V(D)J recombination, a programmed DNA rearrangement process for assembling antigen receptors during the development of lymphocytes, poses a potential threat to genomic stability. The long-term objective is to understand the mechanism of cell cycle control of V(D)J recombination and suppression of tumorigenesis in developing lymphocytes. The key hypothesis is that the gene mutated in ataxia telangiectasia (ATM) plays a key role in cell cycle control of V(D)J recombination, lack of this control may directly lead to chromosomal translocations and lymphoma. The hypothesis is based on 1) ATM is physically located at the V(D)J recombination sites, 2) ATM-/- mice succumb to early thymic lymphomas with chromosomal translocations and 3) eliminating V(D)J recombination eradicates chromosomal translocation in ATM-/- mice and delays the onset of lymphoma.
Our specific aims are: 1) to establish the role of ATM in containing V(D)J recombination related DNA breaks within the G07G1 phase of the cell cycle. We will use specialized assay for V(D)J specific DNA breaks in cells at different cell cycle status, purified from wild-type and ATM-/- mice, and during T and B lymphocyte development;2) to understand how ATM functions in containing DNA breaks during V(D)J recombination, we will compare regulation of Rag nuclease, and histone H2AX phosphorylation in wild-type and ATM-/- mice, both factors are key regulators in generating, containing and/or efficient joining of DNA breaks: 3) to understand the tumorigenesis in ATM deficient mice, we will fully characterize ATM-/- lymphomas, to establish cytogenetic profiles, translocation junctions and to determine the potential oncogenes affected;We will also set to establish a mouse embryonic stem cell ESC) culture, to induce differentiation into T and B lymphocytes, to characterize V(D)j recombination, to compare results from wild-type and ATM-ESC, and to determine how DNA breaks lead to translocation in ATM-/-, and to adoptive transfer both T and B cells into immunodeficient host mice, to examine the tumorigenic potential. Relevance: V(D)J recombination, if not properly controlled, may lead to develop of lymphoma. The study is designed to understand how genome instability occurs, and how this instability leads to tumor, and to understand how ATM functions in maintaining genome stability and suppressing tumorigenesis.
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