Assessing the role of the T cell receptor beta gene enhancer in regulating coding joint formation during V(D)J recombination.
ABSTRACT To assess the role of the T cell receptor (TCR) beta gene enhancer (Ebeta) in regulating the processing of VDJ recombinase-generated coding ends, we assayed TCRbeta rearrangement of Ebeta-deleted (DeltaEbeta) thymocytes in which cell death is inhibited via expression of a Bcl-2 transgene. Compared with DeltaEbeta, DeltaEbeta Bcl-2 thymocytes show a small accumulation of TCRbeta standard recombination products, including coding ends, that involves the proximal Dbeta-Jbeta and Vbeta14 loci but not the distal 5' Vbeta genes. These effects are detectable in double negative pro-T cells, predominate in double positive pre-T cells, and correlate with regional changes in chromosomal structure during double negative-to-double positive differentiation. We propose that Ebeta, by driving long range nucleoprotein interactions and the control of locus expression and chromatin structure, indirectly contributes to the stabilization of coding ends within the recombination processing complexes. The results also illustrate Ebeta-dependent and -independent changes in chromosomal structure, suggesting distinct modes of regulation of TCRbeta allelic exclusion depending on the position within the locus.
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ABSTRACT: Ag receptor allelic exclusion is thought to occur through monoallelic initiation and subsequent feedback inhibition of recombinational accessibility. However, our previous analysis of mice containing a V(D)J recombination reporter inserted into Vbeta14 (Vbeta14(Rep)) indicated that Vbeta14 chromatin accessibility is biallelic. To determine whether Vbeta14 recombinational accessibility is subject to feedback inhibition, we analyzed TCRbeta rearrangements in Vbeta14(Rep) mice containing a preassembled in-frame transgenic Vbeta8.2Dbeta1Jbeta1.1 or an endogenous Vbeta14Dbeta1Jbeta1.4 rearrangement on the homologous chromosome. Expression of either preassembled VbetaDJbetaC beta-chain accelerated thymocyte development because of enhanced cellular selection, demonstrating that the rate-limiting step in early alphabeta T cell development is the assembly of an in-frame VbetaDJbeta rearrangement. Expression of these preassembled VbetaDJbeta rearrangements inhibited endogenous Vbeta14-to-DJbeta rearrangements as expected. However, in contrast to results predicted by the accepted model of TCRbeta feedback inhibition, we found that expression of these preassembled TCR beta-chains did not downregulate recombinational accessibility of Vbeta14 chromatin. Our findings suggest that TCRbeta-mediated feedback inhibition of Vbeta14 rearrangements depends on inherent properties of Vbeta14, Dbeta, and Jbeta recombination signal sequences.The Journal of Immunology 02/2010; 184(3):1369-78. · 5.52 Impact Factor
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ABSTRACT: Allelic exclusion of V(beta)-to-DJ(beta) recombination depends on asynchronous rearrangement of alleles of the gene encoding T cell receptor beta in double-negative thymocytes and feedback inhibition that is maintained in double-positive thymocytes. Feedback is thought to be enforced through downregulation of V(beta) accessibility. In an attempt to override this negative regulation, we introduced the enhancer of the gene encoding T cell receptor alpha into the V(beta) gene cluster downstream of V(beta)12. In double-negative thymocytes, the introduced enhancer had no measurable effect on accessibility, but V(beta)12 rearrangement was stimulated and V(beta)12 allelic exclusion was partially subverted. In contrast, double-positive thymocytes showed increased V(beta) transcription and accessibility, but feedback inhibition of V(beta)-to-DJ(beta) recombination remained intact. Our results indicate additional regulatory constraints on V(beta)-to-DJ(beta) recombination that operate beyond the accessibility barrier.Nature Immunology 03/2005; 6(2):189-97. · 26.20 Impact Factor
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ABSTRACT: V(D)J recombination assembles antigen receptor genes from germline V, D and J segments during lymphocyte development. In αβT-cells, this leads to the subsequent expression of T-cell receptor (TCR) β and α chains. Generally, V(D)J recombination is closely controlled at various levels, including cell-type and cell-stage specificities, order of locus/gene segment recombination, and allele usage to mediate allelic exclusion. Many of these controls rely on the modulation of gene accessibility to the recombination machinery, involving not only biochemical changes in chromatin arrangement and structural modifications of chromosomal organization and positioning, but also the refined composition of the recombinase targets, the so-called recombination signal sequences. Here, we summarize current knowledge regarding the regulation of V(D)J recombination at the Tcrb gene locus, certainly one for which these various levels of control and regulatory components have been most extensively investigated.Seminars in Immunology 12/2010; 22(6):330-6. · 5.93 Impact Factor