Control of spermatogonial stem cell formation

精原干细胞形成的控制

基本信息

批准号：
10323256
负责人：
David A. Zarkower
金额：
$ 30.8万
依托单位：
UNIVERSITY OF MINNESOTA
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-01-01 至 2023-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10323256
关键词：
ATAC-seq Address Affect Binding Birth Body part Cell Fate Control Cells ChIP-seq Chromatin DNA DNA Binding Data Development Diagnosis Distal Enhancers Event Failure Family Female Fertility Gene Expression Genes Genetic Genetic Transcription Germ Cell Cancers Germ Cells Germ Lines Goals Gonadal structure Health Histones Hormones Human Infertility Knowledge Learning Link Maintenance Male Infertility Malignant neoplasm of testis Mammals Maps Mediating Mus Neonatal Nucleic Acid Regulatory Sequences Perinatal Phase Phenotype Process Production Protein Family Proteins Reagent Regulation Regulatory Element Research Role Sexual Reproduction Signal Transduction Site Small Interfering RNA Somatic Cell Specific qualifier value Spermatocytes Spermatogenesis Supporting Cell Testicular malignant germ cell tumor Testing Testis Transcript Transcription Initiation Site Validation Work ZNF145 gene cell behavior cell type experimental study fetal genetic analysis genomic tools in vivo in vivo evaluation insight knock-down male male fertility mutant neonatal mice postnatal response sertoli cell sex sex development disorder single-cell RNA sequencing sperm cell stem cell biology stem cell biomarkers stem cell fate stem cells tool transcription factor transcriptome sequencing

项目摘要

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.

摘要 /项目摘要拟议工作的总体目标是了解DMRT1基因如何控制哺乳动物睾丸中的精子干细胞形成。睾丸具有两个基本功能：生产精子，用作雄性生殖系DNA永生的车辆的细胞；和生产指导身体其他部位以男性特异性发展的激素。这些过程的失败导致性发展的不育症，生殖细胞癌和性疾病（DSD）。 DMRT1属于保守的转录调节剂和它控制哺乳动物睾丸中的多个关键过程生殖细胞和体细胞。最近的发现是需要DMRT1形成精子干细胞（SSC）。该提议的中心假设是DMRT1充当开放的先驱转录因子染色质并允许其他合作的转录因子结合和调节基因表达，从而编排SSC编队。该提议的三个目标集中在更深入地了解如何 DMRT1控制SSC细胞命运。 AIM 1询问DMRT1如何指导SSC组。它检查了DMRT1的损失影响SSC形成中的关键事件，包括SSC调节剂的增殖和表达。然后寻求一个对DMRT1如何调节靶基因转录以控制细胞命运的机械理解，测试 DMRT1是先锋转录因子的假设。实验将采用一系列最新 ART基因组工具在内 SSC中的结合会影响增强剂活性。主题搜索和芯片将用于识别可能的合作转录因子。 AIM 2将在SSC期间直接和间接地识别由DMRT1间接调节的基因形成。规范的成绩单将通过标准和单细胞RNA-seq和Hichip确定用于将监管区域与他们控制的转录启动站点联系起来。 AIM 3将测试选定的DMRT1合作转录因子和靶基因的功能重要性，使用培养的SSC中的慢病毒敲低，然后是对顶级候选者的体内验证。拟议的工作具有直接人类健康相关性：人类中的DMRT1与不育，睾丸生殖细胞癌和 DSD，包括男女性逆转。结果，拟议的工作将有助于发现机械 SSC形成的基础，可以提供对干细胞生物学的一般见解。