The human NHEDC1 (hNHEDC1) protein is thought to be essential for sperm motility and fertility however the mechanisms regulating its gene expression are largely unknown. In this study we have identified multiple DNA regulatory elements in the 5′ end of the gene encoding hNHEDC1 (SLC9B1) and have explored the role that DNA methylation at these elements plays in the regulation of its expression. We first show that the full-length hNHEDC1 protein is testis-specific for the tissues that we tested and that it localizes to the cells of the seminiferous tubules. In silico analysis of the SLC9B1 gene locus identified two putative promoters (P1 and P2) and two CpG islands - CpGI (overlapping with P1) and CpGII (intragenic) - at the 5′ end of the gene. By deletion analysis of P1, we show that the region from −23bp to +200bp relative to the transcription start site (TSS) is sufficient for optimal promoter activity in a germ cell line. Additionally, in vitro methylation of the P1 (the −500bp to +200bp region relative to the TSS) abolishes its activity in germ cells and somatic cells strongly suggesting that DNA methylation at this promoter could regulate SLC9B1 expression. Furthermore, bisulfite-sequencing analysis of the P1/CpGI uncovered reduced methylation in the testis vs. lung whereas CpGII displayed no differences in methylation between these two tissues. Additionally, treatment of HEK 293 cells with 5-Aza2-Deoxycytidine led to upregulation of NHEDC1 transcript and reduced methylation in the promoter CpGI. Finally, we have uncovered both enhancer and silencer functions of the intragenic SLC9B1 CpGII. In all, our data suggests that SLC9B1 gene expression could be regulated via a concerted action of DNA methylation-dependent and independent mechanisms mediated by these multiple DNA regulatory elements.

The α4 Na,K-ATPase is a sperm-specific protein essential for sperm motility and fertility yet little is known about the mechanisms that regulate its expression in germ cells. Here, the potential involvement of DNA methylation in regulating the expression of this sperm-specific protein is explored. A single, intragenic CpG island (Mα4-CGI) was identified in the gene encoding the mouse α4 Na,K-ATPase (Atp1a4), which displayed reduced methylation in mouse sperm (cells that contain α4) compared to mouse kidney (tissue that lacks α4 expression). Unlike the intragenic CGI, the putative promoter (the −700 to +200 region relative to the transcriptional start site) of Atp1a4 did not show differential methylation between kidney and sperm nevertheless it did drive methylation-dependent reporter gene expression in the male germ cell line GC-1spg. Furthermore, treatment of GC-1spg cells with 5-Aza2-Deoxycytidine led to upregulation of the α4 transcript and decreased methylation of both the Atp1a4 promoter and the Mα4-CGI. In addition, Atp1a4 expression in mouse embryonic stem cells deficient in DNA methytransferases suggests that both maintenance and de novo methylation are involved in regulating its expression. In an attempt to define the regulatory function of the Mα4-CGI, possible roles of the Mα4-CGI in regulating Atp1a4 expression via methylation-dependent transcriptional elongation inhibition in somatic cells and via its ability to repress promoter activity in germ cells were uncovered. In all, our data suggests that both the promoter and the intragenic CGI could combine to provide multiple modes of regulation for optimizing the Atp1a4 expression level in a cell type-specific manner.