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.

Public Health Relevance

B lymphocytes undergo two distinct programmed gene rearrangements affecting immunoglobulin (Ig) genes. The first is termed VDJ joining and occurs early in B cell development while the second is called class switch recombination and occurs in mature B cells. Here we examine the spatial organization of the chromatin landscape across the IgH gene locus to determine unique chromosomal structure/function relationships and to understand how these collaborate to facilitate the use of the distal VH genes and provide for the full complement of antibody gene rearrangements. These studies are key for understanding how antibodies are formed during normal and disease processes.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Exploratory/Developmental Grants (R21)
Project #
5R21AI117687-02
Application #
9026565
Study Section
Cellular and Molecular Immunology - B Study Section (CMIB)
Program Officer
Nasseri, M Faraz
Project Start
2015-03-15
Project End
2017-02-28
Budget Start
2016-03-01
Budget End
2017-02-28
Support Year
2
Fiscal Year
2016
Total Cost
Indirect Cost
Name
University of Illinois at Chicago
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
098987217
City
Chicago
State
IL
Country
United States
Zip Code
60612
Gürsoy, Gamze; Xu, Yun; Kenter, Amy L et al. (2017) Computational construction of 3D chromatin ensembles and prediction of functional interactions of alpha-globin locus from 5C data. Nucleic Acids Res 45:11547-11558
Kenter, Amy L; Kumar, Satyendra; Wuerffel, Robert et al. (2016) AID hits the jackpot when missing the target. Curr Opin Immunol 39:96-102
Montefiori, Lindsey; Wuerffel, Robert; Roqueiro, Damian et al. (2016) Extremely Long-Range Chromatin Loops Link Topological Domains to Facilitate a Diverse Antibody Repertoire. Cell Rep 14:896-906