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

Abstract

Recombination of Tcrb gene segments in DN thymocytes is subject to allelic exclusion, such that only a single functional Vβ - DJβ rearrangement is generated per T cell. For Tcrb 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 in situ hybridization to directly visualize the nuclear localization of Tcra and Tcrb, pericentromeric heterochromatin, and the nuclear lamina. Here we provide evidence for a fundamentally different basis for Tcrb allelic exclusion. We demonstrate that Tcrb is highly associated with pericentromeric heterochromatin and the nuclear lamina in pro-B cells and in DN and DP thymocytes. We also find that Tcrb does not associate with peri-centromeric heterochromatin and the nuclear lamina in a strict monoallelic fashion. Rather, Tcrb 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 Tcrb 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 Tcrb, led to both a repositioning of Tcrb 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 Tcrb alleles with repressive nuclear compartments bias initial Tcrb recombination to be monoallelic in developing thymocytes and that such interactions are essential for Tcrb allelic exclusion.