Integration of transcriptional responses in macrophages

描述（由申请人提供）：拟进行研究以检验Ets转录抑制因子的诱导在巨噬细胞分化的终末期和永久性细胞周期停滞之间提供分子联系的假设。这一假设是基于我们最近的研究表明，Ets阻遏物METS/PE 1在终末巨噬细胞分化过程中显著上调，可以阻断Ras依赖性细胞增殖，而不抑制Ras依赖性巨噬细胞特异性基因的表达。建议选择性抑制增殖的能力，METS结合作为一个单体的Ets网站在一系列的E2 F依赖性细胞周期调控基因，但不Ets激活剂和AP-1蛋白的三元复合物识别的网站。假设细胞增殖的METS依赖性抑制需要含DEAD盒的蛋白质DP 103，其也已被鉴定为EB病毒感染后B淋巴细胞永生化所需的EBNA 2和EBNA 3c蛋白质的靶标。DP 103被认为是METS的辅阻遏物，它通过使一种新的辅阻遏物复合物的组装成核而起作用。生物化学、细胞和分子遗传学方法将用于检验以下假设，即METS/DP 103复合物和E2 F/pRB家族蛋白质复合物之间的功能相互作用涉及在终末巨噬细胞分化期间指导永久退出细胞周期。提出了五个具体目标：具体目标1将在全基因组水平上测试以下假设：在终末巨噬细胞分化期间，METS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。具体目标2将测试的假设，METS/DP 103介导的生长抑制需要与Rb家族成员和相关的辅阻遏复合物，港口组蛋白脱乙酰酶和组蛋白甲基转移酶活性的相互作用。具体目标3将通过分析METS和ERF缺陷型巨噬细胞来检验METS/PE 1和相关Ets阻遏物ERF负调节巨噬细胞增殖的假设。具体目标4将通过分析DP 103缺陷型巨噬细胞来检验METS和ERF介导的生长抑制需要与DP 103相互作用的假设。具体目标5将检验细胞可以通过从细胞核输出METS和/或通过抑制与DP 103的功能相互作用而对METS介导的生长抑制产生抗性的假设。这些研究将为巨噬细胞和其他细胞类型在终末分化期间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.