The Mechanism Of Histone Methyltransferase ESET In The Survival Of Murine Spermatogonial Stem Cells

Spermatogonial stem cells (SSCs), which are located in the basement membrane ofseminiferous tubule, are adult stem cells that transmit genetic information to the nextgeneration. SSCs maintain cells number by self-renewal as well as produce spermatozoa bydifferentiation. The self-renewal and differentiation of SSCs are regulated by mutiplesignaling pathways (such as Src signaling pathways, Smad signaling pathway, etc.) andtranscription factors (such as PLZF, BCL6B, etc.). To the date, most studies have beenfocused on the classic genetic field, such as the signaling pathways and proteins functions.However, what role histone modification plays in the maintenance of SSCs remains unknown.Methyltransferase ESET (ERG-associated protein with a SET domain) participates in theformation of heterochromatin, and plays essential roles both in the formation of ERVmethylation, maintenance and survival of articular cartilage and neurons. In this study, RNAinterference, the microarray analysis, spermatogonial stem cell transplantation, and ChIP wereused to detect the distribution, function and regulation mechanism of H3K9me3and ESET inSSCs. The main observations were as follows:(1) The expression of ESET was highest in mouse testis tissue compared with the othertissues (heart, liver, spleen, lung, kidney, ovaries and testes), and was gradually increasingduring testis development, in parallel to the global level of H3K9me3. The ESET andH3K9me3were expressed or distributed in SSCs.(2) The effective shRNA against for Eset gene was selected and the lentiviral vector forRNAi was constructed. The titer of Eset RNAi lentivirus reached to1.7x107CFU/ml. Highpurity of SSCs were obtained by magnetic-activated cell sorting. The purity reached91.5%.After lentiviral transduction, the RNAi efficiency was68.3%in SSCs.(3) For microarray analysis,16611genes were analyzed in ESET-KD SSCs. Comparedwith the control group, the expression of2490genes were up-regulated more than two fold,accounts for15.0%; the expression of2425genes were down-regulated more than two fold,accounts for14.6%. GO analysis showed that ESET has catalytic activity and was relatedwith the combination of protein and DNA in mouse SSCs. Pathway analysis further suggested that ESET can influence the expression of a variety of genes in P53pathways, suggesting thatESET may be associated with SSC apoptosis in mice.(4) After two weeks culture, the number of SSCs in ESET-KD group was significantlyreduced compared with control groups. TUNEL analysis showed that the number of apoptoticcells in ESET-KD group were increased. The expression of P53, Apaf1, Caspase9, andCaspase3(P17) in ESET-KD SSCs were significantly increased, while the expression ofBcal2l1and XIAP were significantly reduced. In addition, the cleavage of PARP was alsodetected in the ESET-KD SSCs. These result showed that SSCs which lack of ESETexpression were under apoptosis.In SSC transplantation experiments, the testes weight and the percentage of seminiferoustubules with spermatogenesis in recipient mice transplanted ESET-KD SSCs, wassignificantly reduced. In addition, the expression of GFP and PLZF in recipient micetransplanted ESET-KD SSCs was significantly lower than the control groups. Theseobservations suggest that ESET regulates SSC apoptosis.(5) ESET-ChIP analysis showed that ESET directly binded with the promoter of Sohlh2,Nobox, Dazl, Foxn1, and Cox4i2. H3K9me3-ChIP analysis further showed that H3K9me3level in the promoter of the aforementioned5genes was significantly decreased in ESET-KDgroup. The expression of Caspase9was decreased in cells co-transfected with Cox4i2-siRNAand ESET-siRNA compared with that transfected with ESET-siRNA. These data suggest thatCox4i2is a target gene of ESET that regulates the SSC apoptosis.Co-Immunoprecipitation showed ESET could bind with DNMT3A In C18-4cells. ESETwere The DNA methylation in the promoter of Cox4i2gene was decreased in ESET-KDgroup, while the DNA methylation in the promoter of Dazl gene was not change.In conclusion, this research studied the function and regulatory mechanism of H3K9me3and its methyltransferase ESET in mouse SSCs. This research expanded the research fields,and provided a new way to study the SSC function.