Antigen receptor genes are assembled from multiple gene segments during early lymphoid cell development in a process termed V(D)J rearrangement. During early B cell development in the bone marrow (BM), V(D)J and VJ joining occurs on the IgH and L chain genes, respectively and is mediated by the RAG recombinase in order to generate a diverse repertoire of antibodies. VH genes are dispersed through 2.5 Mb of the Igh locus, and thus compaction of the Igh locus serves to facilitate spatial proximity between the rearranged DHJH join and distal VH genes. Furthermore, V genes rearrange with very different intrinsic frequencies. The pioneering studies of mammalian chromosome structure using chromosome conformation capture (3C) methods have revealed that chromosomes are organized into a hierarchy of units that include topologically associating domains (TADs). Long range chromatin looping interactions within TADS link promoters (Pr) with enhancers (E) and regulate gene expression. However, little is known about the precise looping structure of the Igh locus that leads to locus contraction. We undertook an analysis of the entire Igh locus using chromosome conformation capture (3C) based methodology to systematically characterize three-dimensional (3D) chromatin organization on several genomic scales. We found that the Igh locus is compartmentalized into two unique topological sub-domains separated by a relatively unstructured region. A set of extremely long range looping interactions that bridge the chromatin sub-domains and are pro-B cell-specific and Pax5-dependent. These looping interactions are anchored at Sites termed I, II, II.5 and III and which appear to be critical facilitators of Igh locus contraction. In new studies we identified the DNA motifs underpinning loop-anchor sites at Sites I and II. Our findings have led us to construct a model in which locus compaction may be mediated by specific VH promoter-novel enhancer interactions and CTCF looping. To fully appreciate the importance of these findings it would be very beneficial to visualize the Igh TAD as a 3D structure. This would enable us to directly probe the shape of the chromatin structure and determine the structure-function relationships between key chromatin elements and IgH repertoire. However, the study of TAD structure has been stymied by the lack of powerful modeling prediction tools to convert pairwise chromatin interactions discovered using 3C technologies into high resolution ensembles of 3D chromatin structures. We have addressed these issues as a collaborative team and have designed and implemented the novel Constrained-Self Avoiding Chromatin (C- SAC) chain algorithm that reconstructs large ensembles of 3D chromatin chains from 5C and HiC data sets. We propose to test our computational pipeline on new capture Hi-C data sets from the Igh locus and probe the relationship between specific chromatin loops, TAD structure and its impact on VH gene usage during V(D)J recombination. Our effort is not limited to the Ig loci, as our method would be generally applicable and permit a systematic interrogation of the biological underpinnings of TAD formation in mammalian genomes.
Antibodies are assembled via a DNA gene rearrangement process termed VDJ joining. The stretch of chromosomal DNA responsible for antibody heavy chain V genes is huge. Here we propose to characterize at high resolution and predict key elements in the three dimension chromosomal architecture that spatially organizes the V gene segments and makes them available for use during VDJ joining. These studies address basic questions of how antibody genes are used during humoral immune responses and will help us understand how to fine tune vaccine development to ultimately provide the most efficient immune responses.
