Transcriptional regulatory networks in spermatogonial stem cells

项目总结/摘要 精原干细胞（spermatogonialstemcells，SSCs）是哺乳动物精子发生的基础，维持着精子的增殖和分化， 在整个成年生活中的精子生产。控制关键自我更新的分子机制与 SSC的分化命运决定在很大程度上是未知的。假设转录因子主要 仅限于干细胞和祖细胞精原细胞形成调控网络执行基因表达 对SSC命运决定和精子发生重要的程序。三个具体目标将检验这一假设。 具体目标1的实验将采用功能遗传学来确定是否有特定的转录因子 在体外SSC培养中使用敲低策略， 移植以评估干细胞和生精活性。特异性目标2将识别靶基因 通过ChIP-Seq分析SSC转录因子库，并揭示保守和独特的靶点集。 具体目标3中的实验将通过EMSA和瞬时表达证实靶基因调控的机制。 转染转录分析。这些结果将构成基因调控网络模型的基础 整合了来自多种因素的复杂相互作用，这些因素有助于基因调控和SSC潜力。 拟议的研究将通过以下方式增强PI（Brian Hermann）的独立职业轨迹 建立了一个新的研究重点，研究啮齿动物睾丸中精原干细胞的基本调控， 凯尔·奥维格博士的指导这是PI的一个新的研究方向， 支持细胞基因调控与灵长类精子发生和干细胞。知识和技术 马吉妇女研究所的生殖生物学和干细胞研究环境， 匹兹堡大学是杰出的，完成研究所需的所有资源都可用， 在促进新研究人员的发展方面享有很高的声誉。这种PI与 具有独立性的杰出潜力、创新的导师和上级环境将有助于所有人 建议的研究和促进PI的职业生涯走向独立的各个方面。 PI的长期职业目标是在终身制轨道上建立一个独立的研究计划 教师职位，对理解精原干细胞和精子发生做出重大学术贡献， 并开发治疗男性不育症的新方法。促进过渡的三个近期目标 职业发展奖的目标是：1）发展独立的研究领域， 2)表现出出色的生产力，3）获得额外的独立资金。职业 发展计划包括功能遗传学的概念指导，与文化有关的技术培训， 和小鼠模型中的SSC移植，生物信息学数据分析指导，非正式和 在与运行成功的独立研究项目相关的领域进行结构化指导，并进行评估 职业发展的进展，由外部咨询委员会。 最终，这些研究可能会提供有关启动和维持这些机制的见解。 精子发生，这对治疗男性不育症有意义。研究的生物特性 SSCs还可以扩展对干细胞如何行为的理解，并有助于细胞的正常功能。 各种成人组织。此外，对候选人职业发展的额外培养将促进 具有竞争力的，独立的研究生涯，为生物医学科学做出实质性贡献。

Project Summary/Abstract 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 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.