Cyclin D3 and DNA double strand breaks: genome stability and lymphomagenesis

描述（由申请人提供）：本申请的目的是阐明发育中的淋巴细胞协调DNA双链断裂（DSB）修复和细胞周期进展的机制。这些过程的适当协调是维持细胞活力和预防可能导致癌症的基因组不稳定所必需的。淋巴细胞是研究细胞对dsb生理水平反应的一个很好的模型系统，因为发育中的淋巴细胞在V(D)J重组和类开关重组（CSR）过程中必须诱导和修复多个dsb。在B细胞的IgH位点和T细胞的TCR¿位点上完全的V(D)J重组可上调细胞周期蛋白D3，从而诱导细胞快速增殖。暴露于次级淋巴器官抗原的成熟B细胞同样诱导D3表达、增殖并开始CSR过程。在IR诱导的DSBs中，原代小鼠淋巴细胞中D3蛋白和mRNA的表达下调，其机制需要肿瘤抑制因子ATM和p53以及microRNA （miR）加工酶Dicer。初步数据表明，D3表达的抑制主要是通过增加mRNA的周转。这与ATM和p53调节参与DSB反应的mir表达的几种已知途径是一致的。Aim 1旨在揭示dsb下调D3表达的ATM和p53依赖机制，特别强调mir介导的途径。为了实现这一目标，3'UTR的突变分析将与miR过表达和敲低试验相结合，以鉴定针对D3 mRNA的特异性miR，并确认这些miR的功能活性。对于发现靶向D3的miRs，生化研究将会

DESCRIPTION (provided by applicant): 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.