Accessibilty Control of Antigen Receptor Gene Assembly

描述（由申请人提供）：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重组从不同的编码段组装免疫球蛋白（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，以确保适当的淋巴细胞发育，并避免导致淋巴样肿瘤的染色体易位。V、D和J片段的染色质可及性由组织/阶段特异性增强子控制，这些增强子影响V(D)J重组酶的适当靶向。申请人的实验室最近发现，未重排基因片段两侧的种系启动子也是重组酶可及性所必需的。TCRbeta胚系启动子通过一种独立于靶基因片段转录的空间受限机制调节可及性。结合之前对增强子的研究，这两项基本发现突出了种系启动子作为抗原受体基因组装过程中的第二个可及性控制元件（ACE）的新作用。我们假设ace介导了一系列生化事件，最终导致重组酶可接近的位点，包括(i)增强子导向的局部染色质打开，（ii）高阶增强子/启动子复合物（即增强体）的形成，（iii）启动子的激活，以及（iv）启动子-近端核小体的染色质重塑。为了验证这一中心假设，我们将定义ACE控制下产生重组酶可及位点的核小体重塑事件（Specific Aim 1）。在特异性目标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代码。Aim 3研究将揭示促进ACE串扰的特定转录因子，并有助于形成一个准备重排的位点。总体而言，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的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.