Role of Chromatin and Gene Poising in T Cell Differentiation and Activation

候选人现为国家自然科学研究院赵克吉博士实验室博士后。该提案描述了一个导致独立的综合研究和培训计划。在此项目的指导阶段，候选人将继续在应用现代高通量技术（如ChIP-Seq和RNA-Seq）所需的计算数据分析方面进行培训，并培养成功学术生涯所需的其他技能。本课题的研究目的是了解染色质和基因定位在辅助性T细胞分化和功能中的作用。在初始抗原刺激下，幼稚T细胞分化为效应T细胞，然后分化为记忆T细胞。由于先前的抗原暴露，记忆T细胞能够产生细胞因子并比初始T细胞更快地对抗感染。T细胞的这种记忆能力可能是由表观遗传机制介导的。最近，我们开发了ChIP-Seq，一种研究染色质修饰全基因组分布的新方法。将这种方法应用于静止T细胞，我们发现一些沉默基因具有通常与表达基因相关的“活性”染色质修饰，甚至在其启动子处具有RNA聚合酶II （Pol II）。我们称这些基因为“蓄势待发”，因为在T细胞激活过程中被诱导的大多数基因都属于这组基因。为了测试中毒是从头发生还是必须是先前表达的结果，我们建议在体外幼稚T辅助细胞向Th1/Th2细胞分化的过程中跟踪染色质和Pol II结合以及基因表达的变化。此外，我们假设效应记忆T辅助细胞快速产生细胞因子的能力是由这些基因的介导的。我们将把幼稚和记忆T细胞亚群中细胞因子和其他重要基因的染色质结构与这些基因在这些细胞类型中快速表达的能力联系起来（目的1）。平衡基因在其启动子处具有Pol II和所有必要的染色质修饰，但不转录。当T细胞激活时，只有少数的平衡基因被表达。我们将剖析平衡基因的转录抑制机制，并确定在T细胞激活时以基因特异性方式释放这种抑制的因素（目的2）。综上所述，这些研究结果将有助于我们对获得性免疫反应的理解。

DESCRIPTION (provided by applicant): Summary The candidate is currently a postdoctoral fellow at NHLBI in the laboratory of Dr. Keji Zhao. The proposal describes a combined research and training program leading to independence. During the mentored phase of this project, the candidate will continue training in computational data analysis necessary for the application of modern high- throughput techniques such as ChIP-Seq and RNA-Seq, as well as develop other skills necessary for a successful academic career. The research goal of this project is to understand the role of chromatin and gene poising in T helper cell differentiation and function. Upon initial antigen stimulation, naive T cells differentiate into effector and then into memory T cells. As a result of previous antigen exposure, memory T cells are able to produce cytokines and fight infection more rapidly than naive T cells. This ability of memory T cells is likely mediated by the epigenetic mechanisms. Recently, we developed ChIP-Seq, a novel method to study genome-wide distribution of chromatin modifications. Applying this method to resting T cells, we discovered that some silent genes posses "active" chromatin modifications normally associated with expressed genes and even have RNA Polymerase II (Pol II) at their promoters. We called these genes poised, because the majority of the genes that were actually induced during T cell activation belonged to this gene set. To test whether poising may occur de novo or has to be a result of prior expression we propose to follow changes in chromatin and Pol II binding and gene expression during in vitro differentiation of naive T helper cells into Th1/Th2 cells. Further, we hypothesize that the ability of effector memory T helper cells to quickly produce cytokines is mediated by poising of these genes. We will correlate chromatin structure at cytokine and other important genes in naive and subsets of memory T cells with the ability of these genes to be rapidly expressed in these cell types (Aim 1). Poised genes possess Pol II and all the necessary chromatin modifications at their promoters, but are not transcribed. Upon T cell activation only a minority of poised genes are expressed. We will dissect the mechanism of transcription inhibition at the poised genes and identify factors responsible for the release of this inhibition in a gene specific manner upon T cell activation (Aim 2). Taken together, results of these studies will contribute to our understanding of acquired immune response.