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.
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