Immunoglobulin (Ig) heavy chain locus (IgH) VH, D, and JH segments are clustered over megabase (Mb) regions of the mouse IgH locus. Their assembly via V(D)J recombination is regulated in multiple contexts, including ordered D to JH joining prior to VH to DJH joining, with the latter step regulated to promote utilization of proximal and distal VHs to promote diverse repertoires. The IGCR1 control region in VH to D inter-genic region mediates V(D)J recombination control via two CTCF-binding elements (CBEs) proposed to mediate regulatory chromatin loops. To initiate V(D)J recombination, the RAG1/2 endonuclease (RAG) cleaves V(D)J gene segments flanked by complementary recombination signal sequence (RSS) pairs and mediates their subsequent joining. RAG can directionally track across Mb chromosomal loops, which could allow it to locate proper RSS pairs for cleavage activities. RAG tracking was discovered with our new LAM-HTGTS technique, which is so sensitive that it reveals multitudes of low frequency RAG-initiated cleavage/joining events invisible to prior assays. We have adapted the LAM-HTGTS for V(D)J repertoire sequencing (?HTGTS-Rep-seq?). We propose four specific aims designed to build on these findings and new technologies to elucidate mechanisms that promote joining of IgH Vs, Ds, and Js across Mb distances to generate diverse antibody repertoires. We also propose to extend these studies to other mouse Ig loci and to human antigen receptor loci. Many proposed studies will employ G1-arrested, RAG-inducible v-Abl transformed pro-B cell lines as a test system, but key results will be confirmed or extended by studies of normal B lineage cells.
Aim 1 proposes to elucidate the mechanism and functional consequences of RAG tracking by using a large c-Myc locus loop domain as a test system to obviate potential confounding effects of numerous RSSs and other regulatory elements in antigen receptor loci. Proposed studies will evaluate contribution of RAG tracking versus other mechanisms for capture of distant RSS pairs and elucidate factors that propel RAG tracking.
Aim 2 proposes to elucidate mechanisms that regulate IgH D to JH recombination. In IgH, Ds are located upstream of JHs within an IGCR1- dependent domain that isolates them from upstream Vs. In this domain, D to JH recombination initiates from a RAG-bound recombination center (RC) comprising JHs and the most proximal D (DQ52). We will test the hypothesis that RAG is activated in the RC by pairing of DQ52 and JH RSSs, with convergent 12RSSs of other Ds then captured by other mechanisms, potentially including tracking. We also propose to define cis elements required for RC formation.
Aim 3 proposes to elucidate mechanisms that bring distant VHs into V(D)J recombination domains, including unique aspects of IgH CBE organization across the IgH locus and potential roles of VH-associated CBEs.
This aim also proposes to extend studies to the Ig? light chain locus.
Aim 4 proposes to extend Aim 2 and 3 studies to human antigen receptor loci, which is important because V(D)J recombination control mechanisms of human Ig or TCR loci have not been studied directly.

Public Health Relevance

Unlike most of our genes, antibody genes are assembled from gene segments to allow the generation of B lymphocytes that 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, both in mice and in humans. 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 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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Liu, Qian
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Boston Children's Hospital
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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
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
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
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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