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

确定 DNA 甲基化在 Na,K-ATPase-Na/H 交换 pH 调节系统基因的组织特异性表达中的作用。

• 批准号: 10202996
• 负责人: PAUL F JAMES
• 金额: $ 42.33万
• 依托单位: MIAMI UNIVERSITY OXFORD
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-06-01 至 2024-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10202996
• 关键词: Address; Affect; Automobile Driving; Bioinformatics; Biological Assay; CRISPR/Cas technology; Cell Line; Cell membrane; Cells; CpG Islands; CpG dinucleotide; Cultured Cells; DNA; DNA Methylation; DNA sequencing; Development; Disease; Elements; Epigenetic Process; Family; Future; Gene Expression; Gene Expression Regulation; Genes; Genomics; Germ Cells; Germ Lines; Human; In Situ; Individual; Infertility; Ions; Knock-in; Lead; Male Infertility; Mammalian Cell; Mediating; Membrane Transport Proteins; Methylation; Modification; Mus; Na(+)-K(+)-Exchanging ATPase; Nucleic Acid Regulatory Sequences; Physiology; Play; Protein Isoforms; Proteins; Regulation; Regulator Genes; Regulatory Element; Reporter Genes; Repression; Reproductive Biology; Role; Site-Directed Mutagenesis; Somatic Cell; Sperm Motility; Spermatocytes; Spermatogenic Cell; System; Testis; Tissue-Specific Gene Expression; Tissues; Up-Regulation; bisulfite; cell motility; cell type; data mining; demethylation; functional group; gene repression; human male; imprint; in silico; interest; male; male fertility; promoter; sperm cell; sperm function

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.

抽象的： 表观遗传机制对于调节组织特异性基因表达和 因此，包括男性生殖系在内的所有组织的发育和功能。脱氧核糖核酸 甲基化是一种重要的表观遗传机制，与调节有关 几种睾丸/精子特异性基因的表达。此外，异常的DNA甲基化 精子与男性不孕有关。但是，几乎没有进展 确定各个CpG的因果关系和功能意义 二核苷酸和产生的基因活性，限制了表观遗传学的领域。在提出的研究中 在这里，我们将研究DNA甲基化/去甲基化在调节的作用 一组功能相关的Na+转移膜的组织特异性基因表达 蛋白质，Na，K-ATPase和Na/H交换器（NHES），在精子中很重要 生理学和男性生育能力。 Na，K-ATPase建立了跨等离子体的Na+梯度 膜为NHE提供了从细胞导出H+并调节的能量 细胞内pH。一个Na，K-ATPase Alpha亚基（alpha4）和三个NHES（NHA1/NHEDC1， NHA2/NHEDC2和NHE10）在精子中起重要作用，因为这些转运蛋白的损失 在不育雄性小鼠中。 Na，K-ATPase alpha4同工型和三个NHE（NHA1/NHEDC1， NHE10和NHE11）仅在睾丸/精子中表明这些转运蛋白 包括睾丸/精子特异性Na，K-ATPase/NHE功能网络来调节细胞内 pH和精子功能。使用生物信息学，报告基因测定和目标 甲基化/脱甲基化，我们将探讨甲基化在表达中起作用的作用 睾丸/精子特异性Na，K-ATPase/NHE功能网络基因，将确定 每个基因中介导组织特异性表达的特定CpG。两个异常的DNA 印迹和非刻印基因的甲基化以及Na，K-ATPase的表达降低 已经发现α4亚基和NHE10与不育的人类男性有关。 因此，除了提供有关DNA甲基化依赖性基因的基本信息外 表达，了解DNA甲基化依赖性调节的表达 睾丸/精子特异性Na，K-ATPase/NHE功能网络基因可能会影响我们的 了解人类生殖生物学并影响其将来的治疗 - 识别 调节这些基因表达的特定元素将提供表观遗传学 路线图允许在因不育而靶向上调这些基因的靶向上调 失去表达。