Transcriptional regulatory networks in spermatogonial stem cells

描述(申请人提供)：精原干细胞(SSCs)是哺乳动物精子发生的基础，在成年生活中维持精子的产生。控制SSCs关键的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，转录因子主要局限于茎和祖精原细胞，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。在特定目标1的实验中，将使用功能遗传学来确定特定的转录因子在体外SSC培养中是否对精子发生至关重要，然后进行移植，以评估干细胞和精子发生的活性。特异性靶点2将通过CHIP-SEQ鉴定SSC转录因子的靶基因，揭示保守和独特的靶点集。特定目的3的实验将通过EMSA和瞬时转染转录分析来确认靶基因的调控机制。这些结果将构成基因调控网络模型的基础，该模型融合了多种因素的复杂相互作用，这些因素对基因调控和SSC潜力有贡献。拟议的研究将通过建立一个新的研究重点，在Kyle Orwig博士的指导下，研究啮齿动物睾丸中SSCs的基本调控，从而增强PI(Brian Hermann)的独立职业轨迹。对于已经在支持细胞基因调控、灵长类精子发生和干细胞方面建立了专长的PI来说，这是一个新的研究方向。马吉妇女研究所和匹兹堡大学在生殖生物学和干细胞研究方面的智力和技术环境非常出色，完成研究所需的所有资源都可用，而且在促进新研究人员发展方面享有很高的声誉。具有突出的独立潜力、创新的导师和优越的环境的PI的这种组合将促进拟议学习的各个方面，并推动PI的职业生涯走向独立。PI的长期职业目标是在终身教职的基础上建立一个独立的研究项目，对SSCs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的新方法。职业发展奖解决了促进向独立过渡的三个直接目标：1)发展独立研究利基，2)展示杰出的生产力，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，与小鼠模型中的SSCs培养和移植有关的技术培训，生物信息学数据分析的指导，与成功开展独立研究计划相关的领域的非正式和结构化指导，以及外部顾问委员会对职业发展进展的评估。最终，这些研究可能会为启动和维持精子发生的机制提供见解，这对治疗男性不育具有启示意义。研究SSCs的生物学特性也可能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进一步培养将促进具有竞争力的、独立的研究职业生涯，从而为生物医学科学做出实质性贡献。 公共卫生相关性：精原干细胞(SSCs)负责成人一生中精子的产生，对男性生育能力至关重要。指导这些细胞表现为干细胞并维持精子产生的方法尚不清楚。SSCs表达的基因和打开和关闭这些基因的蛋白质的独特组合可能控制SSC的行为。这一应用将提供关于基因调控在正常SSC行为中的作用的关键见解，这可能揭示男性不育的潜在原因。

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.