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 multiple DNA double strand breaks (DSBs) and resultant genomic instability. It has been known for 30 years that complete assembly of most Ig/TCR genes occurs on one allele at a time, indicating fundamental importance of mechanisms that control V(D)J recombination between alleles. Asynchronous initiation of V rearrangements, such that each allele recombines one at a time, and Ig/TCR-mediated feedback inhibition of further V rearrangements to maintain allelic exclusion are two important facets of this control. It was proposed in 1980 that V recombination also must activate more immediate signals that transiently prevent V rearrangements, providing time for assembled genes to be expressed and signal Ig/TCR-mediated feedback inhibition. However, evidence for this additional long-predicted facet of V(D)J recombination control has been lacking. The applicant's lab recently showed that RAG DSBs induced during Ig? recombination signal through the Ataxia Telangiectasia mutated (ATM) kinase to transiently inhibit additional Ig? rearrangements. They demonstrated that ATM both inhibits Rag1 and Rag2 mRNA and protein levels in response to RAG DSBs and enforces mono-allelic Ig? expression. Based on their discoveries, the applicant hypothesizes that RAG DSBs transiently inhibit additional V recombination through multiple independent mechanisms to safeguard against oncogenic Ig translocations and ensure monospecificity of Ig-bearing B cells. These complementary mechanisms are hypothesized to generate a failsafe regulatory strategy that includes suppression of: 1) nuclear RAG expression by DSB response pathways that are ATM-dependent but restricted to developing B cell precursors, 2) recombination in trans at other Ig alleles, and 3) sequential rearrangements in cis on the cleaved Ig allele. The applicant proposes to dissect the underlying molecular mechanisms for these ATM-dependent restrictions and determine their independent contributions to enforcement of mono-allelic Ig expression, suppression of Ig translocations, and shaping Ig repertoire. Knowledge acquired from this project will define molecules and pathways that protect us from lymphoid cancers and production of B lymphocytes with multiple antigen specificities, resulting in autoimmunity. In the long-term, such knowledge will foster the development of novel prognostics 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.
