Study On The Regulation Of Sox30 Gene Expression By Mouse Germ Cell Specific Transcription Factor SOHLH1

Objective:The process by which male germ cells develop from mature primordial germ cells with mitotic activity to mature sperm is called spermatogenesis.Spermatogenesis originates from spermatogonial stem cells and is divided into three stages:the mitosis phase of spermatogonia,the meiosis phase of spermatocytes,and the spermatogenesis phase.After undergoing mitosis,spermatogonia undergo a series of complex structural remodeling during spermatogenesis to form mature sperm.Many genes associated with spermatogenesis have begun to be transcribed in the early stages of male germ cell development,such as spermatogonia,where transcribed mRNA is translated into protein and functions when the cell develops to a later stage.Spermatogenesis-and oogenesis-specific bHLH transcription factor-1（Sohlh1）is mainly expressed in A₁-A₄ spermatogonia in male germ cells,and the encoded protein contains a bHLH structure.The domain,which can form a heterodimer or a homodimer,binds to the E-box sequence CANNTG on DNA to regulate the expression of downstream genes.Our previous study found that Sohlh1 knockout male mice are infertile,spermatogonial cell development and differentiation abnormalities lead to reduced spermatocytes into meiosis,no mature sperm production.The results of microarray showed that there were differences in the expression of key genes in Sox30 and Rnf17 in the testis tissues of wild-type males and Sohlh1 knockout males 7 days after birth.It is speculated that SOHLH1 may directly or indirectly regulate Sox30 gene transcription.Sox30 is the only member of the Sox family H subfamily,and the encoded protein has an HMG box domain that is involved in gonadal development and spermatogenesis.Sox30 knockout male mice have no fertility,and the acrosome particles on the surface of the round spermatozoa do not participate in acrosome formation,indicating that spermatogenesis is arrested in the spermatogenesis stage.It is speculated that Sox30may be related to acrosome formation in spermatogenesis.There are no reports of transcription factors regulating Sox30 expression at the stage.SOHLH1 is mainly expressed in differentiated spermatogonia in the early stage of spermatogenesis.It has been found that Sox30 mRNA is transcribed and persists in spermatocytes,but SOX30protein first appeared in round sperm cells in the late spermatogenesis.Informatics predicted that there is a SOHLH1 transcription factor binding sequence upstream of the Sox30 gene,suggesting that the Sox30 gene may be regulated by SOHLH1transcription.Normal Sox30 mRNA has a complete HMG domain and a C-terminal domain,but the Sox30 mRNA variant lacks a complete C-terminal domain.Studies have shown that the C-terminal domain is associated with delayed translation of mRNA.This study was to analyze the transcription of Sox30 gene mRNA in wild-type mice and Sohlh1^-/-mouse male germ cells,and to explore the expression pattern of Sox30 gene in male germ cells and important transcription factors related to early development of spermatogonia.SOHLH1 is involved in the transcriptional regulation mechanism of the related gene Sox30 in the late spermatogenesis,and further screens the DNA sequence of the SOHLH1-specific binding site on the Sox30 gene promoter sequence.Methods:Wild-type C57BL/6J male and Sohlh1 knockout males were used as subjects in this study.Different transcripts of Sox30 mRNA in PD7（7 days after birth）and adult mouse testicular tissue RNA were detected by qRT-PCR.The expression level of SOHLH1 was determined by constructing the Sox30 promoter expression vector and the dual luciferase reporter gene.The main binding site of SOHLH1 on the Sox30 promoter sequence was screened by ChIP assay.Results:The results of qRT-PCR showed that the expression of Sox30 mRNA was significantly down-regulated in Sohlh1^-/-mouse testis tissue compared with wild-type C57B6L/J mice 7 days after birth（P<0.01）,Sox30 mRNA variant expression.The amount was adjusted downward（P<0.05）.Compared with adult wild-type C57B6L/J mice,the expression of Sox30 mRNA in Sohlh1^-/-mice was significantly down-regulated（P<0.01）,and the expression of mRNA variants was up-regulated（P<0.01）due to the loss of Sohlh1 gene.The expression levels of both transcripts of Sox30 were abnormal,and the Sox30 mRNA variants in adult Sohlh1^-/-mice showed abnormal transcription and accumulation.The results of dual luciferase assay showed that when the Sohlh1 eukaryotic expression plasmid was co-transfected with the promoter fluorescent expression vector,the fluorescence activity of the Sox30promoter was the strongest in the-876 bp region,and the core promoter fragment was located.Within this fragment,the germ cell-specific expression of the transcription factor SOHLH1 activates the transcription of the Sox30 gene.ChIP assay showed that the transcription factor SOHLH1 can bind to the upstream of the Sox30 transcription start site-544bp E-box（CAACTG）,-489bp E-box（CAGGTG）,-166bp E-box（CAACTG）,-101bp E-box（CATATG）These four E-box sites,but bind strongly to the E-box site at the-489 bp（CAGGTG）position,which is the major transcriptional activation site on the Sox30 gene promoter.Conclusion:1.The transcription factor SOHLH1 has transcriptional activation on the expression of testis tissue-specific gene Sox30.The transcriptional activation of Sox30by SOHLH1 proves that the gene has delayed translation.2.In the mouse testis,the transcription factor SOHLH1 can bind to the-544 bp,-489bp,-166 bp and-101 bp E-box domains upstream of the Sox30 gene transcription initiation site,and activate transcriptional activation of Sox30 at-489 bp.The E-box locus at the（CAGGTG）position is more strongly associated with the transcription factor SOHLH1,which is the major transcriptional activation site on the Sox30 gene promoter.