Non-Canonical Roles for RAG1 in Lymphocyte Development

The RAG1/RAG2 (RAG) endonuclease recombines accessible antigen receptor (AgR) genes through DNA double strand break (DSB) intermediates to generate a diverse AgR repertoire. RAG-mediated DSBs signal changes in expression of genes encoding proteins involved in cellular survival, lymphocyte differentiation, and AgR selection. RAG proteins are each comprised of "core" endonuclease domains and dispensable "non-core" regions. Humans with mutations in non-core RAG1 regions exhibit fatal primary immunodeficiencies, and mice expressing truncated core, but not full-length, Rag1 protein (Rag1C/C mice) exhibit impaired early lymphocyte development associated with reduced levels of AgR gene rearrangements. In addition to serving with RAG2 as the V(D)J endonuclease, the RAG1 protein has been proposed to utilize non-core regions to regulate V(D)J recombination by enhancing AgR locus accessibility, promoting efficient RAG endonuclease activity, and/or generating RAG DSB-induced survival and differentiation signals. The experiments described in this thesis use a variety of mouse genetic models to investigate how mutations in RAG1 protein that preserve RAG endonuclease activity impair V(D)J recombination efficiency and lymphocyte development. First, these studies have identified roles for non-core Rag1 regions in regulating normal Alpha/Beta T cell development and TCRB recombination by promoting VB rearrangements and diverse usage of VB gene segments in both primary and secondary VB rearrangements. Second, these studies show that non-core Rag1 regulates normal B cell development by inducing transcriptional activation of the pro-survival kinase Pim2 in response to RAG cleavage and by promoting the survival of developing IgKappa+ and IgLambda+ B cells. These studies have also identified roles for Rag1 in enhancing IgKappa and IgLambda locus accessibility in pre-B cells prior to RAG cleavage. Collectively, the data presented in this thesis demonstrate that RAG1 has critical functions outside of V(D)J recombination that enhance AgR gene segment accessibility, promote V(D)J recombination at multiple AgR loci, and transduce pro-survival signals during AgR recombination to establish a broad AgR repertoire and thereby foster normal lymphocyte development. Degree Type Dissertation Degree Name Doctor of Philosophy (PhD) Graduate Group Immunology First Advisor Craig H. Bassing