Transcriptional Networks Regulating Luminal Environment in the Epididymis

描述(申请人提供)：囊性纤维化跨膜电导调节器(CFTR)的功能失活(通过突变)，这是一种小电导、cAMP激活的氯离子通道，会导致发育缺陷，导致人类男性不育。Cftr基因失活导致不育的机制尚不清楚，但人们认为部分原因是男性生殖道系统的畸形或病理变化。这项研究的目的是通过我们目前对cftr基因表达调控机制的理解，破译调控附睾腔环境的转录网络。CFTR的突变导致了毁灭性的遗传性疾病囊性纤维化(CF)。CFTR通道对附睾腔内的离子转运和管腔环境有重要作用，它的缺失会导致男性生殖道的阻塞和/或丢失，进而导致不育。附睾是男性生殖道排泄管系统的近端部分，是分泌和吸收离子、水和无机溶质的复杂器官。重要的是，它为正常的精子成熟提供了正确的管腔环境。在正常附睾中，管腔环境的建立和维持是通过多个离子通道、离子交换器、溶质载体和转运体的功能协作来实现的，但这些基因的表达协调机制尚未得到很好的研究。在第一个特定的目标中，我们将确定CFTR基因座染色质结构的修改如何控制其在附睾中的表达。我们将评估可能建立活性位点环状构象的顺式作用增强剂和绝缘体元件，并确定反式作用因子如CTCF和粘附素如何影响这种空间组织。此外，我们将研究在CFTR活性较低或不活跃的细胞中可能抑制基因表达的机制和DNA结合蛋白。第二个特定目标的实验将确定转录途径，以协调离子通道、离子交换器、转运体和溶质载体的表达，这些离子通道、离子交换器、转运体和溶质载体与附睾上皮中的CFTR相互作用，以建立和维持管腔环境。我们将使用全基因组方法(DNase-seq)来确定基因的顺式作用调控元件，这些基因编码的蛋白质有助于离子和溶质通过附睾上皮的运输。在绘制这些元素的图谱之后，我们将使用生物信息学和实验相结合的方法来确定介导基因表达协调调节的反式作用因子。这些研究的成功进行将为改变男性生育能力提供独特的翻译机会。在疾病状态下，我们可能能够恢复正常的附睾腔环境，从而恢复生育能力。通过调节这种环境，我们也可能抑制正常的精子成熟，从而开发出新的节育方法。 与公共卫生相关：囊性纤维化跨膜电导调节器(CFTR)的功能失活(通过突变)，这是一种小电导，cAMP激活的氯离子通道，导致生殖管系统发育缺陷，导致人类男性不育。这项拟议的研究的目标是通过我们目前对CFTR基因表达调控机制的理解，破译调控生殖管管腔环境的转录网络。这些研究的成功进行将为改变男性生育能力提供独特的翻译机会。在不育症状态下，我们可能能够恢复正常的附睾腔环境，从而恢复生育能力。这种环境的调节可能会提供抑制正常精子成熟的方法，从而开发出新的节育方法。

DESCRIPTION (provided by applicant): Functional inactivation (by mutation) of the cystic fibrosis transmembrane conductance regulator (CFTR), a small conductance, cAMP activated chloride ion channel, leads to developmental defects that cause male infertility in humans. The mechanism by which inactivation of the CFTR gene causes infertility is poorly understood but is believed to be due in part to malformations or pathological changes in the male genital duct system. The goals of the proposed research are to decipher the transcriptional networks that regulate luminal environment in the epididymis, by building on our current understanding of the mechanisms regulating expression of the CFTR gene. Mutations in CFTR cause the devastating inherited disorder cystic fibrosis (CF). The CFTR channel makes an essential contribution to ion transport and the luminal environment in the epididymis and its loss causes obstruction and/or loss of the male genital ducts and subsequent infertility. The epididymis forms the proximal part of the excretory duct system of the male reproductive tract and is a complex organ that secretes and absorbs ions, water and inorganic solutes. Critically, it provides the correct luminal environment for normal sperm maturation. In normal epididymis the luminal environment is established and maintained through the functional cooperation of multiple ion channels, ion exchangers, solute carriers and transporters, though the mechanisms that coordinate the expression of these genes has not been well studied. In the first specific aim we will determine how modifications in the chromatin structure of the CFTR locus control its expression in the epididymis. We will evaluate the cis-acting enhancers and insulator elements that likely establish the looped conformation of the active locus and determine how trans-acting factors such as CTCF and cohesin influence this spatial organization. Moreover, we will investigate mechanisms and DNA- binding proteins that may suppress gene expression in cells where CFTR is less active or inactive. Experiments in the second specific aim will determine the transcriptional pathways that coordinate expression of ion channels, ion exchangers, transporters and solute carriers that interact with CFTR in the epididymis epithelium to establish and maintain the luminal environment. We will use a genome-wide approach (DNase- seq) to identify the cis-acting regulatory elements for genes encoding the proteins that contribute to ion and solute transport across the epididymal epithelium. Following mapping of these elements we will use combined bioinformatic and experimental approaches to identify the trans-acting factors that mediate the coordinated regulation of gene expression. Successful conduct of these studies will provide unique translational opportunities to modify male fertility. In disease states we may be able to reinstate the normal epididymal luminal environment and thereby restore fertility. Through modulation of this environment we may also be able to inhibit normal sperm maturation and thereby develop novel methods of birth control. PUBLIC HEALTH RELEVANCE: Functional inactivation (by mutation) of the cystic fibrosis transmembrane conductance regulator (CFTR), a small conductance, cAMP activated chloride ion channel, leads to developmental defects in the genital duct system that cause male infertility in humans. The goals of the proposed research are to decipher the transcriptional networks that regulate luminal environment in the genital ducts, by building on our current understanding of the mechanisms regulating expression of the CFTR gene. Successful conduct of these studies will provide unique translational opportunities to modify male fertility. In infertile disease states we may be able to reinstate the normal epididymal luminal environment and thereby restore fertility. Modulation of this environment may provide ways to inhibit normal sperm maturation and thereby develop novel methods of birth control.