Biochemical reconstitution of antigen receptor gene regulation

National Institute On Aging

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Abstract

Mutagenic activity of the V(D)J recombinase is controlled by regulated access to target antigen receptor loci and generation of double-strand DNA breaks only after joint recognition of paired recombination signal sequences (RSS). We have recapitulated both key aspects of recombinase regulation in a cell-free system using plasmid substrates assembled into chromatin. During the 2009 fiscal year, we accomplished the following: 1. Generated VDJ recombination substrates under control of Tet0 regulatory sequences. These plasmids will be used to recruit histone modifying enzymes to the substrates. 2. Demonstrated that two new components of the Fanconia anemia complex interact with mononucleosomes. These observations provide a plausible model for the recruitment of Fanconia complex to sites of DNA breakage. (Collaboration with the laboratory of Dr. Weidong Wang, LG, NIA/IRP.) 3. Constructed expression vectors for recruitment of chromatin remodeling and histone modifying enzymes to Tet0 and Gal4 binding sites. These vectors fuse Tet repressor or Gal4 DNA binding domains to various chromatin modifying enzymes. 4. Initiated collaboration with Dr. Michael Lieber's laboratory to study the role of H3K4me3 in altering RAG activity in vitro. Continued collaboration with Dr. Steve Desiderios laboratory to use modified histone templates in VDJ recombination assays in vitro.