Elucidating CTCF-Mediated Regulation of V(D)J Recombination at the Immunoglobulin Heavy Chain Locus

Abstract

Immunoglobulin heavy (IgH) and light (IgL) chains associate to form the B cell receptor (BCR), which is secreted as an antibody upon B cell activation. Each B cell generates and expresses a unique set of IgH and IgL variable regions generated via V(D)J recombination from multiple germline V, D, and J segments. V(D)J recombination at IgH is ordered: D to JH joining occurs first followed by VH to DJH; lineage-specific: VH to DJH rearrangements do not occur in T cells; and feedback-regulated: productive IgH chains down-regulate further VH to DJH rearrangements. Intergenic Control Region 1 (IGCR1) within the IgH VH-D intergenic region contains two CTCF-binding elements ("CBEs") required for order, lineage-specificity, and feedback-regulation. IGCR1 also diversifies VH usage by negatively regulating dominant D-proximal VH81X rearrangements. Beyond IGCR1 CBEs, there are ~60 CBEs throughout the VH region and ten CBEs just downstream the IgH locus (“3’CBEs”). IGCR1 CBEs form chromatin loops with the IgH intronic enhancer located between the variable and constant regions, and the 3’CBEs. Specific roles for each IGCR1 CBE and their loop interactions in regulating V(D)J rearrangement were unknown. We investigated the individual contribution of the two IGCR1 CBEs and found that mutation of either CBE led to partial defects in order, lineage-specificity, feedback-regulation, and negative regulation of VH81X usage. However, mutation of both CBEs had a greater impairment on IGCR1 function compared to mutation of either single CBE. Thus, IGCR1 CBEs cooperate for full V(D)J recombination regulation. IgH VDJH junctions are processed through the addition or deletion of nucleotides to generate a complementary determining region 3 (CDR3) for antigen contact that greatly contributes to antibody repertoire diversity. We adapted the high-throughput genome-wide translocation sequencing (HTGTS) assay to characterize antibody repertoires (HTGTS-Rep-seq). Using J primers, HTGTS-Rep-seq quantitatively identifies VDJH and VJκ exons and their CDR3 sequences from progenitor and mature mouse B cells. HTGTS-Rep-seq also identifies both productive and non-productive V(D)J configurations, as well as DJH intermediates. We have used this assay to assess impact on IgH V(D)J repertoires with respect to individual contributions of IGCR1 CBEs and their orientation in the IgH locus.