Delineation of a cytokine gene regulatory network and rewiring in disease

 描述（由申请人提供）：我的长期马驹是成为一个独立的研究人员在一个主要的美国研究机构，在免疫学和系统生物学之间的接口开发尖端研究。为了实现这一目标，我的近期目标是：（1）进行创新，高质量的研究，这将导致高影响力的出版物，独立于我的导师的研究路线，（2）获得一套新的技能，在生物信息学，复杂的数据分析，免疫学和基因组编辑方法，以补充我现有的背景。获得著名的NIH独立途径奖将加强我的职业机会，并为我提供发展和成熟自己的研究计划的手段，再加上获得作为独立研究人员成功所需的额外培训。拟议的研究包括我的主要科学兴趣：整合免疫调节与系统生物学方法来研究基因表达。细胞因子是细胞与细胞之间的信号蛋白，在描述免疫反应中发挥着基本作用。细胞因子的产生在转录水平上受到高度调节，并且不能适当地表达它们通常会导致几种病理，例如自身免疫性疾病、癌症和对感染的易感性。经过三十多年的研究，细胞因子表达的许多方面仍然没有得到很好的理解，细胞因子基因表达的转录调控尚未在系统水平上探索。本研究将通过全面鉴定调控细胞因子基因的转录因子，构建第一个以基因为中心的细胞因子基因调控网络。为此，我将首先通过使用Walhout实验室最近开发的高通量酵母单杂交（eY 1H）测定法直接测试蛋白质-DNA相互作用（PDI）来确定可以与所有人类细胞因子的启动子和其他已知调控区域结合的Ts。我将把检测到的PDIs建模为GRN，并将该GRN与公开可用的表达数据整合，以定义哪些TF正或负调节细胞因子表达。这些分析与敲除细胞系中的功能验证试验一起将鉴定将它们与疾病中的功能作用和相关性联系起来的细胞因子调节剂。非编码突变是许多人类遗传疾病的核心，占全基因组关联研究中确定的所有突变的90%。例如，细胞因子调节区的突变与癌症、对感染的更高易感性、慢性关节炎和系统性红斑狼疮有关。这些突变可导致转录因子结合位点的丧失或获得。测试PDI变化的测定是劳动密集型的，并且限于几十个TF。因此，我建议使用一种新的eY 1H管道系统地询问PDI的变化，导致疾病相关的细胞因子调控区的突变的结果。这种实验方法的发现将在免疫细胞系中得到进一步验证，并将为许多免疫相关疾病提供机制见解和治疗靶点。我提出的跨学科计划要求我在多个研究领域获得额外的培训，这将补充我的背景，并为我未来的研究提供最先进的工具。我已确定瓦胡特博士和菲茨杰拉德博士为理想的导师，与沃尔夫博士和迈尔斯博士合作，开展我提出的工作，具有高度互补的专业知识。与这些世界知名的研究人员在一个刺激的环境中合作，如UMMS，以及我的指导委员会和我为该奖项的指导阶段计划的培训活动，将使我能够（1）更加精通最先进的免疫学技术并学习最新的免疫学概念，（2）获得生物信息学和复杂的数据分析技能，以及（3）学习基因组编辑方法，所有这些都是解释和验证拟议工作所必需的。这一科学专业知识将与职业发展研讨会和活动相补充，以获得指导，领导力，granische和写作技能，我将需要成功地作为一个独立的调查员。获得这个奖项将使我处于一个理想的位置，利用我在瓦尔豪特实验室迄今为止的经验，同时允许我定义自己的研究利基。该奖项将使人们更好地了解免疫基因在正常和病理条件下是如何调节的，这将对公共卫生产生广泛的影响。

 DESCRIPTION (provided by applicant): My long term foal is to become an independent investigator at a major American research institution, developing cutting-edge research at the interface between immunology and systems biology. To achieve this goal my immediate objectives are: (1) to produce innovative, high quality research that will result in high impact publications that are independent of my mentor's lines of research, (2) to acquire a new set of skills in bioinformatics, complex data analyses, immunology, and genome editing approaches to complement my existing background. Obtaining the prestigious NIH pathway to independence award would bolster my career opportunities and provide me with the means to develop and mature my own research program, plus acquire the additional training I need to succeed as an independent researcher. The proposed research encompasses my major scientific interests: integrating immune regulation with systems biology approaches to study gene expression. Cytokines are cell-to-cell signaling proteins that play fundamental roles in delineating immune responses. Cytokine production is highly regulated at the transcriptional level, and failure to express them appropriately often results in several pathologies such as autoimmune diseases, cancer and susceptibility to infections. After more than three decades of research, many aspects of cytokine expression are still not well understood, and transcriptional regulation of cytokine gene expression has not been explored on a systems level. My research will delineate the first gene-centered cytokine gene regulatory network (GRN) by comprehensively identifying the transcription factors (TFs) that regulate cytokine genes. To do so, I will first identify the Ts that can bind to the promoter and other known regulatory regions for all human cytokines by directly testing for protein-DNA interactions (PDIs) using high-throughput yeast one-hybrid (eY1H) assays, recently developed in the Walhout lab. I will model the PDIs detected into a GRN and will integrate this GRN with publicly available expression data to define which TFs positively or negatively regulate cytokine expression. These analyses together with functional validation assays in knockout cell lines will identify cytokine regulators linking them to their functional role and relevance in disease. Non coding mutations are at the core of many human genetic diseases and constitute 90% of all mutations identified in genome-wide association studies. For instance, mutations in cytokine regulatory regions have been associated with cancer, higher susceptibility to infections, chronic arthritis and systemic lupus erythematosus. These mutations can result in loss or gain of transcription factor binding sites. Assays to test th PDI changes are labor intensive and limited to a few dozen TFs. Thus, I propose to use a novel eY1H pipeline to systematically interrogate for PDI changes that result as a consequence of disease-associated mutations in cytokine regulatory regions. Findings from this experimental approach will be further validated in immune cell lines and will provide mechanistic insights and therapeutic targets for many immune related diseases. The cross-disciplinary program I propose requires that I obtain additional training in multiple areas of research that will complement my background and will provide me with state-of-the-art tools for my future research as an independent investigator. I have identified Dr. Walhout and Dr. Fitzgerald as ideal mentors to carry out my proposed work, together with Dr. Wolfe and Dr. Myers as collaborators, with highly complementary expertise. Working in collaboration with these world-renowned researchers in a stimulating environment such as that of UMMS, together with the training activities my mentoring committee and I have planned for the mentored phase of the award, will enable me to (1) become more proficient in state-of-the-art immunology techniques and learn up-to-date immunology concepts, (2) acquire bioinformatics and complex data analysis skills, and (3) learn genome editing approaches, all necessary for the interpretation and validation of the proposed work. This scientific expertise will be complemented with career development seminars and activities to acquire the mentoring, leadership, grantsmanship and writing skills I will need to succeed as an independent investigator. Securing this award would place me in an ideal position to capitalize on my experience to date in the Walhout laboratory, while allowing me to define my own research niche. This award will lead to a better understanding of how immune genes are regulated in normal and pathologic conditions, which will have a broad impact on public health.