Integration of transcriptional responses in macrophages

描述(由申请人提供)：有研究建议检验这样一种假设，即ETS转录抑制因子的诱导在巨噬细胞分化的终末阶段和永久性细胞周期停滞之间提供了分子联系。这一假说是基于我们最近的研究表明，ETS抑制因子Mets/PE1在巨噬细胞终末分化过程中显著上调，并可以在不抑制RAS依赖的巨噬细胞特异性基因表达的情况下阻止RAS依赖的细胞增殖。选择性抑制增殖被认为是由于Mets能够作为单体与一系列依赖E2F的细胞周期调节基因中的Ets位点结合，而不是与Ets激活剂和AP-1蛋白的三元复合体识别的位点结合。蛋氨酸依赖的细胞增殖抑制被认为需要含有DP103蛋白的DEAD-box，DP103也被确定为EB病毒感染后B淋巴细胞永生化所需的EBNA2和EBNA3c蛋白的靶点。DP103被认为通过核组装一种新的辅阻遏子复合体来发挥甲硫氨酸的辅阻遏子的作用。生化、细胞和分子遗传学方法将被用来检验这一假设，即Mets/DP103复合体和E2F/PRB家族蛋白复合体之间的功能相互作用参与了在巨噬细胞终末分化过程中引导细胞周期永久退出的假设。提出了五个具体目标：特定目标1将在全基因组水平上测试这一假设，即在终端巨噬细胞分化过程中，蛋氨酸选择性地取代细胞周期控制基因上的ETS激活剂，而不是细胞类型特异性基因。特定目标2将测试假设，即Mets/DP103介导的生长抑制需要与Rb家族成员以及具有组蛋白去乙酰酶和组蛋白甲基转移酶活性的相关辅阻遏子复合体相互作用。具体目标3将通过对Mets和ERF缺陷的巨噬细胞的分析，验证Mets/PE1和相关的ETS抑制因子ERF负向调节巨噬细胞增殖的假设。特定目标4将通过对DP103缺失的巨噬细胞的分析，验证Mets和ERF介导的生长抑制需要与DP103相互作用的假设。特殊目标5将测试这样一个假设，即细胞可以通过从细胞核输出Met和/或通过抑制与DP103的功能相互作用来抵抗Mets介导的生长抑制。这些研究应该为巨噬细胞和其他类型细胞的终末分化过程中实现永久退出细胞周期的机制提供重要的新见解。

DESCRIPTION (provided by applicant): Studies are proposed to test the hypothesis that the induction of Ets transcriptional repressors provides a molecular link between the terminal phase of macrophage differentiation and permanent cell cycle arrest. This hypothesis is based on our recent studies demonstrating that the Ets repressor METS/PE1 is markedly up regulated during terminal macrophage differentiation and can block Ras-dependent cell proliferation without inhibiting Ras-dependent expression of macrophage-specific genes. Selective inhibition of proliferation is proposed to result from the ability of METS to bind as a monomer to Ets sites in a series of E2F-dependent cell cycle-regulatory genes, but not to sites recognized by ternary complexes of Ets activators and AP-1 proteins. METS-dependent inhibition of cell proliferation is hypothesized to require the DEAD-box containing protein DP103, which has also been identified as a target of the EBNA2 and EBNA3c proteins that are required for immortalization of B lymphocytes following Epstein-Barr virus infection. DP103 is proposed to function as a corepressor of METS by nucleating the assembly of a novel co-repressor complex. Biochemical, cellular and molecular genetic approaches will be used to test the hypothesis that functional interactions between the METS/DP103 complex and E2F/pRB family protein complexes are involved in directing permanent exit from the cell cycle during terminal macrophage differentiation. Five Specific Aims are proposed: Specific Aim 1 will test at a genome-wide level the hypothesis that METS selectively replaces Ets activators on cell cycle control genes, but not cell type-specific genes, during terminal macrophage differentiation. Specific Aim 2 will test the hypothesis that METS/DP103-mediated growth inhibition requires interactions with members of the Rb family and associated corepressor complexes that harbor histone deacetylase and histone methyltransferase activities. Specific Aim 3 will test the hypothesis that METS/PE1 and the related Ets repressor ERF negatively regulate macrophage proliferation by analysis of METS and ERF-deficient macrophages. Specific Aim 4 will test the hypothesis that METS and ERF-mediated growth inhibition requires interactions with DP103, through the analysis of DP103- deficient macrophages. Specific Aim 5 will test the hypothesis that cells can become resistant to METS-mediated growth inhibition by exporting METS from the nucleus and/or by inhibiting functional interactions with DP103. These studies are should provide significant new insights into mechanisms by which permanent exit from the cell cycle is achieved during terminal differentiation of macrophages and other cell types.