Regulation of V(D)J Recombination by Transcriptional Promoters

Abstract

Enhancer elements potentiate the rearrangement of antigen receptor loci via changes in the accessibility of gene segment clusters to V(D)J recombinase. Here, we show that enhancer activity per se is insufficient to target T-cell receptor ␤ miniloci for D␤J␤ recombination. Instead, a promoter situated 5 to D␤1 (PD␤) was required for efficient rearrangement of chromosomal substrates. A critical function for promoters in regulating gene segment accessibility was further supported by the ability of heterologous promoters to direct rearrangement of enhancer-containing substrates. Importantly, activation of a synthetic tetracycline-inducible promoter (Ptet) positioned upstream from the D␤ gene segment was sufficient to target recombination of miniloci lacking a distal enhancer element. The latter result suggests that DNA loops, generated by interactions between flanking promoter and enhancer elements, are not required for efficient recognition of chromosomal gene segments by V(D)J recombinase. Unexpectedly, the Ptet substrate exhibited normal levels of rearrangement despite its retention of a hypermethylated DNA status within the D␤J␤ cluster. Together, our findings support a model in which promoter activation, rather than intrinsic properties of enhancers, is the primary determinant for regulating recombinational accessibility within antigen receptor loci.Precursor lymphocytes diversify immunoglobulin (Ig) and T-cell receptor (TCR) variable-region genes via a program of DNA recombination involving large arrays of variable (V), diversity (D), and joining (J) gene segments. All rearrangement events are mediated by a common V(D)J recombinase activity that targets conserved recognition sequences flanking each gene segment (32,39). Despite these shared features, the rearrangement of antigen receptor loci proceeds in a tissue-, stage-, and allele-specific manner (39). For example, thymocytes specifically target TCR D␤ and J␤ gene segments for recombination upon commitment to the T-cell lineage. In turn, D␤J␤ joins rearrange with one of 30 upstream V␤ elements to complete assembly of a variable-region coding exon. The resultant expression of TCR␤ protein signals for a cessation of TCR␤ recombination and the initiation of TCR␣ gene assembly (42). Likewise, precursor B cells execute an ordered program of rearrangements at the Ig heavy-chain (IgH) and lightchain loci (8,22). These observations indicate that precursor lymphocytes must direct and then redirect V(D)J recombinase activity to specific regions within antigen receptor loci at distinct stages of their development.Recent studies have shown that the tissue-and stage-specific aspects of V(D)J rearrangement are governed by changes in the accessibility of gene segment clusters to recombinase proteins RAG-1 and RAG-2 (25, 41). An important role for enhancer elements in regulating the recombinational accessibility of linked gene segments has been deduced from numerous experimental approaches (reviewed in reference 39). For example, targeted deletion of Ig or TCR enhancers se...

Regulation of IgH Gene Assembly: Role of the Intronic Enhancer and 5′DQ52 Region in Targeting DHJH Recombination

“…DNA lysates were prepared at final concentrations of 1 ϫ 10 3 cells/l. The relative levels of D H 3 J H and V H 3 D H J H rearrangements (31), as well as total DNA content (C) (22), were measured using published PCR assays. To detect D Q52 3 J H3 rearrangements, PCR amplifications were performed for 32 cycles consisting of 1 min at 94°C, 1 min at 56°C, and 1.5 min at 72°C, followed by a single 10-min period at 72°C.…”

“…Deletion of transcriptional enhancers from the TCR␣, TCR␤, or the Ig loci significantly reduces both germline transcription and recombination of gene segments in cis- (18 -21). Likewise, deletion of a germline promoter associated with the D␤1 gene segment severely impairs D␤13 J␤ recombination in thymocytes or in model recombination substrates (22,23). One apparent exception to enhancer-mediated control of recombinase accessibility was thought to be the IgH locus.…”

Cancer predisposition and hematopoietic failure in Rad50S/S mice

“…Semiquantitative PCR and Southern blot analysis of immunoglobulin and TCR␤ gene segment rearrangements were as described (Schlissel et al 1991;Sikes et al 1999). Thymus DNA from three 1-wk-old Rad50 +/+ , Rad50 S/S , and scid mice was prepared and pooled for use in the ligation-mediated PCR assay as described (Zhu and Roth 1995).…”

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

“…Within this region, we assayed the conserved decamer sequence (45,46) in several V␤ promoters (V␤12P, V␤11P, V␤9P), the RSS elements flanking V␤ segments (V␤13R, V␤12R, V␤11R, V␤9R, V␤6R), and several intergenic sites located between V␤ segments. To assay D␤J␤ chromatin we analyzed a site in PD␤1 (47,48), just upstream of the D␤1 gene segment. Acetylation at these sites was compared with that of Oct-2 as a negative control not expressed in thymocytes (30), and to CD3⑀ as a positive control expressed at high levels in thymocytes.…”