Definition of a T-Cell Receptor β Gene Core Enhancer of V(D)J Recombination by Transgenic Mapping

Abstract

V(D)J recombination in differentiating lymphocytes is a highly regulated process in terms of both cell lineage and the stage of cell development. Transgenic and knockout mouse studies have demonstrated that transcriptional enhancers from antigen receptor genes play an important role in this regulation by activating cisrecombination events. A striking example is the T-cell receptor ␤-chain (TCR␤) gene enhancer (E␤), which in the mouse consists of at least seven nuclear factor binding motifs (␤E1 to ␤E7). Here, using a well-characterized transgenic recombination substrate approach, we define the sequences within E␤ required for recombination enhancer activity. The E␤ core is comprised of a limited set of motifs (␤E3 and ␤E4) and an additional previously uncharacterized 20-bp sequence 3 of the ␤E4 motif. This core element confers cell lineage-and stage-specific recombination within the transgenic substrates, although it cannot bypass the suppressive effects resulting from transgene integration in heterochromatic centromeres. Strikingly, the core enhancer is heavily occupied by nuclear factors in immature thymocytes, as shown by in vivo footprinting analyses. A larger enhancer fragment including the ␤E1 through ␤E4 motifs but not the 3 sequences, although active in inducing germ line transcription within the transgenic array, did not retain the E␤ recombinational activity. Our results emphasize the multifunctionality of the TCR␤ enhancer and shed some light on the molecular mechanisms by which transcriptional enhancers and associated nuclear factors may impact on cis recombination, gene expression, and lymphoid cell differentiation.Immunoglobulin (Ig) and T-cell receptor (TCR) genes are assembled from separate variable (V), diversity (D), or joining (J) gene segments in a process known as V(D)J recombination (4, 44, 55). Normally, V(D)J recombination is restricted to, and required for, early B and T lymphocyte development. It depends on a unique activity, called the V(D)J recombinase, which targets recombination signal sequences (RSSs; consisting of a heptamer, a spacer of 12 or 23 bp, and a nonamer) flanking the rearranging sides of all V, D, and J segments. Rearrangement events primarily involve pairs of segments with RSSs of asymmetrical spacer length (12/23 rule).The functional core of the V(D)J recombinase consists of the lymphoid-restricted RAG-1 and RAG-2 gene products which recognize, pair off, and cleave the RSSs from two rearranging segments, a step also involving architectural proteins from the high-mobility-group family (22, 61). These cleavages consist of DNA double-strand breaks (DSBs) introduced precisely at the junction between the heptamer and adjacent coding sequences, yielding two distinct products: the hairpinsealed coding ends (CEs) and the phosphorylated, blunt-ended signal ends (SEs). Eventually, the cleaved products are assembled together, a process which requires, in addition to the RAG factors, components of the general DNA DSB repair machinery (26, 54). The two CEs are assembled to form a ...

Functions of E2A-HEB Heterodimers in T-Cell Development Revealed by a Dominant Negative Mutation of HEB

“…The defect in V(D)J recombination at the TCR␤ gene locus indicates a possible role of E2A-HEB heterodimers in regulating TCR␤ gene expression and/or rearrangement. This result substantiates recent work by Tripathi et al showing that the TCR␤ gene rearrangement is critically dependent on two E-box sites present in the TCR␤ gene enhancer (37). However, our finding did not rule out the possibility that the defect in TCR␤ gene rearrangement is due to altered expression of other genes, such as RAG1 and RAG2, required for normal TCR␤ gene activation and rearrangement.…”

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

“…The targeting vector was similar to the one described by Huang et al (22), with the exception that the HpaI-NcoI DNA fragment containing E␤ was replaced by a 169-bp NlaIV DNA subfragment (21). C57BL/6J wild-type (WT), E␤-deleted (E␤ Ϫ/Ϫ ) (14), Rag2-deficient (R Ϫ/Ϫ ) (23), TCR␦-deficient (TCR␦ Ϫ/Ϫ ) (24), and SJL mice (25) were also used in this study.…”

“…) formerly defined using a transgenic approach (21). We report that Tcs from homozygous E␤ 169/169 knockin mice readily develop into mature TCR␤ ϩ T cells.…”

Distinct Control of the Frequency and Allelic Exclusion of the Vβ Gene Rearrangement at the TCRβ Locus

“…In experiments using a TCR␤ minilocus in transgenic mice, a V␤ was shown to recombine in the absence of germline transcription (21), although the observed rearrangement may have been promoted by a V␤ promoter present 2.5 kb upstream in the same construct. It is also possible that different cis-regulatory elements in the promoter may function in transcription and recombination as observed for enhancers (22,23). Similarly, the dependence on E␤ for V␤ to D␤J␤ rearrangement in recombination substrates may have been complicated by integration site influences, concatenation of substrate, and changes in distance and configuration of V␤ to D␤ and E␤ in the substrate (24).…”