Regulation of TCRBeta Chain Gene Rearrangement

描述（由申请人提供）：在胸腺细胞发育过程中，T细胞受体（TCR）基因的组装在许多情况下受到精确调控。这通过调节V（D）J重组反应和/或通过调节V（D）J重组酶对特异性抗原受体基因的可及性而发生。TCR β链基因以等位基因内和等位基因间有序的方式由V β、D β和J β基因片段组装而成。在这里，我们建议阐明调节TCR β链基因组装的等位基因内和等位基因间排序的机制。我们将检验这一假设，即D β基因片段侧翼的重组信号（RS）在调节D β至J β和V β至DJ β重排中起关键作用（具体目标1和2）。我们设想，3'和5' Db RS通过RAG-1/2蛋白的稳定结合，分别为D β到J β和V β到DJ β重排的突触复合物形成成核。此外，我们假设RAG-1/2优先在3'D β RS上加载，并且一旦加载，3' D β RS抑制RAG-1/2在5'D β RS上的结合。这样的过程将通过防止V β基因片段重排为种系D β基因片段来强制TCR β链基因组装的等位基因内排序。将通过产生和分析修饰的TCR β等位基因以及通过分析RAG-1/2与体内染色体RS的结合来检验这些假设。TCR β等位基因处的可变区基因组装以允许在替代等位基因上开始重排之前测试完整VDJP重排的方式在等位基因间排序，以确定其是否是非生产性的。我们将检验这样的假设，即非生产性TCR β链基因重排的产生导致促进替代TCR β等位基因重排的信号（具体目标三）。我们假设，这是通过激活无义介导的衰变（NMD）的RNA监视的转录模板从非生产性途径。TCR β链基因重排。该假设将通过产生修饰的TCR β等位基因来测试，当非生产性重排时，该等位基因将不模板化能够激活无义介导的衰变的转录物。

DESCRIPTION (provided by applicant): The assembly of T cell receptor (TCR) genes is precisely regulated in many contexts during thymocyte development. This occurs through modulation of the V(D)J recombination reaction and/or through modulation of accessibility of the V(D)J recombinase to specific antigen receptor genes. TCRbeta chain genes are assembled from Vbeta, Dbeta and Jbeta gene segments in a manner that is both intra- and inter- allelically ordered. Here we propose to elucidate the mechanisms that regulate the intra- and inter- allelic ordering of TCRbeta chain gene assembly. We will test the hypothesis that the recombination signals (RSs) flanking the Dbeta gene segment serve a pivotal role in regulating Dbeta to Jbeta and Vbeta to DJbeta rearrangement (Specific aims one and two). We envision that the 3' and 5' Db RSs nucleate synaptic complex formation for Dbeta to Jbeta and Vbeta to DJbeta rearrangement, respectively, through stable binding of the RAG-1/2 proteins. In addition, we hypothesize that RAG-1/2 is loaded preferentially at the 3' Dbeta RS and that, once loaded, the 3' Dbeta RS inhibits RAG-1/2 binding at the 5' Dbeta RS. Such a process would enforce intra-allelic ordering of TCRbeta chain gene assembly by preventing Vbeta gene segment rearrangement to a germline Dbeta gene segment. These hypotheses will be tested through the generation and analysis of modified TCRbeta alleles and through analyses of RAG-1/2 binding to chromosomal RSs in vivo. Variable region gene assembly at the TCRbeta alleles is inter-allelically ordered in a manner that permits the testing of a complete VDJP rearrangement, to determine if it is nonproductive, before rearrangement is initiated on the alternate allele. We will test the hypothesis that the generation of a non-productive TCRbeta chain gene rearrangement results in signals that promote rearrangement on the alternate TCRbeta allele (Specific aim three). We hypothesize that this occurs through activation of the nonsense mediated decay (NMD) pathway of RNA surveillance by transcripts templated from non-productive. TCRbeta chain gene rearrangements. This hypothesis will be tested through the generation of a modified TCRbeta allele which, when non-productively rearranged, will not template transcripts that are able to activate nonsense mediated decay.