The Role of Nuclear Position and Locus Conformation in Regulating V(D)J Recombination of the Tcrb Locus

<p>Recombination of <em>Tcrb</em> gene segments in DN thymocytes is subject to allelic exclusion, such that only a single functional V_β - DJ_β rearrangement is generated per T cell. For <em>Tcrb</em> to be allelically excluded the two alleles must initiate recombination asynchronously and once a β-protein is selected, feedback signals must suppress further recombination. Earlier studies of antigen-receptor loci implicated directed monoallelic association with pericentromeric heterochromatin in the initiation or maintenance of allelic exclusion. In this study we used three-dimensional fluorescent <em>in situ</em> hybridization to directly visualize the nuclear localization of <em>Tcra</em> and <em>Tcrb</em>, pericentromeric heterochromatin, and the nuclear lamina. Here we provide evidence for a fundamentally different basis for <em>Tcrb</em> allelic exclusion. We demonstrate that <em>Tcrb</em> is highly associated with pericentromeric heterochromatin and the nuclear lamina in pro-B cells and in DN and DP thymocytes. We also find that <em>Tcrb</em> does not associate with peri-centromeric heterochromatin and the nuclear lamina in a strict monoallelic fashion. Rather, <em>Tcrb</em> alleles independently associate with the two compartments, leading to a stochastic distribution of nuclei containing both, one, or neither allele associated. In the subset of DN thymocyte nuclei with monoallelically associated <em>Tcrb</em> alleles, the non-rearranged allele is most often associated with repressive compartments. This suggests that association with these compartments inhibits recombination prior to β-selection. This inhibition occurs without altering the conformation of the locus. Moreover, the introduction of an ectopic enhancer into <em>Tcrb</em>, led to both a repositioning of <em>Tcrb</em> away from these repressive compartments. This repositioning was correlated with an increase in the frequency of recombination and a break in allelic exclusion. These data lead us to propose that stochastic rather than directed interactions of <em>Tcrb</em> alleles with repressive nuclear compartments bias initial <em>Tcrb</em> recombination to be monoallelic in developing thymocytes and that such interactions are essential for <em>Tcrb</em> allelic exclusion.</p>Dissertatio