Tumor suppressor role of the DREAM complex

DREAM 复合物的肿瘤抑制作用

• 批准号: 9392358
• 负责人: Amy E Schade
• 金额: $ 3.16万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-01 至 2019-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9392358
• 关键词: Alleles; Amino Acids; Binding; CCNE1 gene; CDK2 gene; CDK4 gene; Cell Cycle; Cell Cycle Regulation; Cells; ChIP-seq; Chromatin; Clinical; Complex; Cyclin A; Cyclin D1; Cyclins; Data; E2F1 gene; Fibroblasts; Gene Expression; Genes; Growth Factor; Kinetics; Knock-out; Mass Spectrum Analysis; Mediating; Molecular Conformation; Oncogenic; Phosphorylation; Phosphorylation Site; Phosphotransferases; Process; Proteins; RB1 gene; Regulation; Repression; Research; Rest; Retinoblastoma Protein; Role; Site; Stable Isotope Labeling; TP53 gene; Testing; Therapeutic; Transactivation; Tumor Suppressor Proteins; cdc Genes; cell transformation; gene repression; inhibitor/antagonist; insight; member; metaplastic cell transformation; mutant; neoplastic cell; novel; promoter; protein complex; small molecular inhibitor; tumor

The DREAM complex (DP, Rb-like p130/p107, repressor E2F4/5, and MuvB) establishes quiescence (G0) by binding and repressing cell cycle-regulated promoters. Rb, a protein related to p130, controls E2F-dependent genes at the G1/S boundary by inhibiting activator E2Fs promoter of G1/S early cell cycle gene expression. The distinct contributions of p130 and Rb to regulation of this overlapping set of cell cycle E2F-regulated promoters are not well understood. Cyclin-CDK complexes phosphorylate Rb to relieve repression of activator E2Fs to allow transactivation of E2F cell cycle regulated promoters. Little is known about the mechanism of relief of DREAM repression of cell cycle genes, and whether it is required for activator E2F transactivation of gene expression. Our data indicates that Cyclin D-CDK4 promotes DREAM complex disruption. E2F4 persists on promoters without phosphorylated p130 or MuvB. We seek to determine the mechanism of E2F4 release from chromatin and its relationship to the occupancy of activating E2Fs during G1. The sufficiency of DREAM to regulate E2F promoters during cell cycle reentry remains unknown, but we observed that RB1 knockout HFFs have similar cell cycle gene expression kinetics and retain sensitivity to palbociclib, a Cyclin D-CDK4/6 specific inhibitor. These results suggest that DREAM complex may regulate E2F promoters independently of Rb. We will distinguish the contributions of Rb and DREAM-mediated repression of cell cycle gene expression and specific contribution of Cyclin D-CDK4/6 inactivation of Rb and DREAM to cellular transformation. We propose that DREAM complex disruption by Cyclin D-CDK4/6 promotes cell cycle gene expression and transformation independently of pRB. In Aim 1, we will determine whether Cyclin D-CDK4/6 phosphorylation of p130 is sufficient to disrupt the DREAM complex by causing release of p130 from E2F4 and cell cycle regulated promoters. We will determine which phosphorylation sites on p130 contribute to its binding to E2F4 and regulate disassociation from early cell cycle gene promoters. In Aim 2, we will determine the mechanism of loss of E2F4 from cell cycle regulated promoters during G1. We will obtain a global view of chromatin association kinetics of DREAM members and E2F1 during G1. We will determine the mechanism of co-regulation of repressor and activator E2Fs of cell cycle genes during G1. In Aim 3, we will determine if Cyclin D-CDK4/6 mediated disruption of the DREAM complex is required for cellular transformation. We will distinguish pRB and DREAM-mediated repression of cell cycle gene expression and specific contributions as tumor suppressors. We will determine if DREAM restricts cell cycle gene expression independent of Rb and if disruption of DREAM is required to transform fibroblasts. Distinguishing Rb and DREAM cell cycle control will elucidate the previously unappreciated tumor suppressor role of p130. If DREAM is sufficient to repress cell cycle gene expression in Rb null transformed cells, the therapeutic range of CDK4/6 inhibitors may be expanded to tumors with normal DREAM to promote cell cycle exit.

DREAM复合物（DP、Rb样p130/p107、阻遏物E2 F4/5和MuvB）通过以下方式建立静止期（G 0）： 结合和抑制细胞周期调节启动子。Rb，一种与p130相关的蛋白，控制E2 F依赖性 通过抑制G1/S早期细胞周期基因表达的激活剂E2 Fs启动子， p130和Rb对E2 F调控的这组重叠的细胞周期调控的不同贡献是： 启动子还没有被很好地理解。细胞周期蛋白-CDK复合物磷酸化Rb以解除激活剂的阻遏 E2 F，以允许E2 F细胞周期调节启动子的反式激活。对这种机制知之甚少。 缓解细胞周期基因的DREAM抑制，以及是否需要激活剂E2 F反式激活 基因表达。我们的数据表明，细胞周期蛋白D-CDK 4促进DREAM复合物的破坏。E2 F4持续存在 在没有磷酸化p130或MuvB的启动子上。我们试图确定E2 F4释放的机制， 从染色质和它的关系，在G1期激活E2 Fs的占用。梦想的充分性 调控E2 F启动子在细胞周期折返仍然未知，但我们观察到，RB 1基因敲除， HFF具有相似的细胞周期基因表达动力学，并保持对Palbociclib（一种细胞周期蛋白D-CDK 4/6）的敏感性 特异性抑制剂这些结果表明DREAM复合物可能独立于E2 F启动子调节E2 F启动子， RB.我们将区分Rb和DREAM介导的细胞周期基因表达抑制的作用 以及细胞周期蛋白D-CDK 4/6对Rb和DREAM的失活对细胞转化的特异性贡献。我们 提出通过细胞周期蛋白D-CDK 4/6破坏DREAM复合物促进细胞周期基因表达， 转化独立于pRB。在目标1中，我们将确定细胞周期蛋白D-CDK 4/6是否 p130的磷酸化足以通过引起p130从E2 F4释放而破坏DREAM复合物， 细胞周期调控启动子。我们将确定p130上哪些磷酸化位点有助于其结合 到E2 F4并调节与早期细胞周期基因启动子的分离。在目标2中，我们将确定 G1期细胞周期调控启动子E2 F4缺失的机制。我们将获得一个全球视野， G1期DREAM成员和E2 F1的染色质缔合动力学。我们将确定 G1期细胞周期基因的阻遏物和激活物E2 Fs的共同调节。在目标3中，我们将确定 细胞周期蛋白D-CDK 4/6介导的DREAM复合物的破坏是细胞转化所必需的。我们将 区分pRB和DREAM介导的细胞周期基因表达抑制和特异性贡献， 肿瘤抑制剂。我们将确定DREAM是否独立于Rb限制细胞周期基因表达，以及是否 DREAM的破坏是转化成纤维细胞所必需的。区分Rb和DREAM细胞周期控制 将阐明p130以前未被重视的肿瘤抑制作用。如果梦想足够， 抑制Rb缺失转化细胞的细胞周期基因表达，CDK 4/6的治疗范围 抑制剂可以扩展到具有正常DREAM的肿瘤以促进细胞周期退出。