The goal of this application is to elucidate mechanisms by which developing lymphocytes coordinate DNA double strand break (DSB) repair and cell cycle progression. Proper coordination of these processes is required for maintenance of cellular viability and prevention of genomic instability that can cause cancer. Lymphocytes are an excellent model system to study the response of cells to physiologic levels of DSBs, since developing lymphocytes must induce and repair several DSBs during the processes of V(D)J recombination and class switch recombination (CSR). Complete V(D)J recombination at the IgH locus in B cells, and the TCR? locus in T cells upregulates cyclin D3, which then induces a burst of rapid proliferation. Mature B cells exposed to antigen in secondary lymphoid organs similarly induce D3 expression, proliferate, and begin the process of CSR. D3 protein and mRNA expression is downregulated in primary mouse lymphocytes in response to DSBs induced by IR, through a mechanism that requires the tumor suppressors ATM and p53, and the microRNA (miR) processing enzyme Dicer. Preliminary data suggest that D3 expression is repressed primarily through increased mRNA turnover. This is consistent with several known pathways by which ATM and p53 regulate the expression of miRs involved in the DSB response.
Aim 1 seeks to uncover the ATM and p53-dependent mechanisms through which DSBs downregulate D3 expression, with a particular emphasis on miR-mediated pathways. To achieve this goal, mutational analysis of the 3'UTR will be combined with miR overexpression and knockdown assays to identify specific miRs that target the D3 mRNA, and to confirm functional activity of these miRs. For miRs found to target D3, biochemical studies will investigate how ATM and p53 may enhance miR biogenesis.
Aim 2 addresses the in vivo relevance of DSB-induced D3 repression by using bone marrow reconstitution to generate mice whose hematopoietic systems express a mutant D3 cDNA that cannot be normally regulated by breaks. Taking advantage of several techniques available to study lymphocyte development, genome stability, and malignancy, the proposed experiments will test the hypothesis that developing lymphocytes with unrepaired breaks must downregulate D3 expression to avoid S phase entry, in order to prevent genome instability that would cause apoptosis or lymphoma. Flow cytometry will address whether D3 dysregulation disrupts normal lymphocyte development, and PCR-based assays will determine whether unrepaired breaks pass into S phase aberrantly in the presence of dysregulated D3. Finally, chimeras will be aged to monitor for tumor development, and resulting tumors will be molecularly characterized. Because defective coordination of DSB repair and cell cycle progression underlie many human immunodeficiencies and lymphomas, the proposed work will contribute substantially to our knowledge of the etiology and treatment of these pathologies.
Uncoupling of DNA double strand break (DSB) responses and cell cycle progression leads to genomic instability and is a causative or tumor-promoting factor in a variety of human cancers. The failure to coordinate DSB repair and proliferation that is hypothesized to result from the mis-regulation of cyclin D3 may serve as a model for cancers that have similarly uncoupled these processes, improving our ability to design and test novel therapeutics. Further, the mechanistic studies investigating the role of ATM and p53 in microRNA biogenesis will extend our knowledge of mechanisms by which these critical tumor suppressors post-transcriptionally regulate gene expression, enhancing our ability to understand and treat cancers with ATM or p53 mutations.