The RAG1/RAG2 endonuclease catalyzes assembly of antigen receptor genes in developing G1 phase lymphocytes through the induction of DNA double-strand breaks (DSBs), which are repaired by non- homologous end-joining (NHEJ) proteins. DSB repair defects and inability to coordinate DSB repair with cell survival and cell cycle progression result in genomic instability that can cause leukemias and lymphomas. The RAG proteins each consist of "core" endonuclease regions, and "non-core" regions required for normal assembly of lymphocyte antigen receptors and normal early lymphocyte development. RAG1 non-core regions include protein domains that ubiquitylate histones and interact with the additional ubuitylating enzymes to prevent aberrant deletion and/or insertion of nucleotides during repair of RAG-mediated DSBs. These phenotypes may result from direct defects in DSB repair in G1 phase cells and/or an inability to coordinate repair with cell survival and cell cycle progression. I have found that mice expressing truncated "core" Rag1 proteins (Rag1C/C mice) exhibit impaired late B cell development and that this defect is tied to decreased transcription of antigen receptor genes and failure to induce survival signals following RAG-mediated DSBs. I have observed that Rag1C/C pre-B cells show loss of coding ends following Rag-generated DSBs, suggesting that non-core regions of Rag1 may have a role in preventing aberrant resection of these DNA ends. Together, these data form the basis of my hypothesis that, in addition to serving with RAG2 as the V(D)J endonuclease, RAG1 functions as a chromatin-modifying enzyme to promote normal DSB repair and as a signaling molecule to coordinate DSB repair with cell survival and cell cycle progression. To test this, I will confirm that non-cor Rag1 regions protect RAG-generated DNA coding ends from aberrant resection in G1 cells by analyzing recombination of chromosomally-integrated substrates in wild-type and Rag1C/C pre-B cells. Further, I will test the ability of non-core Rag1 regions to facilitate DNA repair via promoting H2AX phosphorylation and H2AX, H2A, and H2B ubiquitylation following RAG DSBs. To test the ability of non-core Rag1 to coordinate RAG DSB repair with cell survival and cell cycle regulation, I will first determine how non-core Rag1 regions promote NF?B-dependent transcriptional activation of the pro-survival Pim2 kinase in response to RAG DSBs. I will use retroviruses to investigate if Rag activates NF?B via ubiquitylation of NEMO. I will then investigate if non-core Rag1 regions promote NF?B-dependent activation of the CDK2-AP1 cell cycle checkpoint protein to prevent unrepaired RAG DSBs from entering the cell cycle by knocking down and over-expressing CDK2-AP1. Successful completion of these studies should demonstrate novel functions of Rag1 as a histone-modifying factor and signaling molecule that promotes cell survival and cell cycle regulation, which will further our understanding of how developing lymphocytes integrate DNA repair with cell survival and cell cycle progression to yield a protective adaptive immune system and prevent formation of translocations and tumors.
The aims outlined in this application will elucidate precise molecular mechanisms and signaling pathways for DNA repair in lymphocytes as they assemble antigen receptor genes. Following antigen receptor gene rearrangements, all developing lymphocytes must integrate signals involved in DNA repair, cell cycle control, and cell survival so that they can produce antigen receptors that protect host organisms from allergens and pathogens and do not develop oncogenic antigen receptor locus translocations that cause lymphoid cancers. I therefore propose to study these mechanisms, as they have broad applicability to human health and the prevention of immunodeficiency, autoimmunity and lymphomas.
|Majumder, Kinjal; Koues, Olivia I; Chan, Elizabeth A W et al. (2015) Lineage-specific compaction of Tcrb requires a chromatin barrier to protect the function of a long-range tethering element. J Exp Med 212:107-20|