Assessing the Role of the T Cell Receptor Gene Enhancer in Regulating Coding Joint Formation during V(D)J Recombination

Centre d'Immunologie de Marseille-Luminy, INSERM, CNRS, Université de la Méditerranée, 13288 Marseille, France.
Journal of Biological Chemistry (Impact Factor: 4.57). 06/2003; 278(20):18101-9. DOI: 10.1074/jbc.M212647200
Source: PubMed


To assess the role of the T cell receptor (TCR) β gene enhancer (Eβ) in regulating the processing of VDJ recombinase-generated
coding ends, we assayed TCRβ rearrangement of Eβ-deleted (ΔEβ) thymocytes in which cell death is inhibited via expression
of a Bcl-2 transgene. Compared with ΔEβ, ΔEβ Bcl-2 thymocytes show a small accumulation of TCRβ standard recombination products,
including coding ends, that involves the proximal Dβ-Jβ and Vβ14 loci but not the distal 5′ Vβ 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 Eβ, 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 Eβ-dependent and -independent changes in chromosomal
structure, suggesting distinct modes of regulation of TCRβ allelic exclusion depending on the position within the locus.

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    ABSTRACT: The precise function of cis elements in regulating V(D)J recombination is still controversial. Here, we determined the effect of inactivation of the TCRbeta enhancer (Ebeta) on cleavage and rearrangement of Dbeta1, Dbeta2, Jbeta1, and Jbeta2 gene segments in CD4-CD8- [double-negative (DN)] and CD4+CD8+ [double-positive (DP)] thymocytes. In Ebeta-deficient mice, (i) Dbeta1 rearrangements were more severely impaired than Dbeta2 rearrangements; (ii) most of the Dbeta and Jbeta cleavages and rearrangements occurred in DP, rather than in DN, thymocytes; and (iii) most of the 3' Dbeta1 cleavages were coupled to 5' Dbeta2 cleavages instead of to Jbeta cleavages, resulting in nonstandard Dbeta1-Dbeta2-Jbeta2 joints. These findings suggest that the Ebeta regulates TCRbeta rearrangement by promoting accessibility of Dbeta and Jbeta gene segments in DN thymocytes and proper pairing between Dbeta1 and Jbeta gene segments for cleavage and joining in DP thymocytes.
    Preview · Article · Dec 2003 · Proceedings of the National Academy of Sciences
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    ABSTRACT: To investigate the role of promoters in regulating variable gene rearrangement and allelic exclusion, we constructed mutant mice in which a 1.2-kb region of the Vβ13 promoter was either deleted (P13−/−) or replaced with the simian virus 40 minimal promoter plus five copies of Gal4 DNA sequences (P13R/R). In P13−/− mice, cleavage, rearrangement, and transcription of Vβ13, but not the flanking Vβ gene segments, were significantly inhibited. In P13R/R mice, inhibition of Vβ13 rearrangement was less severe and was not associated with any apparent reduction in Vβ13 cleavage. Expression of a T-cell receptor (TCR) transgene blocked cleavages at the normal Vβ13-recombination signal sequence junction and Vβ13 coding joint formation of both wild-type and mutant Vβ13 alleles. However, a low level of aberrant Vβ13 cleavage was consistently detected, especially in TCR transgenic P13R/R mice. These findings suggest that the variable gene promoter is required for promoting local recombination accessibility of the associated Vβ gene segment. Although the promoter is dispensable for allelic exclusion, it appears to suppress aberrant Vβ cleavages during allelic exclusion.
    Preview · Article · Sep 2004 · Molecular and Cellular Biology
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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.
    Full-text · Article · Mar 2005 · Nature Immunology
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