Role of chromosomal context in IgVk locus novel enhancer effects on individual Vk gene utilization, germline transcription, and long-range interactions within the Igk locus

Scripps Research Institute, The, La Jolla CA

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Abstract

Abstract The Ig? locus produces a wide array of Ig? rearrangements, but how this complex process is regulated is not well understood. The creation of a diverse Ig? repertoire is facilitated by major changes in the 3D structure of the Ig? locus called locus contraction, and this is thought to allow V? genes throughout the locus a chance to come into proximity with the J? genes to which one V? gene will rearrange in each pre-B cell. However, the factors that are important in initiating and orchestrating the changes in the 3D structure and establishing long- range loops within the Ig? locus in pre-B cells are not known. Importantly, how the 3D structure affects V? gene utilization is not known. Hi-C and 4C studies indicate that there are some regions within the V? portion of the locus that are preferential hubs of long-range interactions with iE?, which is located just 2 kb downstream of the J? genes. We found that the most predominant of these interaction hubs within the V? locus demonstrate binding of multiple transcription factors (TF), and they also have broad regions with the epigenetic marks of enhancers, a few even at the pro-B cell stage. Unlike the known Ig? enhancers at the 3' end of the Ig? locus, these novel enhancer-like regions are in the middle and distal parts of the V? locus. No enhancers have been characterized within the large V region portions of any antigen receptor loci, so this is completely novel. We hypothesized that these enhancer elements would be important in orchestrating the 3D configuration of the contracted Ig? locus, and also that the 3D configuration will affect the composition of the Ig? repertoire. Deletion of the most prominent enhancer, which we term E88, in an inducible Abl cell line and in mice showed reduced rearrangement of most V? genes assayed throughout the locus, particularly those in in a broad range surrounding E88, and especially at earlier times after induction of rearrangement. By 4C, we have shown altered long-range interactions in E88-deleted cells. In order to gain insight into the mechanism by which E88 exerts it effect on V gene rearrangement frequency, we will move E88 to a region far from its natural position in the V? locus in both E88-deleted cells and wild type Abl cells. We will determine if the relocated E88 now becomes a hub of interactions like E88 in its proper location, and if the normal E88 will compete with the relocated E88 for long-range interactions. Importantly, we will determine the effects of this ectopic E88 on local and locus-wide individual V? gene rearrangement frequencies. This will provide novel data regarding the relationship of the 3D structure of the Ig? locus with individual V gene usage, which is not know for any of the Ig loci. If ectopic E88 does result in increased rearrangement to nearby genes, it is possible that such manipulations might be of therapeutic use in mice bearing human Ig loci to increase rearrangement of desired V? genes. !