Characterization of chromatin loops responsible for Igh locus contraction

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 DESCRIPTION (provided by applicant): Immunoglobulin (Ig) genes are unique in that they are subject to three different types of gene alterations to achieve a fully functional humoral immune response. During early B cell development in the bone marrow (BM), V(D)J or VJ joining occurs on the IgH and L chain genes, respectively and is mediated by the RAG recombinase. Our new studies describe a stepwise process of chromosomal conformational alterations which collaborate to create conditions amenable for the assembly of V-D-J gene segments into contiguous V(D)J exons that encode the antigen binding portion of IgH molecules. Recent studies indicate that chromatin looping influences partner selection during V(D)J recombination, CSR and may drive specific chromosomal translocation events. Although a small subset of loops have been discerned for the Igh locus an unbiased examination of locus looping was unavailable. We therefore undertook an analysis of the entire Igh locus using 3C based methodology in combination with next generation sequencing technologies. This has permitted us to systematically characterize three dimensional (3D) chromatin organization on several genomic scales. We have found that the Igh locus is compartmentalized into two unique sub-topological domains separated by a relatively unstructured region. Comparison of non-lymphoid MEF cells and pro-B lymphocytes has revealed a set of very-long range looping interactions that serve to bridge the sub- topological domains and are both unique to pro-B cells and Pax5 dependent. Thus, we have identified the Pax5 dependent looping interactions, termed sites I, II, II.5 and III, responsible for Igh locus contraction that serves to create spatial proximity between the rearranged DJH joins and distal VH genes. These findings have implications for IgH repertoire formation under normal conditions and in pathological disease states. We propose to fully characterize looping interactions involving Site I and then to use this information to construct mice in which Site I has been deleted or mutated. The consequences of targeted deletion of Site I will be fully explored using 3C chromatin looping assays, 3D FISH, and analysis of B cell development and VH gene usage during V(D)J joining. These studies will form the basis for new insights regarding development of humoral immunity.