Transcriptional regulatory networks in spermatogonial stem cells

描述（由申请人提供）：精原干细胞（ssc）是哺乳动物精子发生的基础，在整个成年期维持精子的产生。控制ssc自我更新与分化命运决定的分子机制在很大程度上是未知的。该假说认为，主要局限于干细胞和祖精子原细胞的转录因子形成调控网络，执行对SSC命运决定和精子发生重要的基因表达程序。《三个具体目标》将检验这一假设。特异性目标1的实验将采用功能遗传学来确定特定的转录因子是否对精子发生至关重要，在体外SSC培养中使用敲低策略，然后移植来评估干细胞和生精活性。Specific Aim 2将通过ChIP-Seq鉴定SSC转录因子的靶基因库，并揭示保守的和独特的靶标集。Specific Aim 3的实验将通过EMSA和瞬时转染转录分析来确定靶基因的调控机制。这些结果将构成基因调控网络模型的基础，该模型包含多种因素对基因调控和SSC潜力的复杂相互作用。在Kyle Orwig博士的指导下，这些研究将通过建立一个新的研究重点来研究啮齿动物睾丸中ssc的基本调控，从而增强PI （Brian Hermann）的独立职业轨迹。这是PI在支持细胞基因调控和灵长类动物精子发生和干细胞方面建立的专业知识的一个新的研究方向。在magee妇女研究所和匹兹堡大学，生殖生物学和干细胞研究的知识和技术环境是杰出的，完成研究所需的所有资源都是可用的，并且在促进新研究人员的发展方面有着良好的声誉。具有杰出独立潜力的PI，具有创新精神的导师和优越的环境将促进PI在独立职业生涯中的各个方面的研究和进步。PI的长期职业目标是建立一个独立的终身教职研究项目，对ssc和精子发生的理解做出重大的学术贡献，并开发治疗男性不育症的新方法。职业发展奖项旨在实现三项直接目标：1)发展独立的研究领域，2)展示卓越的生产力，3)获得额外的独立资助。职业发展计划包括功能遗传学方面的概念指导，与小鼠模型中ssc培养和移植相关的技术培训，生物信息学数据分析方面的指导，成功运行独立研究项目相关领域的非正式和结构化指导，以及由外部咨询委员会评估职业发展进展。最终，这些研究可能提供关于启动和维持精子发生的机制的见解，这对治疗男性不育症具有重要意义。研究ssc的生物学特性也可以扩大对干细胞行为的理解，并有助于各种成人组织的正常功能。此外，对候选人职业发展的额外培养将促进有竞争力的、独立的研究事业，为生物医学科学做出实质性贡献。

DESCRIPTION (provided by applicant): Spermatogonial stem cells (SSCs) are at the foundation of mammalian spermatogenesis, maintaining sperm production throughout adult life. The molecular mechanisms that control the critical self-renewal vs. differentiation fate decision of SSCs are largely unknown. The hypothesis is that transcription factors primarily restricted to stem and progenitor spermatogonia form regulatory networks to execute gene expression programs important for SSC fate decisions and spermatogenesis. Three Specific Aims will test this hypothesis. Experiments in Specific Aim 1 will employ functional genetics to determine if specific transcription factors are essential for spermatogenesis using a knockdown strategy in ex vivo SSC cultures followed by transplantation to assess stem cell and spermatogenic activity. Specific Aim 2 will identify the target gene repertoire of SSC transcription factors by ChIP-Seq and reveal conserved and unique targets sets. Experiments in Specific Aim 3 will confirm mechanisms of target gene regulation by EMSA and transient transfection transcriptional analysis. These results will form the basis of gene regulatory network models incorporating complex interactions from multiple factors contributing to gene regulation and SSC potential. The proposed studies will be enhance the independent career trajectory of the PI (Brian Hermann) by establishing a new research focus investigating the fundamental regulation of SSCs in the rodent testis under the mentorship of Dr. Kyle Orwig. This is a new research direction for the PI who has established expertise in Sertoli cell gene regulation and primate spermatogenesis and stem cells. The intellectual and technical environment for research in reproductive biology and stem cells at the Magee-Womens Research Institute and University of Pittsburgh is outstanding, all resources required to complete the studies are available, and there is a strong reputation for promoting development of new investigators. This combination of a PI with outstanding potential for independence, an innovative mentor, and superior environment will facilitate all aspects of the proposed studies and advancement of the PI's career towards independence. The long-term career goals of the PI are to establish an independent research program in a tenure-track faculty position, make significant scholarly contributions to the understanding of SSCs and spermatogenesis, and develop new approaches for treating male infertility. Three immediate objectives to promote the transition to independence are addressed by the career development award: 1) develop an independent research niche, 2) demonstrate outstanding productivity, and 3) secure additional independent funding. The career development plan involves conceptual mentoring in functional genetics, technical training relating to culture and transplantation of SSCs in the mouse model, instruction in bioinformatics data analysis, informal and structured mentoring in areas relevant to running a successful independent research program, and evaluation of career development progress by an external advisory board. Ultimately, these studies may provide insights about the mechanisms that initiate and maintain spermatogenesis, which has implications for treating male infertility. Investigating the biological properties of SSCs may also expand the understanding of how stem cells behave and contribute to the normal function of a variety of adult tissues. Moreover, additional nurturing of the candidate's career development will promote a competitive, independent research career to contribute substantively to the biomedical sciences. PUBLIC HEALTH RELEVANCE: Spermatogonial stem cells (SSCs) are responsible for sperm production throughout adult life and are essential for male fertility. The methods which instruct these cells to behave like stem cells and maintain sperm production are unknown. The unique combination of genes expressed by SSCs and the proteins that turn these genes on and off may control of SSC behavior. This application will provide key insights about the role of gene regulation in normal SSC behavior, which may reveal underlying causes of male infertility.