The diverse repertoire of antigen receptors is initiated during lymphocyte development by V(D)J recombination. This genetic mechanism rearranges germline antigen receptor loci by joining component gene segments, which together encode for the antigen specificity of the receptor. The initial site-specific DNA cleavage steps are catalyzed by the recombination activating gene proteins, RAG1 and RAG2. Together the RAG proteins introduce DNA double strand breaks at sequence-specific sites within the antigen receptor loci. Subsequently, non-homologous end joining factors join the appropriate DNA ends together to yield functional antigen receptor genes. While the RAG proteins can erroneously cleave at off-target sites, their activity is typically restricted to the antigen receptor loci. Yet, the RAG proteins are also known to bind to modified histone proteins that are enriched at open chromatin sites, placing them in close proximity to numerous non-antigen receptor DNA sites. These counterintuitive results highlight that much is still unknown regarding how the RAG proteins are regulated in developing lymphocytes. Understanding regulation of the RAG proteins will be facilitated by single cell analysis of RAG protein-chromatin interactions that occur throughout the nucleus relative to their interactions at specific DNA recognition sites in the antigen receptor loci. However, the tools to visualize single complexes containing RAG-bound histones and, in particular, specific DNA sites with bound RAG proteins are lacking. Our goal in this project is to develop new methods to measure RAG-chromatin interactions in cell nuclei and to visualize localized RAG interactions at specific genomic DNA sites in single cells. Development and optimization of these methods will be important in addressing questions related to the regulation of V(D)J recombination, and how defects in this process can lead to disease.
Functional immunoglobulin and T cell receptor genes are generated during lymphocyte development by V(D)J recombination, a cut-and-paste DNA rearrangement mechanism. The lymphoid-specific proteins, RAG1 and RAG2, create the initial DNA double strand breaks in V(D)J recombination at specific DNA sequences. Given that the RAG proteins can cleave at incorrect sites and lead to genomic instability, it is essential to investigate the interaction of RAG proteins with genomic DNA in lymphocyte nuclei. In this project, we will develop new methods to visualize RAG protein complexes within the nucleus and their interaction at specific genomic sites, with the goal that these methods will facilitate research on the activity and regulation of V(D)J recombination.
| Rodgers, Karla K (2017) Riches in RAGs: Revealing the V(D)J Recombinase through High-Resolution Structures. Trends Biochem Sci 42:72-84 |
