The goal of the research proposed in this application is to explore the mechanisms that underpin antigen receptor gene rearrangement. The antigen receptor loci are organized in distinct domains that contain multiple variable (VH), diversity (DH), joining (JH) and constant (CH) coding elements. To probe the topography of the Igh locus, we as well as others, have determined the spatial distances separating genomic markers that span the entire locus. The spatial distance measurements were compared to computer simulations of distinct chromatin topologies. These studies predicted that the Igh locus is structured into domains consisting of clusters of loops. Using computational geometric approaches we showed that the Igh locus is organized as individual domains that merge upon commitment to the B cell fate. More recent observations have identified potential anchors, proposed to sequester the proximal variable coding elements to lie in orbit at the base of a rosette, surrounding the DHJH coding element complexed to the recombinase. Taken together, these studies have suggested that during early B cell development, the entire repertoire of VH regions (2.5 Mbp) is merged and juxtaposed to the DH elements, allowing the VH regions to encounter DHJH coding elements with relatively high and similar frequencies. Here we propose to continue these studies and determine the spectrum of 3D-conformations of the Igh locus adopted by the Igh locus fiber during the developmental progression of B cell progenitors. We would describe at high resolution the trajectories adopted by the Igh locus in interphase chromatin. We would examine how the topology of the Igh locus fiber is regulated during developmental progression. We would examine how VH regions separated by large genomic distances have equal opportunities to encounter DHJH elements in developing B cell progenitors. In summary, these studies would provide mechanistic insight into how antibody diversity is generated.
Recent studies have demonstrated that the antigen receptor loci are organized as clusters of loops. The studies proposed in this application are aimed to provide insight into how genome topology allows the generation of a diverse antigen receptor repertoire in order to mount an efficient and rapid immune response to invading pathogens.
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