Impact of novel enhancers on Igh repertoire diversity

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ABSTRACT Antigen receptor genes are assembled from several gene segments via V(D)J rearrangement during early lymphoid cell development to generate a diverse repertoire of antibodies. 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. VH genes are dispersed through 2.5 Mb of the Igh locus. Locus compaction 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. 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 a topologically associated domain (TAD) that is partitioned into three sub-domains. A set of pro-B cell- specific very-long range looping interactions bridge the sub-domains and are Pax5-dependent. These looping interactions are anchored at Sites I, II, II.5 and III and which are critical facilitators of Igh locus contraction. We have now defined the DNA motifs in these loop anchors and discovered a series of pro-B cell specific novel enhancers (NEs) that participate in a NE-NE-VH gene promoter interactome. We have systematically characterized locus compaction using specific KO mice in combination with chromatin-loop mapping methods and newly constructed NE1 KO mice and cell lines. To begin to understand NE interactome function we asked whether those NEs engaged in E-E and E-Pr looping are in an active state as defined by the H3K27Ac histone marks in single cells. We discovered that the NEs are marked by remarkably large H3K27Ac foci. Here we will 1) systematically characterize NEs individually and in the NE interactome as it relates to VH gene usage during repertoire formation, and 2) examine the relationship between the NE interactome and H3K27Ac foci. The presence of H3K27Ac foci on NEs may indicate the participation of the Igh locus in transcriptional condensates which may define the molecular environment for V(D)J recombination.