Differential Regulation of Proximal and Distal Vβ Segments Upstream of a Functional VDJβ1 Rearrangement upon β-Selection

Abstract

Developmental stage-specific regulation of transcriptional accessibility helps control V(D)J recombination. Vβ segments on unrearranged TCRβ alleles are accessible in CD4−/CD8− (double-negative [DN]) thymocytes, when they recombine, and inaccessible in CD4+/CD8+ (double-positive [DP]) thymocytes, when they do not rearrange. Downregulation of Vβ accessibility on unrearranged alleles is linked with Lat-dependent β-selection signals that inhibit Vβ rearrangement, stimulate Ccnd3-driven proliferation, and promote DN-to-DP differentiation. Transcription and recombination of Vβs on VDJβ-rearranged alleles in DN cells has not been studied; Vβs upstream of functional VDJβ rearrangements have been found to remain accessible, yet not recombine, in DP cells. To elucidate contributions of β-selection signals in regulating Vβ transcription and recombination on VDJβ-rearranged alleles, we analyzed wild-type, Ccnd3−/−, and Lat−/− mice containing a preassembled functional Vβ1DJCβ1 (Vβ1NT) gene. Vβ10 segments located just upstream of this VDJCβ1 gene were the predominant germline Vβs that rearranged in Vβ1NT/NT and Vβ1NT/NTCcnd3−/− thymocytes, whereas Vβ4 and Vβ16 segments located further upstream rearranged at similar levels as Vβ10 in Vβ1NT/NTLat−/− DN cells. We previously showed that Vβ4 and Vβ16, but not Vβ10, are transcribed on Vβ1NT alleles in DP thymocytes; we now demonstrate that Vβ4, Vβ16, and Vβ10 are transcribed at similar levels in Vβ1NT/NTLat−/− DN cells. These observations indicate that suppression of Vβ rearrangements is not dependent on Ccnd3-driven proliferation, and DN residence can influence the repertoire of Vβs that recombine on alleles containing an assembled VDJCβ1 gene. Our findings also reveal that β-selection can differentially silence rearrangement of germline Vβ segments located proximal and distal to functional VDJβ genes.