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

摘要： 表观遗传机制对于组织特异性基因表达的调节至关重要， 因此，包括雄性生殖细胞在内的所有组织的发育和功能。DNA 甲基化是一种重要的表观遗传学机制， 几种睾丸/精子特异性基因的表达。此外，异常的DNA甲基化， 精子与男性不育有关。然而，在以下方面进展甚微： 确定个体CpG甲基化的因果关系和功能意义 二核苷酸和由此产生的基因活性，限制了表观遗传学领域。在拟议的研究中， 在这里，我们将研究DNA甲基化/去甲基化在调节细胞凋亡中的作用。 一组功能相关Na+转运膜组织特异性基因表达 蛋白质，Na，K-ATP酶和Na/H交换剂（NHEs），在精子中很重要 生理学和男性生育力。Na，K-ATP酶在血浆中建立Na+梯度 膜，为NHE提供能量以从细胞中输出H+并调节 一个Na，K-ATP酶α亚基（α 4）和三个NHE（NHA 1/NHEDC 1， NHA 2/NHEDC 2和NHE 10）在精子中起重要作用，因为这些转运蛋白的丢失导致 在不育的雄性小鼠中。Na，K-ATP酶α 4同种型和三种NHE（NHA 1/NHEDC 1， NHE 10和NHE 11）仅在睾丸/精子中表达，表明这些转运蛋白 包括睾丸/精子特异性Na，K-ATP酶/NHE功能网络，以调节细胞内 pH值和精子功能利用生物信息学、报告基因分析和靶向 甲基化/去甲基化，我们将探讨甲基化在表达中的作用， 睾丸/精子特异性Na，K-ATP酶/NHE功能网络基因，并将确定 在每个基因中介导组织特异性表达的特异性CpG。两个异常的DNA 印迹和非印迹基因的甲基化以及Na，K-ATP酶表达的降低 已经发现α 4亚基和NHE 10与人类男性不育有关。 因此，除了提供有关DNA甲基化依赖基因的基本信息外， 表达，理解DNA甲基化依赖的表达调控， 睾丸/精子特异性Na，K-ATPase/NHE功能网络基因可能影响我们的研究。 了解人类生殖生物学并影响其未来的治疗-鉴定 调节这些基因表达的特定元件，将提供表观遗传 路线图，以使这些基因在男性谁是不育，由于有针对性的上调 失去了表达。

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.