Control of TCR V Beta Rearrangement & Allelic Exclusion

描述（申请人提供）：免疫系统产生大量克隆分布抗原受体库的能力取决于V（D）J重组过程，该过程在T和B淋巴细胞发育的早期阶段以高度调节的方式发生。发育调控的一个关键方面是等位基因排斥过程，其限制某些抗原受体基因座仅在一个等位基因上产生功能重排的基因。等位基因排斥部分地通过反馈抑制来实施，其中一个等位基因上的生产性VDJ重组事件进一步阻断第二个等位基因上的V至DJ重组。对此负责的机制尚不清楚。我们认为，与TCR β等位基因排斥相关的反馈抑制是多因素的，有几种影响汇聚到抑制V β到DJ β的重组。反馈被认为是通过下调V β可及性来加强的。然而，我们发现，即使在克服了可访问性障碍之后，反馈仍然完好无损。我们建议，这种“超越可及性”的调节可能反映了位点构象的变化，抑制远端重组信号序列（RSS）之间的重组事件，位点亚核位置的变化，可能会限制重组酶的可用性，和独特的属性的RSS或因素招募到他们的附近，可能提供发育特异性RSS的使用。在具体目标I中，我们将使用三维荧光原位杂交方法鉴定TCR β基因座亚核位置和构象的发育变化。在特定目标II和III中，我们将使用互补的基因靶向方法来直接测试反馈抑制的机制。首先，我们将创建和研究嵌入TCR β和TCR α基因座的紧凑型V（D）J重组盒，这些重组盒应覆盖基因座构象和亚核位置的任何贡献。其次，我们将修改TCR β基因座内的局部元素，这些元素应该覆盖来自局部监管的潜在贡献。最后，在第四章中，我们将定义将TCR β基因座分离成不同染色质调控单位的机制。这些研究的成功完成应提供关于V（D）J重组的控制和指导克隆分布和非自身反应性抗原受体库形成的机制的一般经验。

DESCRIPTION (provided by applicant): The ability of the immune system to generate immense repertoires of clonally distributed antigen receptors depends on the process of V(D)J recombination, which occurs in a highly regulated fashion during the early stages of T and B lymphocyte development. A critical aspect of developmental regulation is the process of allelic exclusion, which limits certain antigen receptor loci to produce a functionally rearranged gene on only one allele. Allelic exclusion is enforced in part through feedback inhibition in which a productive VDJ recombination event on one allele blocks further V to DJ recombination on the second allele. The mechanisms responsible are not well understood. We propose that feedback inhibition associated with TCR beta allelic exclusion is multifactorial, with several influences converging to suppress V beta to DJ beta recombination. Feedback was thought to be enforced by downregulation of V beta accessibility. However, we found that feedback is intact even after the accessibility barrier has been overcome. We propose that this "beyond accessibility" regulation may reflect changes in locus conformation that inhibit recombination events between distal recombination signal sequences (RSSs), changes in locus subnuclear position that may restrict the availability of the recombinase, and unique properties of RSSs or factors recruited to their vicinity that may provide developmental specificity to RSS usage. In Specific Aim I we will identify developmental changes in TCR beta locus subnuclear position and conformation using the approach of three dimensional fluorescence in situ hybridization. In Specific Aims II and III we will use complementary gene- targeting approaches to directly test mechanisms of feedback inhibition. First, we will create and study compact V(D)J recombination cassettes embedded in the TCR beta and TCR alpha loci that should override any contributions of locus conformation and subnuclear position. Second, we will modify local elements within the TCR beta locus that should override potential contributions from local regulation. Finally, in Specific Aim IV we will define the mechanisms that segregate the TCR beta locus into distinct chromatin regulatory units. Successful completion of these studies should provide general lessons regarding the control of V(D)J recombination and the mechanisms that guide the formation of a clonally distributed and non-autoreactive antigen receptor repertoire.