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节。这是通过调节V(D)J重组反应和/或通过调节V(D)J重组酶对特定抗原受体基因的可及性来实现的。TCRbeta链基因由Vbeta、Dbeta和Jbeta基因片段以等位基因内和等位基因间有序的方式组装而成。在此，我们建议阐明调节tcrβ链基因组装的等位基因内和等位基因间排序的机制。我们将验证Dbeta基因片段两侧的重组信号（RSs）在调节Dbeta到Jbeta和Vbeta到DJbeta重排中起关键作用的假设（具体目标1和2）。我们设想，3‘和5’ Db RSs通过稳定结合RAG-1/2蛋白，分别形成Dbeta到Jbeta和Vbeta到DJbeta重排的突触复合体。此外，我们假设RAG-1/2优先装载在3' Dbeta RS上，一旦装载，3' Dbeta RS就会抑制RAG-1/2在5' Dbeta RS上的结合，这一过程将通过防止Vbeta基因片段重排到种系Dbeta基因片段来强制TCRbeta链基因组装的等位基因内排序。这些假设将通过修饰的TCRbeta等位基因的产生和分析，以及通过分析ag -1/2与染色体RSs的结合来验证。TCRbeta等位基因的可变区基因组装是在等位基因间有序进行的，这种方式允许测试完整的VDJP重排，以确定它是否是非生产性的，然后在备用等位基因上开始重排。我们将验证非生产性TCRbeta链基因重排的产生导致信号促进替代TCRbeta等位基因重排的假设（特定目标3）。我们假设这是通过从非生产性转录本中模板化的RNA监视的无义介导的衰变（NMD）途径的激活发生的。tcrβ链基因重排。这一假设将通过产生一个修饰的TCRbeta等位基因来验证，当非生产性重排时，该等位基因将不会模板转录本，从而能够激活无义介导的衰变。

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.