The TCRβ Enhancer Is Dispensable for the Expression of Rearranged TCRβ Genes in Thymic DN2/DN3 Populations but Not at Later Stages

Abstract

The Eβ enhancer has been shown to be dispensable for germline transcription of nonrearranged TCRβ segments but appears to be required for TCRβ V to DJ rearrangement. Eβ dependency of the subsequent expression of VDJ-rearranged TCRβ genes in thymic subpopulations has so far not been analyzed. We generated transgenic mice, using a Vβ8.2Dβ1Jβ1.3-rearranged TCRβ bacterial artificial chromosome, which lacked Eβ, and monitored transgene expression by flow cytometry using Vβ-specific mAbs and an IRES-eGFP reporter. Transgene expression was found in double negative (DN)2 and DN3 but not at later stages of thymopoesis. There was no toxicity associated with the transgene given that apoptosis in DN3, DN4 was not increased, and the number of DN4 cells generated from DN3 cells in reaggregate thymic organ cultures was not diminished. The transgenic TCRβ gave rise to a pre-TCR, as suggested by its ability to suppress endogenous TCRβ rearrangement, to facilitate β-selection on a TCRβ-deficient background and to inhibit γδ T cell lineage development. The results suggest that the Vβ8.2 promoter is sufficient to drive expression of rearranged TCRβ VDJ genes Eβ independently in DN2/DN3 but not at later stages.

Transcriptional regulation and spatial organisation of the human AML1/RUNX1 gene

“…The transient transfection experiments cannot fully reproduce the conditions in which promoters and regulatory elements operate when they are placed in the chromosomal context. In the Jurkat cells, the lymphoid cellspecific TCRb enhancer [Krimpenfort et al, 1988;Levanon et al, 1998;Busse et al, 2005] displayed nearly the same activity as RE1 (Fig. 3).…”

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

“…In this context, CpG methylation may function to restrict the level of Vβ10 transcription and rearrangement in DN cells when factors that promote juxtaposition of Vβ10 with DJβ2 complexes are expressed, but not be needed to regulate Vβ10 rearrangements in DP thymocytes when such factors are not expressed (20, 25). Vβ promoters drive transcription independent of Eβ in DN thymocytes (13), but require Eβ to maintain expression of VDJCβ genes in DP cells (43). In this context, a consequence of functional interactions between Eβ and the Vβ1 promoter on Vβ1 NT alleles in DP cells may be sustained transcription of Vβ10 segments located ~1kb upstream of the Vβ1 promoter.…”

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

“…Instead, weakened enhancer activity, as is the case following the E␤ 169 knockin technique, might expand differential cell expression, intensify phenotypic traits, and unmask the primary activation mode. Thus, in a framework in which TCR␤ gene expression relies on enhancer inputs in both developing and mature T cells (13,14,20), E␤ 169 -driven alleles yielded a reduced and variegated TCR␤ chain phenotype from the ␤-selection step onward, redolent of a graded conduct. In contrast, in earlier DN Tcs, E␤ 169 appears to behave in an exceedingly stochastic fashion, engendering conditions for TCR␤ gene recombination in a limited number of alleles only, more consistent with a binary mode of action.…”

“…Additional evidence implicates E␤ in subsequent expression of VDJ-rearranged TCR␤ genes in developed T cells having passed the ␤-selection checkpoint (20). Knowledge of widespread E␤ activity throughout the T cell lifespan with an apparent requirement in both the early onset and late maintenance of ␤ gene cis expression (including, possibly, fine-tuning in connection to cell selection and/or activation processes) revives the question of whether E␤ function primarily involves a binary or graded mode of gene activation.…”