CONTROL OF TCR V BETA REARRANGEMENT & ALLELIC EXCLUSION

描述(申请人提供)：某些等位基因排斥功能 T细胞受体(TCR)和免疫球蛋白基因座限制发育 淋巴细胞产生一次有效的重排。在TCR测试版 这涉及到一个反馈信号，它抑制V-β到DJ-β 面对重组酶的持续表达进行重排。这表明 在基质可获得性方面的监管，但到目前为止，这还没有 已经得到证实，机械论的见解一直有限。通过比较 双阴性TCRβ基因座染色质中组蛋白3的乙酰化 尚未收到等位基因排斥信号的胸腺细胞 已经收到这个信号的阳性(DP)胸腺细胞，我们证明 与等位基因相关的V-β染色质结构变化 排除。我们建议评估V-β染色质结构的调节 染色质结构在V-β重排和等位基因中的作用 排除。在特定目标I中，我们将表征整个染色质结构 检测糖尿病肾病和糖尿病肾病患者胸腺细胞中TCRβ基因的表达，以评价全球和非糖尿病患者胸腺细胞中TCRβ基因的作用。 等位基因排斥中的局部染色质调节。我们将绘制乙酰化的地图， 可及性、亚核组织和启动子结构。染色质 V-β启动子功能所创造的环境可能是关键的重组 可能是等位基因排除的关键参数。具体目标二，我们将 检测受调控的V-β启动子活性在V-β重排中的作用 通过检测转基因报告缺失的启动子功能来排除等位基因 并通过以下方式从内源基因座中删除启动子 同源重组。尽管存在V-β染色质结构的变化 这与等位基因排斥有关，理论上这个过程可能是 由基于非染色质的机制强制执行。在第三个具体目标中，我们将 通过覆盖结构来解决染色质结构的因果作用 转换并询问我们是否同时覆盖等位基因排斥。我们 将通过提供生殖系V-beta推广副本来实现这一点 使用敲入方法的E-beta。等位基因排斥必须抑制重排 甚至在VDJ-β重排等位基因中，上游V-β的启动子 节段位于E-β的附近。在特定的目标四中，我们将研究染色质 结构的VDJ-beta重排等位基因，并将评估任何作用 重排的V-β片段的启动子，通过 带有或不带有与其相关的重排的V-β片段的敲入 推动者。这些研究应该为发展中的角色提供洞察力 V-β调节染色质开放、增强子活性和启动子功能 重排和等位基因排斥。

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.