Delineation of a cytokine gene regulatory network and rewiring in disease

我的长期目标是成为美国一家主要研究机构的独立研究员， 在免疫学和系统生物学之间开展前沿研究。要做到这一点 目标我目前的目标是：(1)产生创新的、高质量的研究成果，从而产生巨大的影响 独立于我导师的研究领域的出版物，(2)在以下方面掌握一套新技能 生物信息学、复杂数据分析、免疫学和基因组编辑方法，以补充我的 现有背景。获得享有盛誉的NIH独立之路奖将会提振我的事业 机会，并为我提供了开发和成熟我自己的研究计划的手段，外加获得 作为一名独立研究员，我需要接受额外的培训才能取得成功。 这项拟议的研究包含了我的主要科学兴趣：将免疫调节与 系统生物学研究基因表达的方法。细胞因子是细胞间的信号蛋白，发挥作用 在描述免疫反应方面的基本作用。细胞因子的产生在 转录水平，如果不能适当地表达它们，通常会导致几种病理现象，例如 自身免疫性疾病、癌症和感染易感性。经过三十多年的研究， 细胞因子表达的许多方面仍不清楚，而细胞因子的转录调控 基因表达还没有在系统水平上被探索。我的研究将描绘出第一个以基因为中心的 细胞因子基因调控网络(GRN)通过综合鉴定转录因子 调节细胞因子基因。为此，我将首先确定可以与启动子结合的转录因子和其他已知的 通过使用高通量直接测试蛋白质-DNA相互作用(PDI)来调节所有人类细胞因子的区域 沃尔胡特实验室最近开发的酵母单杂交(EY1H)分析。我将为PDI建模 检测到GRN中，并将该GRN与公开可用的表达数据集成以定义哪些TF 正向或负向调节细胞因子的表达。这些分析以及功能验证 在基因敲除细胞系中的检测，将识别将它们与其功能角色联系起来的新的细胞因子调节因子，并 与疾病的相关性。 非编码突变是许多人类遗传病的核心，占所有疾病的90% 全基因组关联研究中发现的突变。例如，细胞因子调节区的突变 与癌症、感染易感性、慢性关节炎和系统性红斑狼疮有关 红斑狼疮。这些突变会导致转录因子结合位点的丢失或增加。待检测的化验方法 对于PDI来说，更改是劳动密集型的，并且仅限于几十个TF。因此，我建议使用一种新的eY1H 系统地询问因疾病相关导致的PDI变化的管道 细胞因子调节区的突变。此实验方法的结果将在以下方面得到进一步验证 免疫细胞系，并将为许多免疫相关的机制洞察和治疗靶点 疾病。 我提出的跨学科计划要求我在以下多个领域获得额外的培训 研究将补充我的背景，并将为我的未来提供最先进的工具 以独立调查员的身份进行研究。我认为沃尔胡特博士和菲茨杰拉德博士是 执行我提议的工作，与沃尔夫博士和迈尔斯博士合作，与高度 互补的专业知识。与这些世界知名的研究人员合作，在一个令人兴奋的 环境，以及我和我的指导委员会所进行的培训活动 为奖项的指导阶段计划的，将使我能够：(1)精通最先进的技术 免疫学技术和学习最新的免疫学概念，(2)获得生物信息学和复合体 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 对提议的工作进行验证。这一科学专长将与职业发展相辅相成。 研讨会和活动，以获得我将需要的指导、领导力、资质和写作技能 成功地成为一名独立的调查员。获得这一奖项将使我处于一个理想的位置来利用 根据我在沃尔胡特实验室迄今的经验，同时允许我定义自己的研究利基。这 获奖将有助于更好地理解免疫基因在正常和病理状态下是如何调节的。 条件，这将对公共卫生产生广泛影响。

My long-term goal 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 TFs that can bind to the promoters 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 novel cytokine regulators linking them to their functional role and relevance in disease. Noncoding 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 for 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 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.