Expression, regulation, and role of enhancer RNAs in T helper cells

描述（由申请人提供）：项目摘要/摘要本K22提案详细描述了我的计划，以获得必要的生物和教育技能，并将其应用于一个重要的健康相关主题，以发展我的独立研究实验室的基础。我在本科和研究生学习期间获得了广泛的计算训练。我已经使用这些计算技能来改善蛋白质二级结构预测，预测增强子的位置，增强子功能模型，并揭示调控语法。到目前为止，我为期一年的博士后培训拓宽了我对基因调控的看法，特别是在T细胞发育期间。我研究了非编码RNA及其在T辅助细胞可塑性和分化中的作用。使用计算技术，我已经确定了T辅助细胞中的几个谱系特异性非编码RNA。我已经开始学习如何进行某些生物实验来验证我的发现，以及如何遵循某些文库制备方案。在这个奖项的指导阶段，我打算扩大我的生物技能。为此，我制定了自己的职业发展计划，包括几个实验室培训研讨会。我要求我的生物学家实验室伙伴让我观察他们做实验，并在某些实验中指导和监督我。我已经要求我现在的导师为我提供适当的材料和设备来进行实验。在这个奖项的指导阶段，我将掌握的预期技能包括实时PCR，流式细胞术，细胞培养和转染，以及高通量测序文库制备。获得这些动手生物技能将使我能够建立我理想的干湿混合实验室，并有效地指导来自计算和生物科学的研究生和博士后学生。我的研究计划将解决几个重要的生物学问题，关于一类新的非编码RNA称为增强子RNA。它将量化这些非编码RNA（Aim 1）并表征它们在T辅助细胞调节（Aim 2）中的作用。T辅助细胞的调节与我们对抗病原体的能力有关，异常可能导致严重的免疫疾病。因此，表征新的调节剂是重要的，并可能开辟新的治疗途径。为了确保成功完成这一提案，我召集了一个专家和合作者委员会，他们将在这一提案的各个方面帮助我。T细胞发育方面的专家伦纳德博士将为我提供必要的生物学发现方面的建议。作为我的主要导师，他还将为我提供该奖项指导阶段所需的空间，设备和材料。Jun Zhu博士是一位测序技术专家，他将帮助我进行方法开发。斯塔布斯博士是转录调控机制方面的专家，他将就增强子RNA的作用向我提供建议。NHLBI的教育主任盖勒博士将为我提供求职、面试和过渡到独立阶段的建议。Spolski博士和Liao博士将帮助我学习如何进行实验。

DESCRIPTION (provided by applicant): Project Summary/Abstract This K22 proposal describes in detail my plan to obtain the necessary biological and educational skills and apply them in a significant health related subject to develop the foundation of my independent research laboratory. I have obtained an extensive computational training during my undergraduate and graduate studies. I have used these computational skills to improve protein secondary structure prediction, to predict enhancer locations, to model enhancer function, and to uncover regulatory grammar. My one-year postdoctoral training so far, has broadened my view of gene regulation, specifically during T cell development. I have studied noncoding RNAs and their role in T helper cell plasticity and differentiation. Using computational techniques, I have identified several lineage-specific noncoding RNAs in T helper cells. I have started to learn how to perform certain biological experiments to experimentally validate my findings and how to follow certain library preparation protocols. During the mentored phase of this award, I intend to expand my biological skills. To this end, I have tailored my career development plan to include several workshops with laboratory trainings. I have asked my biologist lab-mates to let me observe them performing experiments and to teach and supervise me during certain experiments. I have asked my current advisor to provide me with proper material and equipment to perform experiments. The intended skills that I will master during the mentored phase of this award include real-time PCR, flow cytometry, cell culture and transfection, and high-throughput sequencing library preparation. Obtaining these hands-on biological skills will enable me to setup my ideal mixed dry and wet lab and to effectively mentor graduate and postdoctoral students from both computational and biological sciences. My research plan will address several important biological questions regarding a novel class of noncoding RNAs referred to as enhancer RNAs. It will quantify these noncoding RNAs (Aim1) and characterize their role in T helper cell regulation (Aim2). The regulation of T helper cells is associated with our ability to fight against pathogens, and abnormalities may lead to severe immune diseases. Thus, characterizing novel regulators is important and may open new therapeutic avenues. To ensure the successful completion of this proposal, I have assembled a committee of experts and collaborators that will help me on various aspects of this proposal. Dr. Leonard, an expert in T cell development, will provide me the necessary advice on biological findings. As my primary mentor, he will also provide me the space, equipment, and material needed for the mentored phase of this award. Dr. Jun Zhu, an expert in sequencing technologies, will help me on method development. Dr. Stubbs, an expert in mechanisms of transcriptional regulation, will advice me on the role of enhancer RNAs. Dr. Geller, the director of education at NHLBI, will provide me advice on job search, interview, and transition to the independent phase. Dr. Spolski and Dr. Liao will help me to learn how to perform experiments.