CHROMATIN-BASED DISCOVERY AND FUNCTION OF NOVEL TCR REGULATORY ELEMENTS

 描述(申请人提供)：哺乳动物发育过程中基因的调控表达在很大程度上是通过近端启动子和一个或多个组织特异性增强子之间的通信来协调的，这些启动子和一个或多个组织特异性增强子之间可能有数百个千碱基的距离。基因调控研究中的一个主要挑战是识别活跃的顺式启动子集合。 每种细胞类型(其细胞周期)中的元素，并建立启动子-增强子连接，对健康和疾病中的基因调控至关重要。在这一点上，抗原受体(AGR)基因座是破译哺乳动物调控电路许多方面的优秀模型，因为它们含有在淋巴细胞发育过程中差异表达的多个启动子。此外，每个基因座的节段性对于研究启动子-增强子通信的局部、区域和远程控制是理想的。这些调控电路通过V(D)J重组引导AGR基因的逐步组装，从而产生哺乳动物免疫所需的各种免疫球蛋白(Ig)和T细胞受体(TCR)。已经确定了在每个AGR基因座上控制初始、短程重组事件活性的主要增强子。然而，协调大V段阵列的激活和失活以进行远程重组的元件仍然是个谜。因此，在淋巴细胞发育的早期阶段，控制AGR基因组装和表达的环路是不完整的。申请人的实验室最近使用全基因组染色质和B细胞前体的计算分析相结合的方法，在三个免疫球蛋白基因座的每一个中识别新的增强子。目前的项目旨在了解在胸腺细胞发育过程中，未知的顺式元件如何激活TCR基因座内的退化区，从而产生一个主要的TCR谱系。为此，提出了以下实验：(I)收集和计算破译染色质数据，精确定位所有四个TCR基因座中的新调节区；(Ii)表征这些元件的调节功能及其在胸腺细胞发育过程中的波动；以及(Iii)定义TCR基因组装和表达中两个新元件的活性。该项目将为免疫学家提供宝贵的资源，以破译在T淋巴细胞关键发育转变过程中改变的基因电路。此外，这些研究将揭示TCR基因座的全面循环，其基础是空间、遗传和表观遗传机制，以塑造不同的抗原受体谱系。基于这些发现，未来的研究将提供一组动物模型，每个模型都有不同的TCR谱系，这将使理解V段使用变化如何影响对病原体的免疫反应的全新方法成为可能。

 DESCRIPTION (provided by applicant): The regulated expression of genes during mammalian development is coordinated, in large part, by communication between proximal promoters and one or more tissue-specific enhancers, which may be separated by hundreds of kilobases. A major challenge in studies of gene regulation is to identify collections of active cis elements in each cell type (its cistrome) and establish promoter-enhancer connections critical for gene regulation in health and disease. In this regard, antigen receptor (AgR) loci serve as excellent models for deciphering many facets of mammalian regulatory circuits because they harbor multiple promoters that are differentially expressed during lymphocyte development. Moreover, the segmented nature of each locus is ideal for studies of local, regional, and long-range control by promoter-enhancer communication. These regulatory circuits guide the stepwise assembly of AgR genes by V(D)J recombination, which yields the diverse repertoire of immunoglobulin (Ig) and T cell receptors (TCRs) required for mammalian immunity. Major enhancers governing the activity of initial, short-range recombination events at each AgR locus have been identified. However, elements that coordinate activation and inactivation of large V segment arrays for long- range recombination remain enigmatic. As such, the cistrome governing AgR gene assembly and expression during the early stages of lymphocyte development are incomplete. The applicants' laboratories recently used a combination of genome-wide chromatin and computational analyses in B cell precursors to identify novel enhancers in each of the three Ig loci. The current project aims to understand how yet unidentified cis elements activate then decommission regions within TcR loci during thymocyte development, generating a primary TCR repertoire. For this purpose, experiments are proposed to (i) collect and computationally decipher chromatin data, pinpointing new regulatory regions within all four TcR loci, (ii) characterize the regulatory function of such elements and their fluctuations during thymocyte development, and (iii) define the activity of two novel elements in the assembly and expression of TcR genes. This project will provide a valuable resource for immunologists to decipher gene circuits altered during a key developmental transition of T lymphocytes. Moreover, the studies will reveal a comprehensive cistrome for TcR loci that underlies spatial, genetic, and epigenetic mechanisms to sculpt diverse repertoires of antigen receptors. Based on these discoveries, future studies will provide a panel of animal models, each with distinct TCR repertoires, which will enable completely new approaches to understand how altered V segment usage impacts immune responses to pathogens.