Control of spermatogonial stem cell formation

摘要/项目摘要 这项工作的总体目标是了解Dmrt 1基因如何控制 哺乳动物睾丸中精原干细胞的形成。睾丸有两个基本功能：生产 精子，作为男性生殖系DNA永生的载体的细胞; 指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 导致不育、生殖细胞癌和性发育障碍（DSD）。Dmrt 1属于一个家族， 保守的转录调节因子，它控制着哺乳动物睾丸中的多个关键过程， 生殖细胞和体细胞。最近发现DMRT 1是形成精原干细胞所必需的 （SSC）。该建议的中心假设是DMRT 1作为先驱转录因子打开了 染色质，并允许其他协作转录因子结合和调节基因表达，从而 协调SSC的形成。该提案有三个目标，重点是更深入地了解如何 DMRT 1控制SSC细胞命运。目的1询问DMRT 1如何指导SSC形成。它研究了Dmrt 1的丢失如何 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，它寻求一个 了解DMRT 1如何调节靶基因转录以控制细胞命运的机制， DMRT 1是一个先驱转录因子的假说。实验将采用一系列的国家的- 最先进的基因组工具，包括ChIP-seq和ATAC-seq，以找到关键的调控靶点，并了解DMRT 1 SSC中的结合影响增强子活性。Motif搜索和ChIP将用于识别可能的合作 转录因子目的二是鉴定在SSC中DMRT 1直接或间接调控的基因 阵将通过标准和单细胞RNA-seq和HiChIP鉴定受调控的转录物。 用于将调控区彼此连接并连接到它们控制的转录起始位点。目标3将测试 选择DMRT 1合作转录因子和靶基因的功能重要性，使用 在培养的SSC中进行慢病毒敲减，然后对最佳候选物进行体内验证。拟议工作 与人类健康有直接关系：DMRT 1与人类不育、睾丸生殖细胞癌和 DSD包括男性到女性的性别逆转。因此，所提出的工作将有助于揭示 SSC形成的基础，并可能提供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.