Chromatin infrastructure of the Igh locus and its influence on VH gene usage

Project: Research projectResearch Project

Description

ABSTRACT
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.
StatusActive
Effective start/end date12/15/1511/30/20

Funding

  • National Institutes of Health: $866,303.00

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Chromatin
B-Lymphoid Precursor Cells
Genes
VDJ Exons
B-Lymphocytes
Recombinases
Gene Rearrangement
Gene Frequency
Bone Marrow
Gene Expression
Cell Line
Antibodies

Keywords

  • Medicine(all)
  • Immunology and Microbiology(all)