CONTROL OF TCR V BETA REARRANGEMENT & ALLELIC EXCLUSION

描述（由申请人提供）：在某些情况下， T细胞受体（TCR）和免疫球蛋白基因座限制发育 淋巴细胞产生一个单一的生产性重排。在TCR β 基因座，这涉及到一个反馈信号，抑制V-β到DJ-β 重组酶的持续表达。这表明 在底物可及性水平的监管，但迄今为止，这还没有 已经得到证实，机械的见解是有限的。通过比较 双阴性（DN）TCR β基因座染色质中组蛋白3的乙酰化 胸腺细胞尚未接收到等位基因排斥信号， 阳性（DP）胸腺细胞已经收到了这个信号，我们证明 与等位基因相关的V-β染色质结构的变化 排斥。我们建议评估V-β染色质结构的调节 以及染色质结构在V-β重排和等位基因 排斥。在具体目标I中，我们将描述染色质结构， 在DN和DP胸腺细胞中的TCR β基因座，以评估整体与 等位基因排斥中的局部染色质调节。我们将绘制乙酰化， 可及性、亚核组织和启动子结构。染色质 由V-β启动子功能产生的环境可能是关键的重组 并且可能是等位基因排除的关键参数。具体目标二，我们将 测试调节V-β启动子活性在V-β重排中的作用， 通过测定缺乏转基因报告基因的启动子功能进行等位基因排除 和通过从内源基因座缺失启动子， 同源重组虽然V-β染色质结构发生了变化， 与等位基因排斥有关，理论上， 由基于非染色质的机制强制执行。在第三阶段，我们将 解决了染色质结构的因果作用， 转换并询问我们是否同时推翻等位基因排斥。我们 将通过提供一个本地拷贝的生殖系V-β启动拷贝来做到这一点， 使用敲入方法的E-β。等位基因排斥必须抑制重排 甚至在VDJ-β重排的等位基因上，其中上游V-β的启动子 片段都在E-β附近。在第四章中，我们将研究染色质 VDJ-β重排等位基因的结构，并将评估任何作用， 重排的V-β片段的启动子在建立该结构中，通过 具有或不具有其相关的重排的V-β片段的敲入 启动子这些研究应该为发展中国家的作用提供见解。 调节V-β中的染色质开放、增强子活性和启动子功能 重排和等位基因排除。

DESCRIPTION (provided by applicant): Allelic exclusion functions at certain T-cell receptor (TCR) and immunoglobulin loci to restrict developing lymphocytes to produce a single productive rearrangement. At the TCR beta locus, this involves a feedback signal that inhibits V-beta to DJ-beta rearrangement in the face of continued expression of recombinase. This suggests regulation at the level of substrate accessibility, but to date, this has not been confirmed and mechanistic insights have been limited. By comparing the acetylation of histone 3 in TCR beta locus chromatin of double negative (DN) thymocytes that have yet to receive an allelic exclusion signal and double positive (DP) thymocytes that have already received this signal, we demonstrate a change in the structure of V-beta chromatin associated with allelic exclusion. We propose to evaluate the regulation of V-beta chromatin structure and the role of chromatin structure in V-beta rearrangement and allelic exclusion. In Specific Aim I, we will characterize chromatin structure across the TCR beta locus in DN and DP thymocyte to evaluate the role of global vs. local chromatin regulation in allelic exclusion. We will map acetylation, accessibility, subnuclear organization, and promoter structure. The chromatin environment created by V-beta promoter function may be critical recombination and may be a critical parameter for allelic exclusion. Specific Aim II, we will test a role for regulated V-beta promoter activity in V-beta rearrangement and allelic exclusion by assaying promoter function in a transgenic reporter devoid of enhancer elements, and by deleting promoters from the endogenous locus by homologous recombination. Although there is a change V-beta chromatin structure that is associated with allelic exclusion, the process could in theory be enforced by a non-chromatin based mechanism. In Specific Aim III we will address a causal role for chromatin structure by overriding the structural transition and ask whether we simultaneously override allelic exclusion. We will do so by supplying a germline V-beta promote copy of a local copy of E-beta using a knock-in approach. Allelic exclusion must inhibit rearrangement even on VDJ-beta rearranged allele in which the promoters of upstream V-beta segments are in proximity of E-beta. In Specific Aim IV we will study chromatin structure on a VDJ-beta rearranged allele, and will evaluate any role for the promoter of the rearranged V-beta segment in establishing that structure, by knock-in with or the rearranged V-beta segment with or without its associated promoter. These studies should provide insights into roles for developmentally regulated chromatin opening, enhancer activity and promoter function in V-beta rearrangement and allelic exclusion.