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. However, little is known about the precise looping structure of the Igh locus that leads to locus contraction. Although a small subset of loops have been discerned for the Igh locus, an unbiased examination of locus looping has been unavailable. We, therefore, undertook an analysis of the entire Igh locus using chromosome conformation capture (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. 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. We find that the Igh locus is compartmentalized into two unique sub-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 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 addition, we provide new epigenetic and chromatin studies that identify a novel Igh enhancer that interacts with Site I and may play a critical role in locus compaction and/or VH gene expression. Examination of the loop- anchor sites we identified has led to the recognition that locus compaction may be mediated by specific VH promoter-novel enhancer interactions and transcriptionally active regions. We also postulate the non-mutually exclusive proposition that the 3D structure of the Igh locus will influence individual VH gene rearrangement frequencies, favoring VH genes that are brought into close proximity with the rearranged DHJH segments. We propose to systematically characterize locus compaction using specific KO mice in combination with chromatin-loop mapping methods. We will construct pro-B cell lines and mice in which specific sites have been deleted or mutated. The consequences of targeted deletion of loop-anchor sites 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 a diverse antibody repertoire.
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 the chromosomal architectural infrastructure that spatially orients 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.
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