Assembly of lymphocyte antigen receptor genes from variable (V), diversity (D), and joining (J) gene segments by the RAG1/RAG2 (RAG) nuclease is vital for adaptive immunity. However, this process also confers risk as evidenced by assembly of auto-reactive receptors and by lymphoid cancers with oncogenic translocations involving immunoglobulin (Ig) or T cell receptor (TCR) loci. While studies of Ig/TCR assembly have focused on how RAG cleavage is initiated, RAG activity also must be restrained to limit DNA double strand breaks (DSBs) and resultant genomic instability. It has been recognized for 34 years that complete assembly of most Ig/TCR genes occurs on one allele at a time, enforced by a process called allelic exclusion that is conserved among all organisms with adaptive immunity. Two facets of this fundamental control mechanism are asynchronous initiation of V rearrangements between alleles and Ig/TCR-mediated feedback to permanently block further V recombination after a productive join is formed. In addition, it was proposed in 1980 that V recombination must activate more immediate signals to transiently block V recombination, providing the time necessary for coding joins to be repaired, expressed, and then signal feedback inhibition. However, evidence for this long-predicted transient aspect of regulation has been lacking. The applicant's lab recently showed that RAG DSBs induced during V?-to-J? recombination signal via the Ataxia Telangiectasia mutated (ATM) protein kinase to transiently inhibit additional V? rearrangements. They demonstrated that ATM down-regulates RAG1 and RAG2 mRNA and protein levels in response to RAG DSBs, and helps enforce mono-allelic Ig? expression. Based on their findings, the applicant hypothesizes that RAG and other types of DSBs transiently suppress V rearrangements via multiple independent but complementary mechanisms to safeguard from oncogenic Ig translocations and ensure mono-specificity of Ig-bearing B lymphocytes. These mechanisms are hypothesized to create a failsafe regulatory strategy that includes the suppression of: 1) RAG expression by DSB response pathways that are ATM-dependent but restricted to developing B lymphocytes, 2) recombination potential in trans at the second non-cleaved Ig allele, and 3) sequential recombination in cis on the cleaved Ig allele. The applicant proposes to dissect underlying mechanisms for these ATM-dependent restrictions of V(D)J recombination and determine their independent contributions to enforcement of mono-allelic Ig expression, suppression of Ig translocations, and shaping Ig repertoire. The knowledge acquired from this research will define molecules and pathways that protect us from lymphoid cancers, as well as production of B cells with multiple antigen specificities, resulting in autoimmunity. In the long-term, such knowledge will foster the development of novel prognostics, diagnostics, and therapeutics for specific human immunological disorders.
The proposed studies will provide novel insights and greater understanding of molecular mechanisms through which antigen receptor gene assembly is controlled between and along alleles to suppress translocations while ensuring proper assembly and selection of antigen receptor genes. Knowledge acquired from this project will lead to increased understanding of how defects in V(D)J recombination control cause lymphoid malignancies, immunodeficiency, and autoimmunity. In the long-term, such knowledge should identify novel diagnostic and therapeutic targets for humans afflicted with these immunologic disorders.
