Control of spermatogonial stem cell formation

摘要/项目摘要 拟议工作的总体目标是了解DMRT1基因是如何控制 哺乳动物睾丸内精原干细胞的形成。睾丸有两个基本功能：生产 精子，作为雄性生殖系DNA永生的载体的细胞；以及产生 引导身体其他部位以男性特有的方式发育的激素。这些过程的失败 导致不孕、生殖细胞癌和性发育障碍(DSD)。DMRT1属于一个家族 保守的转录调节因子，并控制哺乳动物睾丸中的多个关键过程，两者都在 生殖细胞和体细胞。最近的一项发现是DMRT1是形成精原干细胞所必需的 (SSCS)。这一提议的中心假设是DMRT1作为一个先驱转录因子打开 染色质，并允许其他相互合作的转录因子结合和调节基因表达，从而 编排SSC队形。这项提议有三个目标，重点是更深入地理解 DMRT1控制着SSC细胞的命运。目标1询问DMRT1如何指导SSC的形成。它检查了DMRT1的损失是如何 影响SSC形成中的关键事件，包括SSC调节因子的增殖和表达。然后，它会寻找一个 从机制上理解DMRT1如何调节靶基因转录以控制细胞命运，测试 假设DMRT1是一个先驱转录因子。这些实验将使用一系列最新的 ART基因组工具，包括CHIP-SEQ和ATAC-SEQ，以找到关键的调控目标并了解DMRT1如何 SSCs中的结合影响增强子的活性。Motif搜索和芯片将用于确定可能的合作 转录因子。目标2将确定在SSC过程中受DMRT1直接和间接调控的基因 队形。受调控的转录本将通过标准和单细胞RNA-seq和HiChIP进行鉴定 用于将调控区域彼此联系起来，并与它们控制的转录起始点联系起来。AIM 3将测试 选择DMRT1协同转录因子和靶基因的功能重要性，使用 慢病毒在培养的SSCs中被击倒，随后对最佳候选者进行体内验证。拟议中的工作 与人类健康直接相关：人类的DMRT1与不育症、睾丸生殖细胞癌和 DSD包括男性到女性的性逆转。因此，拟议的工作将有助于揭示 这是干细胞形成的基础，并可能提供对干细胞生物学的一般见解。

Abstract / Project Summary The overall objective of the proposed work is to understand how the Dmrt1 gene controls spermatogonial stem cell formation in the mammalian testis. The testis has two essential functions: production of sperm, the cells that serve as vehicles for the immortality of male germ line DNA; and production of hormones that direct other parts of the body to develop in a male-specific manner. Failures of these processes cause infertility, germ cell cancer, and disorders of sex development (DSD). Dmrt1 belongs to a family of conserved transcriptional regulators and it controls multiple crucial processes in the mammalian testis, both in germ cells and somatic cells. A recent discovery is that DMRT1 is required to form spermatogonial stem cells (SSCs). The central hypothesis of this proposal is that DMRT1 acts as a pioneer transcription factor to open chromatin and allow other cooperating transcription factors to bind and regulate gene expression, thereby orchestrating SSC formation. This proposal has three aims focused on a deeper understanding of how DMRT1 controls SSC cell fate. Aim 1 asks how DMRT1 directs SSC formation. It examines how loss of Dmrt1 affects key events in SSC formation including proliferation and expression of SSC regulators. It then seeks a mechanistic understanding of how DMRT1 regulates target gene transcription to control cell fate, testing the hypothesis that DMRT1 is a pioneer transcription factor. The experiments will employ an array of state-of-the- art genomic tools including ChIP-seq and ATAC-seq to find key regulatory targets and learn how DMRT1 binding in SSCs affects enhancer activity. Motif searches and ChIP will be used to identify likely cooperating transcription factors. Aim 2 will identify the genes regulated directly and indirectly by DMRT1 during SSC formation. Regulated transcripts will be identified by standard and single-cell RNA-seq and HiChIP will be used to link regulatory regions to one another and to the transcriptional start sites they control. Aim 3 will test the functional importance of selected DMRT1 cooperating transcription factors and target genes, using lentiviral knockdown in cultured SSCs followed by in vivo validation for top candidates. The proposed work has direct human health relevance: DMRT1 in humans is linked to infertility, testicular germ cell cancer and DSD including male-to-female sex reversal. As a result, the proposed work will help uncover the mechanistic basis of SSC formation and may provide general insights into stem cell biology.