Mouse Anti-Human TCR V beta 8a monoclonal antibody, clone J7H9

The Tcrb locus is subject to a host of regulatory mechanisms that impart a strict cell and developmental stage-specific order to variable (V), diversity (D), and joining (J) gene segment recombination. The Tcrb locus is also regulated by allelic exclusion mechanisms, which restrict functional rearrangements to a single allele. The production of a functional rearrangement in CD4(-)CD8(-) double-negative (DN) thymocytes leads to the assembly of a pre-TCR and initiates signaling cascades that allow for DN to CD4(+)CD8(+) double-positive (DP) differentiation, proliferation, and feedback inhibition of further V beta to DJ beta rearrangement. Feedback inhibition is believed to be controlled, in part, by the loss of V beta gene segment accessibility during the DN to DP transition. However, the pre-TCR signaling pathways that lead to the inactivation of V beta chromatin have not been determined. Because activation of the MAPK pathway is documented to promote DP differentiation in the absence of allelic exclusion, we characterized the properties of V beta chromatin within DP thymocytes generated by a constitutively active Raf1 (Raf-CAAX) transgene. Consistent with previous reports, we show that the Raf-CAAX transgene does not inhibit Tcrb recombination in DN thymocytes. Nevertheless, DP thymocytes generated by Raf-CAAX signals display normal down-regulation of V beta segment accessibility and normal feedback inhibition of the V beta to DJ beta rearrangement. Therefore, our results emphasize the distinct requirements for feedback inhibition in the DN and DP compartments. Although MAPK activation cannot impose feedback in DN thymocytes, it contributes to feedback inhibition through developmental changes that are tightly linked to DN to DP differentiation.

Accessibility of chromosomal recombination signal sequences to the RAG protein complex is known to be essential for V(D)J recombination at Ag receptor loci in vivo. Previous studies have addressed the roles of cis-acting regulatory elements and germline transcription in the covalent modification of nucleosomes at Ag receptor loci. However, a detailed picture of nucleosome organization at accessible and inaccessible recombination signal sequences has been lacking. In this study, we have analyzed the nucleosome organization of accessible and inaccessible Tcrb and Tcra alleles in primary murine thymocytes in vivo. We identified highly positioned arrays of nucleosomes at D-beta, J(beta), and J(alpha) segments and obtained evidence indicating that positioning is established at least in part by the regional DNA sequence. However, we found no consistent positioning of nucleosomes with respect to recombination signal sequences, which could be nucleosomal or internucleosomal even in their inaccessible configurations. Enhancer- and promoter-dependent accessibility was characterized by diminished abundance of certain nucleosomes and repositioning of others. Moreover, some changes in nucleosome positioning and abundance at J(alpha)61 were shown to be a direct consequence of germline transcription. We suggest that enhancer- and promoter-dependent transcription generates optimal recombinase substrates in which some nucleosomes are missing and others are covalently modified. The Journal of Immunology, 2010, 184: 6970-6977.