Based on a mutational search of the mouse IgH locus, we have implicated the presence of two types of V(D)J recombinational control elements that depend on the integrity of the 100kb intergenic region between the germline IgH VH and DH clusters. One suppresses anti-sense transcription from the downstream DH locus, as deletion of the 100kb VH-D region leads to de novo DH anti-sense transcription that produces long non-coding transcripts in developing B and T cells. This increased transcription is associated with greatly increased D to JH joining in thymocytes implicating a positive role for anti-sense transcription in targeting V(D)J recombination. The second VH-D integenic control element, which we term """"""""5'D4KBS"""""""", is contained with a 4kb sequence just upstream of D cluster. Remarkably, this element mediates ordered IgH variable region exon assembly in B cells by suppressing VH joining to Ds that have not joined to JHs and mediates lineage-specific joining by suppressing VH to DJH joining in thymocytes. The 5'D4KBS also enhances utilization of VHs located 2MB distant and therefore influences primary antibody repertoires. We have found that the 5'D4KBS functionally employs a CTCF looping/insulator factor binding site, at least for its lineage-specific function. We propose 3 specific aims designed to determine how these elements function in mice and in humans and, based on these studies, to identify additional regulatory elements.
Our first aim i s to characterize how the 5'D4KBS controls ordered and lineage specific V(D)J recombination and normalizes VH usage. These studies involve an in depth genetic analysis of the functions of individual factor binding sites within 5'D4KBS. In this regard, our preliminary studies implicate sites that bind CTCF, at least in lineage-specific control. We also will assess how assembly of a DJH intermediate during the V(D)J assembly process inactivates apparently suppressive influences of the 5'D4KBS on VH to DJH joining.
Our second aim seeks to genetically scan the 100kb VH-D intergenic region to locate elements implicated in controlling transcription of the downstream D and JH segments and to test physiological roles for such transcription. Based on the apparent absence of IgH allelic exclusion control elements from the VH-D intergenic region, we also propose to search for such elements elsewhere in the mouse IgH locus.
Our final aim proposes to characterize V(D)J recombination control in mice that have human VH, D, and JH segments in place of the corresponding mouse sequences. These studies will allow us to test the significance of our findings (e.g. the role of VH-D intergenic elements) in the context of whether they are evolutionarily conserved and provide a means to begin an in depth evaluation of the genetic and epigenetic mechanisms involved in generating the human antibody repertoire.)

Public Health Relevance

Unlike most of our genes, antibody genes are assembled from gene segments to allow the generation of B lymphocytes that can produce a vast diversity of different antibodies. Our studies are aimed at discovering how this antibody gene assembly process is carried out and how it is regulated. Knowledge of antibody gene assembly mechanisms will lead to a better understanding of how the diverse sets of antibodies are generated to fight a multitude of different infections and also, of how mistakes in this gene assembly process can predispose to diseases such as immunodeficiency, autoimmunity, and cancer.)

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
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Cellular and Molecular Immunology - B Study Section (CMIB)
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Nasseri, M Faraz
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Children's Hospital Boston
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Lin, Sherry G; Ba, Zhaoqing; Alt, Frederick W et al. (2018) RAG Chromatin Scanning During V(D)J Recombination and Chromatin Loop Extrusion are Related Processes. Adv Immunol 139:93-135
Ru, Heng; Mi, Wei; Zhang, Pengfei et al. (2018) DNA melting initiates the RAG catalytic pathway. Nat Struct Mol Biol 25:732-742
Jain, Suvi; Ba, Zhaoqing; Zhang, Yu et al. (2018) CTCF-Binding Elements Mediate Accessibility of RAG Substrates During Chromatin Scanning. Cell 174:102-116.e14
Chen, Liang; Zhao, Lijuan; Alt, Frederick W et al. (2016) An Ectopic CTCF Binding Element Inhibits Tcrd Rearrangement by Limiting Contact between V? and D? Gene Segments. J Immunol 197:3188-3197
Hu, Jiazhi; Meyers, Robin M; Dong, Junchao et al. (2016) Detecting DNA double-stranded breaks in mammalian genomes by linear amplification-mediated high-throughput genome-wide translocation sequencing. Nat Protoc 11:853-71
Kumar, Vipul; Alt, Frederick W; Frock, Richard L (2016) PAXX and XLF DNA repair factors are functionally redundant in joining DNA breaks in a G1-arrested progenitor B-cell line. Proc Natl Acad Sci U S A 113:10619-24
Zhao, Lijuan; Frock, Richard L; Du, Zhou et al. (2016) Orientation-specific RAG activity in chromosomal loop domains contributes to Tcrd V(D)J recombination during T cell development. J Exp Med 213:1921-36
Lin, Sherry G; Ba, Zhaoqing; Du, Zhou et al. (2016) Highly sensitive and unbiased approach for elucidating antibody repertoires. Proc Natl Acad Sci U S A 113:7846-51
Tian, Ming; Cheng, Cheng; Chen, Xuejun et al. (2016) Induction of HIV Neutralizing Antibody Lineages in Mice with Diverse Precursor Repertoires. Cell 166:1471-1484.e18
Lin, Sherry G; Guo, Chunguang; Su, Arthur et al. (2015) CTCF-binding elements 1 and 2 in the Igh intergenic control region cooperatively regulate V(D)J recombination. Proc Natl Acad Sci U S A 112:1815-20

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