Variegated Transcriptional Activation of the Immunoglobulin κ Locus in Pre-B Cells Contributes to the Allelic Exclusion of Light-Chain Expression

ated at the level of regulated gene rearrangement—an individual B cell makes only one productive (in-frame) gene rearrangement at each locus, the second allele being excluded from expression. One of the outstanding questions in immunology is how productive V(D)J re-Several types of evidence support the hypothesis that V(D)J recombinase activity is regulated by the accessibility of rearranging loci within chromatin structure Summary (Schlissel, 2003). First, rearranging gene segments are transcribed prior to or coincident with their activation for Regulated gene rearrangement is thought to underlie rearrangement (" germline " transcripts) and treatments allelic exclusion, the observation that an individual B that enhance transcription of a locus increase the fre-cell expresses only a single immunoglobulin molecule. quency of its rearrangement. Low levels of germline ␬ Previous data has implicated transcriptional activation transcripts are detected in pro-B cells and much higher of rearranging loci in the regulation of their accessibil-levels in pre-B cells correlating with increased V-to-ity to the V(D)J recombinase. Using homologous re-J␬ rearrangement frequency. Second, mutations that combination in ES cells, we have generated " knockin " disrupt the activity of the enhancers or promoters asso-mice which express a GFP cDNA from an unre-ciated with germline transcription greatly diminish the arranged immunoglobulin ␬ light-chain allele. Surpris-corresponding rearrangement events. Finally, recombi-ingly, we find that only a small fraction of ␬ alleles are nant RAG proteins can recognize and cleave RSSs highly transcribed in a population of pre-B cells, that within any loci in purified genomic DNA templates but such transcription is monoallelic, and that these highly only recognize specific loci in purified chromatin sub-transcribed alleles account for the vast majority of ␬ strates in vitro (Stanhope-Baker et al., 1996). These ac-light-chain gene rearrangement. These data lead us cessible loci correspond to those which would have to suggest that probabilistic enhancer activation and undergone V(D)J recombination in vivo dependent upon allelic competition are part of the mechanism of ␬ the source of chromatin used. locus allelic exclusion and may be a general mecha-While the accessibility hypothesis has garnered nism contributing to cellular differentiation during de-strong experimental support, it remains unclear how velopment. regulated chromatin accessibility might account for certain aspects of the allelic exclusion of Ig gene rearrange-Introduction ment. For example, why don't both Ig␬ alleles in pre-B cells occasionally undergo near-simultaneous V(D)J reDeveloping lymphocytes use a series of site-specific combination resulting in B cells which express two func-DNA recombination reactions known …