Complete cis Exclusion upon Duplication of the Eμ Enhancer at the Immunoglobulin Heavy Chain Locus

Abstract

dDeveloping lymphocytes somatically diversify their antigen-receptor loci through V(D)J recombination. The process is associated with allelic exclusion, which results in monoallelic expression of an antigen receptor locus. Various cis-regulatory elements control V(D)J recombination in a developmentally regulated manner, but their role in allelic exclusion is still unclear. At the immunoglobulin heavy chain locus (IgH), the E enhancer plays a critical role in V(D)J recombination. We generated a mouse line with a replacement mutation in the constant region of the locus that duplicates the E enhancer and allows premature expression of the ␥3 heavy chain. Strikingly, IgM expression was completely and specifically excluded in cis from the mutant allele. This cis exclusion recapitulated the main features of allelic exclusion, including differential exclusion of variable genes. Notably, sense and antisense transcription within the distal variable domain and distal V H -DJ H recombination were inhibited. cis exclusion was established and stably maintained despite an active endogenous E enhancer. The data reveal the importance of the dynamic, developmental stage-dependent interplay between IgH locus enhancers and signaling in the induction and maintenance of allelic exclusion. Developing B and T lymphocytes have the capacity to somatically alter their genomes and diversify their antigen receptor loci through V(D)J recombination. This developmentally regulated process is initiated by the lymphoid-specific RAG1/2 complex, which recognizes conserved recombination signal sequences (RSSs) flanking the V, D, and J segments in the variable domain of antigen receptor (IgH, IgL, and TCR) loci (1, 2). V(D)J recombination correlates with chromatin modifications, germ line transcription of rearranging V, D, and J segments, and large-scale chromosome dynamics within nuclear compartments (3-5).The mouse IgH locus contains ϳ200 V H genes, which are subdivided into V H gene families. The most prominent are the distal V H genes, notably the large V HJ558 gene family, and the proximal V H genes. These are followed by a dozen D segments (ϳ60 kb), 4 J H segments (ϳ2 kb), and 8 constant genes (ϳ200 kb) (6, 7).In B lymphocytes, V(D)J assembly starts at the IgH locus, where D segments are first recombined to J H segments on both alleles. Although they have the potential to undergo V H -DJ H recombination on the two alleles, the vast majority of B lymphocytes are subject to allelic exclusion, i.e., a given B cell expresses only one IgH allele. A productive V(D)J rearrangement allows production of the heavy chain, which associates with surrogate light chains and signals an arrest of V H -DJ H recombination on the second IgH allele. If the first V H -DJ H rearrangement is not productive, then the second allele can undergo V H -DJ H recombination (1, 8). Nonetheless, in rare B cells, productive rearrangements can occur on both alleles (i.e., allelic inclusion), but only one heavy chain from only one allele can associate with surrogate light chains...

Developmental Switch in the Transcriptional Activity of a Long-Range Regulatory Element

“…Thus, recombination of distal but not proximal V H genes is inhibited in mice deficient in the histone-modifying enzyme EZH2 and in different transcription factors involved in V(D)J recombination, such as PAX5, YY1, and Ikaros (35)(36)(37)(38). Mutations targeting various cis-acting elements at the IgH locus similarly showed a differential effect on germ line transcription and recombination of proximal versus distal V H genes (12,16,(39)(40)(41)(42). Importantly, deletion of the 3=RR in the context of the 120-kb deletion had no effect on long-range interactions across the IgH locus in RAG2-deficient pro-B cells (22).…”

“…One prediction of this model is that deletion of the 3=RR will result in increased V H germ line transcription and V H -DJ H frequency, and we found this result in the present study. Another prediction is that if a heavy chain is expressed prematurely [that is, prior to V(D)J recombination], the 3=RR will be instructed to inhibit V H germ line transcription, and the outcome will be an impairment of V H -DJ H recombination; this is indeed the case (42). The wide impact of the 3=RR on sense and antisense germ line transcription within the variable region raises the question of whether the 3=RR targets sense and antisense promoters simultaneously.…”

“…It should be noted that V H -DJ H recombination is the regulated step in allelic exclusion (4,5) and that the control of germ line transcription is likely the primary event during allelic exclusion (42). In the absence of the 3=RR, we found increases of germ line transcription within the distal V H domain (with the exception of PAIR promoter/enhancer-derived transcripts) and of the proportion of distal V H DJ H alleles, with no obvious block at the pro-B cell stage.…”

Switch Tandem Repeats Influence the Choice of the Alternative End-Joining Pathway in Immunoglobulin Class Switch Recombination

“…We have previously shown that replacement of Iγ3 switch promoter by a PV H -VDJ-Eµ cassette (hereafter A150 mutation or mouse line) ( Supplementary Figure 1A ) leads to an accumulation of partially rearranged DJ H alleles and a drastic reduction of V H -DJ H recombination ( 43 ). Consequently, IgM expression is severely impaired in A150 homozygous mice and B cell development is driven by IgG3 ( 43 ) (and Supplementary Figure 1B ). In this study, we used this mouse line to investigate if Sγ3 region, in its new setting, has acquired the recombinational properties of Sµ.…”

“…We reasoned that by putting a downstream S sequence under the control of the known elements that regulate Sµ, we could investigate if that S region can acquire Sµ properties. To this end, we used a mouse line in which Iγ3 promoter was replaced by a pre-rearranged VDJ-Eµ cassette ( 43 ), leaving intact the endogenous Sµ and Sγ3 regions. In this setting, the two S regions have roughly the same size, are almost equally distant from Eµ enhancer, but differ in the nature of their core repeats and the density of AID target motifs.…”