Accessibilty Control of Antigen Receptor Gene Assembly

描述（由申请方提供）：为了产生介导哺乳动物免疫的抗原受体库，淋巴细胞必须通过V（D）J重组从单独的编码片段组装免疫球蛋白（IG）和T细胞受体（TCR）基因。重要的是，必须严格调控V（D）J重组，以确保淋巴细胞正常发育，并避免导致淋巴肿瘤的染色体易位。V、D和J区段的染色质可及性由组织/阶段特异性增强子控制，其影响V（D）J重组酶的适当靶向。申请人的实验室最近发现，未重排基因区段侧翼的种系启动子也是重组酶可及性所需的。TCR β生殖系启动子通过空间限制机制调节可及性，该机制独立于靶基因片段的转录而起作用。再加上以前的增强子研究，这两个基本的发现突出了一个新的作用，生殖系启动子作为第二个可及性控制元件（ACE）在抗原受体基因组装的过程中。我们假设ACE介导了一系列生化事件，最终在重组酶可接近的基因座中达到高潮，包括（i）增强子指导的局部染色质开放，（ii）形成更高级的增强子/启动子复合物（即，增强体），（iii）启动子的激活，和（iv）启动子近端核小体的染色质重塑。为了检验这一中心假设，我们将定义ACE控制下的核小体重塑事件，产生一个重组酶可接近的位点（特异性目的1）。在具体目标2中，我们将确定ACE招募的染色质修饰剂，以建立重塑所需的组蛋白密码。目的3：揭示促进ACE相互作用的特异性转录因子，并有助于重排位点的形成。总的来说，该项目将建立V（D）J重组酶从大量基因片段中选择特定靶点的分子机制，从而产生功能齐全的免疫库。拟议的研究还将为所有真核细胞中基因组信息选择性转录和复制过程中控制染色质可及性的过程提供基本见解。

DESCRIPTION (provided by applicant): In order to generate the antigen receptor repertoire that mediates mammalian immunity, lymphocytes must assemble immunoglobulin (Ig) and T cell receptor (TCR) genes from separate coding segments via V(D)J recombination. Importantly, V(D)J recombination must be tightly regulated to ensure proper lymphocyte development and to avoid chromosomal translocations that cause lymphoid tumors. Chromatin accessibility of V, D, and J segments is governed by tissue/stage-specific enhancers, which affect proper targeting of V(D)J recombinase. The applicant's laboratory has recently discovered that the germline promoters flanking unrearranged gene segments are also required for recombinase accessibility. The TCRbeta germline promoter regulates accessibility via a spatially restricted mechanism that operates independent of transcription through target gene segments. Coupled with prior enhancer studies, these two fundamental discoveries highlight a new role for the germline promoter as a second accessibility control element (ACE) in the process of antigen receptor gene assembly. We hypothesize that ACEs mediate a series of biochemical events that culminate in a recombinase-accessible locus, including (i) enhancer-directed opening of local chromatin, (ii) formation of a higher order enhancer/promoter complex (i.e., an enhanceosome), (iii) activation of the promoter, and (iv) chromatin remodeling of promoter-proximal nucleosomes. To test this central hypothesis, we will define the nucleosome remodeling events under ACE control that yield a recombinase-accessible locus (Specific Aim 1). In Specific Aim 2, we will identify the chromatin modifiers that ACEs recruit to establish the requisite histone code for remodeling. Aim 3 studies will reveal the specific transcription factors that facilitate ACE cross-talk and contribute to the formation of a locus poised for rearrangement. Overall, the project will establish the molecular mechanisms by which V(D)J recombinase selects specific targets from an extremely large array of gene segments, resulting in a fully functional immune repertoire. The proposed research will also provide fundamental insights into the processes that control chromatin accessibility during selective transcription and replication of genomic information in all eukaryotic cells.