AKT/FOXO1 pathway and H3K9me3 level;in addition,SETDB1 cooperated with AKT participate in the regulation of the activity of downstream target protein FOXO1.At the same time,SETDB1-KD also inducesgonocyte apoptosis,but the level of H3K9me3 does not change,whereas is related to the reduction of H3K27me3 catalyzed by histione methyltransferase EZH2,whichinteractes with SETDB1.This study reveals a functional link between the epigenetic machinery and the signaling pathway governing apoptosis,and replenishes the research on the epigenetic regulation of SSC survival,and opens a door for the study of male infertility in the future.