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.
[Show abstract][Hide abstract] 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.
Proceedings of the National Academy of Sciences 12/2003; 100(23):13465-70. DOI:10.1073/pnas.2235807100 · 9.67 Impact Factor
[Show abstract][Hide abstract] 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.
[Show abstract][Hide abstract] 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.
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