Immunoglobulin (Ig) and T cell antigen receptor variable region genes are assembled from V, D, and J segments in developing B and T lymphocytes. These gene segments reside in large and complex chromosomal loci;yet their assembly is tightly regulated in the context of lineage and developmental stage. Also, rearrangements are feedback regulated to ensure generation of one antigen receptor gene for a given locus (allelic exclusion). Such control maintains specificity of the immune response. To effect regulation, multiple cis elements interact to modulate access of a common V(D)J recombinase to the proper substrate gene segments, a process we named """"""""accessibility"""""""" control. Accessibility control, as related to transcription, replication, and repair, is a broadly relevant process;and V(D)J recombination control has provided a most important paradigm. Yet, specific mechanisms remain elusive. Over the past 5 years, we used gene- targeted mutation to """"""""tailor"""""""" endogenous TCR(3 and IgH loci in mice to make amenable experimental systems. Specifically, we mutated various cis-regulatory sequences that alter normal rearrangement patterns with respect to order and, potentially, feedback. Proposed analyses of deregulated recombination events in these and other mutants will provide a basis for elucidating normal mechanisms. In addition, we developed a novel approach to measure """"""""accessibility"""""""", which we will employ to elucidate how accessibility controls endogenous Ig gene assembly. Finally, we have collaborated with Regeneron Pharmaceuticals to use the novel VelociGene method to introduce major modifications over the 3MB endogenous Ig heavy chain locus. This approach has provided alleles that will serve as minimal locus substrates for studies of V(D)J recombination control. With the rapid accumulation of new information and technologies, we argue that the novel endogenous antigen receptor loci that we have generated leave us poised to generate important insights into variable region gene assembly that could not be readily gained by other approaches. Besides relevance to fundamental mechanisms of developmental biology, elucidation of V(D)J recombinational control has substantial implications for health and disease. Thus, the mechanisms we study are required for development of normal antibody and TCR repertoires. Correspondingly, defects in V(D)J recombination underlie immunological diseases ranging from immunodeficiency to autoimmunity. Finally, aberrant V(D)J recombination can unleash oncogenic activities via chromosomal translocations Lay Summary: We have tailored the chromosomes of mice to introduce major changes in genes that encode antibodies. Our studies of altered antibody genes will allow us to understand how the body responds to infections and how mistakes in formation of antibody genes lead to immunodeficiency, autoimmunity and cancer. These studies also may provide insights relevant to generation of antibodies as therapeutic agents for disease.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI020047-28
Application #
7772241
Study Section
Cellular and Molecular Immunology - B Study Section (CMIB)
Program Officer
Nasseri, M Faraz
Project Start
1983-04-01
Project End
2011-02-28
Budget Start
2010-03-01
Budget End
2011-02-28
Support Year
28
Fiscal Year
2010
Total Cost
$601,803
Indirect Cost
Name
Immune Disease Institute, Inc.
Department
Type
DUNS #
059709394
City
Boston
State
MA
Country
United States
Zip Code
02115
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
Hu, Jiazhi; Zhang, Yu; Zhao, Lijuan et al. (2015) Chromosomal Loop Domains Direct the Recombination of Antigen Receptor Genes. Cell 163:947-59

Showing the most recent 10 out of 159 publications