The main goal of this work is to understand the basic mechanisms underlying the developmental regulation of V(D)J recombination. Developmental regulation is thought to depend in part on regulated expression of the RAG1/RAG2 recombinase, and in part on changes in T cell receptor (TCR) locus chromatin structure and organization that allow access to and synapsis of recombination signal sequences. The specific focus of this project is the murine TCRalpha/delta locus, which undergoes recombination according to distinct developmental programs during the double negative (DN) and double positive (DP) stages of thymocyte development.
One aim will be to more fully characterize how local changes in chromatin structure are established and how such changes contribute to the recombination of Vdelta gene segments in DN thymocytes and Jalpha gene segments in DP thymocytes. This will be accomplished through detailed mapping of chromatin disruption, RAG protein binding and V(D)J recombination on genetically manipulated alleles.
A second aim will be to evaluate the hypothesis that developmentally regulated changes in locus conformation are essential to support the developmental program. Developmental changes in locus conformation will be mapped by three-dimensional fluorescence in situ hybridization (3D-FISH) and chromosome conformation capture (3C). Genetically manipulated strains of mice will be used to test the roles of TCR locus enhancers and the roles of proteins CTCF and cohesin in defining locus conformational states. To formally test the significance of locus conformation, the TCRalpha/delta locus will genetically modified to permit trapping of a contracted conformation.
A third aim will be to elucidate the mechanism by which the architectural protein SATB1 regulates recombinase expression in DP thymocytes. The hypothesis to be tested is that SATB1 is required for long distance interactions in the RAG locus that are critical for transcription of the RAG genes.

Public Health Relevance

V(D)J recombination is essential for the development of a functional immune system. Failure of V(D)J recombination can cause immunodeficiency and errors in V(D)J recombination can result in chromosomal translocations that contribute to tumorigenesis. These studies will elucidate basic mechanisms that contribute to normal immune system development and pathologies such as immunodeficiency and cancer.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37GM041052-25
Application #
8515439
Study Section
Special Emphasis Panel (NSS)
Program Officer
Marino, Pamela
Project Start
1997-07-01
Project End
2016-07-31
Budget Start
2013-08-01
Budget End
2014-07-31
Support Year
25
Fiscal Year
2013
Total Cost
$511,320
Indirect Cost
$182,670
Name
Duke University
Department
Neurosciences
Type
Schools of Medicine
DUNS #
044387793
City
Durham
State
NC
Country
United States
Zip Code
27705
Shih, Han-Yu; Krangel, Michael S (2013) Chromatin architecture, CCCTC-binding factor, and V(D)J recombination: managing long-distance relationships at antigen receptor loci. J Immunol 190:4915-21
Seitan, Vlad C; Krangel, Michael S; Merkenschlager, Matthias (2012) Cohesin, CTCF and lymphocyte antigen receptor locus rearrangement. Trends Immunol 33:153-9
Hao, Bingtao; Krangel, Michael S (2011) Long-distance regulation of fetal V(?) gene segment TRDV4 by the Tcrd enhancer. J Immunol 187:2484-91
Shih, Han-Yu; Hao, Bingtao; Krangel, Michael S (2011) Orchestrating T-cell receptor ? gene assembly through changes in chromatin structure and organization. Immunol Res 49:192-201
Seitan, Vlad C; Hao, Bingtao; Tachibana-Konwalski, Kikue et al. (2011) A role for cohesin in T-cell-receptor rearrangement and thymocyte differentiation. Nature 476:467-71
Kondilis-Mangum, Hrisavgi D; Cobb, Robin Milley; Osipovich, Oleg et al. (2010) Transcription-dependent mobilization of nucleosomes at accessible TCR gene segments in vivo. J Immunol 184:6970-7
Ji, Yanhong; Little, Alicia J; Banerjee, Joydeep K et al. (2010) Promoters, enhancers, and transcription target RAG1 binding during V(D)J recombination. J Exp Med 207:2809-16
Shih, Han-Yu; Krangel, Michael S (2010) Distinct contracted conformations of the Tcra/Tcrd locus during Tcra and Tcrd recombination. J Exp Med 207:1835-41
Krangel, Michael S (2009) Mechanics of T cell receptor gene rearrangement. Curr Opin Immunol 21:133-9
Vazquez, Berta N; Laguna, Teresa; Carabana, Juan et al. (2009) CD69 gene is differentially regulated in T and B cells by evolutionarily conserved promoter-distal elements. J Immunol 183:6513-21

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