S-SOX5 Regulates The Expression Of SPAG16L Through Directly Binding To Its Promoter

Objective : The mammalian sperm-associated antigen 16 gene(Spag16)uses alternative promoters to produce two major transcript isoforms(Spag16L and Spag16S)that encode critical proteins involved in the cilia/flagella formation and motility.In silico analysis of both mouse and human SPAG16 L promoters reveals the existence of multiple putative SOX5 binding sites.Given that the SOX5 gene encodes a 48-k Da transcription factor(S-SOX5)and the presence of putative SOX5 binding sites at the SPAG16 L promoter,regulation of SPAG16 L expression by S-SOX5 was studied in the present work,which could provide a theoretical basis for comprehensively exploring of flagella formation during spermatogenesis.Method:1)Con Site program was used to predict transcription factors of human SPAG16 L and found multiple putative SOX5-binding site in the 2-kb proximal promoter region of SPAG16 L.2)The c DNA was used as a template to construct SPAG16 L promoter luciferase reporter fusion by cloning a 2kb human SPAG16 L proximal promoter region including the transcriptional start sit and multiple putative SOX5 binding sites into the Kpn I/Nhe I sites of the p GL3-basic vector(SPAG16L/p GL3).Similary,full-length human S-SOX5 c DNA was cloned into the Kpn I/Xho I sites of pc DNA3 or Bam HI/Not I sites of p Target vector plasmids(S-SOX5/pc DNA3 and S-SOX5/p Target),and the RNAi constructs targeting SOX5 transcripts were generated.3)SPAG16L/p GL3 plasmid was transfected into BEAS-2B cells,with p GL3 empty vector as a control.The expression level of SPAG16 L was assessed by measuring the promoter activity with the Luciferase Reproter Assay system.S-SOX5/pc DNA3 was transfected into BEAS-2B cells,with pc DNA3 vector as a control.WB and RT-PCR analysis were used to investigate endogenous expression of SOX5 in BEAS-2B cells and the effect of S-SOX5 induction on the expression of SPAG16 L,respectively.4)Two human RNAi plasmid constructs that respectively target portion 225-246 and 1109-1130 of the SOX5 transcript were used.The efficiency of the SOX5 RNAi plasmids was examined by WB analysis.5)To study the molecular interaction between S-SOX5 and the SPAG16 L promoter,BEAS-2B cells were infected with S-SOX5 adenovirus(Ad S-SOX5)and Ch IP assay was conducted with an antibody specially against S-SOX5 or rabbit Ig G.6)Two SOX5 binding sites in the SPAG16 L promoter construct were mutated,and effect of these mutations on the expression of SPAG16 L was examined.Result:Multiple putative SOX5-binding sites in the 2-kb proximal promoter region of SPAG16 L were found by using the Con Site program,considering the relative activity of SPAG16 L in BEAS-2B cells,then it is worthy to study transcriptional regulation by S-SOX5.RT-PCR showed that S-SOX5 message is the predominant isoform in BEAS-2B cells and the 48-k Da S-SOX5 was detected in western blot analysis.When co-transfected with S-SOX5,the level of SPAG16 L expression was higher than that of the control empty p GL3-basic plasmids.After BEAS-2B cells was infected with S-SOX5/pc DNA3 or S-SOX5 adenovirus,expression of S-SOX5 was clearly increased,and the relative level of SPAG16 L m RNA was significantly higher than that in the control.To further investigate the regulatory role of S-SOX5 on SPAG16 L expression,RNAi method was used and the data suggest that knockdown of S-SOX5 in BEAS-2B cells reduces expression of SPAG16 L,indicating SPAG16 L expression depends on the activity of S-SOX5 protein.In addition,Ch IP assay showed that S-SOX5 is capable of directly binding to the promoter of SPAG16 L.When putative SOX5 binding sites at the proximal promoter region of SPAG16 L was mutated,S-SOX5 failed to activate SPAG16 L promoter activity.Conclusion:The present study demonstrates that SPAG16 L is one of S-SOX5 target gene.The transcriptional regulation of SPAG16 L by S-SOX5 is achieved by directly binding to putative SOX5 binding sites in the SPAG16 L promoter region.