Gonadal expression of FSH receptor

描述(申请人提供)：FSHR是内分泌轴的重要组成部分，控制性腺功能、配子发生和生育。FSHR转录调控机制的研究为揭示Sertoli和颗粒细胞中参与选择性基因表达的蛋白质提供了机会，并扩大了我们对调节生殖健康因素的理解。到目前为止，FSHR的转录研究仅集中在启动子和5‘侧翼序列上。虽然这些分析对我们理解FSHR至关重要，但似乎该区域以外的调控序列对FSHR的转录调控是重要的。幸运的是，由于基因组研究的广泛进步，我们识别这种调控序列的能力已经显著提高，这提供了丰富的新信息和资源。拟议的研究结合了计算分析、转录分析和转基因方法，以进一步了解控制FSHR转录的过程。目的一是利用小鼠基因敲除模型和染色质免疫沉淀法研究转录因子在体内对FSHR的调控作用，并进一步了解近端启动子调控转录的机制。在AIM II中，将使用比较基因组学、DNase I超敏作图和瞬时转基因分析来确定FSHR基因座内的远端调控区。DNA/蛋白质结合研究将用于识别与重要调控元件相关的蛋白质。目的III建议利用转基因小鼠对FSHR调节元件进行体内鉴定。含有FSHR的酵母人工染色体(YAC)将被用来产生转基因小鼠，这些小鼠将被鉴定为在各种组织中表达。建议的调控元件内的突变将通过同源重组在YAC中产生，并在转基因小鼠中进行正确表达的测试，以评估它们在体内的作用。这项拟议的研究将揭示FSHR表达所需的调节元件和蛋白质，不仅有助于更好地了解其转录调控，而且还将揭示性腺功能、生育和广义基因转录的基本原理。通过增进对这些领域的了解，我们提高了评价人类生殖健康问题和制定协助生育问题和管理的战略的能力。

DESCRIPTION (provided by applicant): Fshr is a critical component of the endocrine axis that controls gonad function, gametogenesis, and fertility. Characterization of the mechanisms regulating Fshr transcription provides the opportunity to reveal proteins involved in selective gene expression in Sertoli and granulosa cells and expand our understanding of factors regulating reproductive health. To date, transcriptional studies of Fshr have focused only on promoter and 5' flanking sequences. While these analyses are critical to our understanding of Fshr, it appears that regulatory sequences outside this region are important for Fshr transcriptional regulation. Fortunately, our ability to identify such regulatory sequences has advanced significantly due to the extensive progress in genome research, which provides a wealth of new information and resources. The proposed studies combine computation analysis, transcriptional assays, and transgenic approaches to further our understanding of the processes controlling Fshr transcription. In Aim I, studies are proposed to use mouse knockout models and chromatin immunoprecipitation to evaluate the in vivo roles of transcription factors implicated in Fshr regulation and expand our understanding of the mechanisms regulating transcription through the proximal promoter. In Aim II, comparative genomics, DNase I hypersensitivity mapping and transient transfection analysis will be used to identify distal regulatory regions within the Fshr locus. DNA/protein binding studies will be used to identify proteins associated with important regulatory elements. Aim III proposes to use transgenic mice for in vivo characterization of Fshr regulatory elements. An Fshr-containing yeast artificial chromosome (YAC) will be used to generate transgenic mice that will be characterized for expression in various tissues. Mutations within proposed regulatory elements will be generated in the YAC by homologous recombination and tested for correct expression in transgenic mice, to evaluate their role in vivo. The proposed studies will reveal regulatory elements and proteins needed for Fshr expression, leading not only to a better understanding of its transcriptional control but to the basic principles governing gonadal function, fertility, and generalized gene transcription. By advancing our understanding in these areas, we enhance our ability to evaluate matters of human reproductive health and to develop strategies to assist fertility problems and management.