Differential Transcriptional Regulation of Individual TCR Vβ Segments Before Gene Rearrangement

Abstract

The promoter sequences of individual murine TCR Vβ segments are dissimilar, but any functional differences between them are masked after productive gene rearrangement by the dominance of the TCRβ 3′ enhancer. However, thymocytes of recombination-activating gene-2 (Rag2)-deficient mice allow the transcriptional activity of Vβ promoters to be studied before rearrangement. Here we report that many Vβ segments are detectably transcribed in Rag2−/− thymocytes and that there are significant differences in expression among different Vβ segments. Primer extension and characterization of cDNA clones from SCID thymocytes suggest that these germline Vβ transcripts generally use the same start sites as those previously determined in mature T cells. The strength of expression before rearrangement does not correlate with proximity to the known enhancer, because members of the most distal Vβ cluster (Vβ2.1, Vβ1.1, Vβ4.1) are relatively strongly expressed and more proximal Vβ segments (Vβ14.1, Vβ3.1, Vβ7.1, Vβ6.1) are only weakly expressed. Different Vβ segments also show different developmental programs of activation in different thymocyte subsets, with the Vβ5.1(L)-8.2(V) spliced transcript expressed earliest as well as most strongly overall. Comparison with Rag+ MHC class I−/− and class II−/− thymocytes confirms that many of these expression differences are leveled by rearrangement and/or by β selection, before MHC-dependent selection. However, the expression pattern of Vβ2.1 is highly distinctive and includes cell types apparently outside the T lineage, suggesting potential acquisition of specialized roles.

Duality of Enhancer Functioning Mode Revealed in a Reduced TCRβ Gene Enhancer Knockin Mouse Model

“…As shown in Fig. 3A, peripheral T cells from WT mice expressed diverse V␤ chains in variable proportions, in agreement with previous studies (34,35). Surprisingly, on E␤ 169/169 T cells, we predominantly detected a small number of distinct V␤ chains in proportions that varied from the WT, suggesting the expression of a biased repertoire in the mutant situation.…”

“…Interestingly, V␤5 and V␤8, found expressed at a relatively elevated frequency on ␣␤T cells, also display high levels of germline transcription (see Ref. 34 and this study), and are located within a relatively relaxed chromatin domain (J. Zacharias, S. Spicuglia, and P. Ferrier, unpublished observations). Proximity to the D␤-J␤ clusters might also contribute to a higher frequency of rearrangements involving the 3Ј V␤14 gene segment in E␤ 169/169 T cells.…”

A Change in the Structure of Vβ Chromatin Associated with TCR β Allelic Exclusion

“…4A). Perhaps consistent with this, recent data indicated that V␤2 displays promiscuous activity in other lineages, as V␤2 germline transcripts were identified in NK cells and in a myeloid-enriched population of bone marrow cells (52).…”

Contribution of TCR-β Locus and HLA to the Shape of the Mature Human Vβ Repertoire

“…The V␤ genes are already transcribed before rearrangement (30), and the transcriptional activity of different V␤s is different. However, Chen et al (30) showed that many of the differences were leveled off by rearrangement and/or ␤-selection, suggesting that other factors change the V␤ usage at different stages during thymocyte development, the most likely candidate being differences in RSS heptamer, nonamer, and 23-bp spacer region (32). Posnett et al (22) have shown that a SNP in the 23-bp spacer region of V␤28 strongly correlated with the usage of this V␤ by T cells.…”

“…Various factors may contribute to the final shape of the TCRV␤ repertoire, such as TCR-␤ locus accessibility (23)(24)(25)(26), accessibility of the recombination signal sequences to V(D)J recombinases (27,28), differential individual V␤ promoter activities (29,30), recombination efficiency which depends on variations in the recombination signal sequence (RSS) (31), proximity to the DJ␤ cluster (16), and pairing efficiency with the pre-T␣ chain (16). Wilson et al (16) showed that DJ␤ proximity and efficiency of pairing with pre-T␣ did not affect murine V␤ usage.…”