Determining the role of DNA methylation in the tissue-specific expression of the Na,K-ATPase-Na/H exchanger pH regulatory system genes.

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Abstract: Epigenetic mechanisms are critical for the regulation of tissue-specific gene expression and therefore the development and function of all tissues including the male germ line. DNA methylation, an important epigenetic mechanism, has been implicated in the regulation of expression of several testis/sperm-specific genes. Furthermore, abnormal DNA methylation in sperm has been associated with infertility in males. However, little progress has been made in determining a causal relationship and functional significance of methylation of individual CpG dinucleotides and resulting gene activity, limiting the field of epigenetics. In the studies proposed here, we will examine the role that DNA methylation/demethylation plays in regulating the tissue-specific gene expression of a group of functionally related Na+-transporting membrane proteins, the Na,K-ATPase and Na/H exchangers (NHEs), that are important in sperm physiology and male fertility. The Na,K-ATPase establishes the Na+ gradient across the plasma membrane that provides the energy for the NHEs to export H+ from the cell and regulate intracellular pH. One Na,K-ATPase alpha subunit (alpha4) and three NHEs (NHA1/NHEDC1, NHA2/NHEDC2, and NHE10) play important roles in sperm as loss of these transporters results in infertile male mice. The Na,K-ATPase alpha4 isoform and three NHEs (NHA1/NHEDC1, NHE10, and NHE11) are expressed only in the testis/sperm suggesting that these transporters comprise a testis/sperm-specific Na,K-ATPase/NHE functional network to regulate intracellular pH and sperm function. Using bioinformatics, reporter gene assays, and targeted methylation/demethylation, we will explore the role that methylation plays in the expression of the testis/sperm-specific Na,K-ATPase/NHE functional network genes and will identify the specific CpGs in each gene which mediate tissue-specific expression. Both abnormal DNA methylation of imprinted and non-imprinted genes and reduced expression of the Na,K-ATPase alpha4 subunit and NHE10 have been found to be associated with infertile human males. Therefore, beyond providing fundamental information about DNA methylation-dependent gene expression, understanding the DNA methylation-dependent regulation of the expression of the testis/sperm-specific Na,K-ATPase/NHE functional network genes could impact our understanding of human reproductive biology and affect its treatment in the future - identification of the specific elements that regulate expression of these genes, will provide the epigenetic roadmap to allow for the targeted upregulation of these genes in males who are infertile due to loss of their expression.