Cyclin-dependent kinase control of cell-division and transcription cycles

细胞分裂和转录周期的细胞周期蛋白依赖性激酶控制

• 批准号: 10559139
• 负责人: ROBERT P FISHER
• 金额: $ 50.7万
• 依托单位: ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-04-01 至 2028-02-29
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10559139
• 关键词: Binding; Cell Cycle; Cell Cycle Regulation; Cell division; Cells; Chromatin; Code; Collaborations; Complex; Coupling; Cyclin-Dependent Kinases; Disease; Dissection; Drug Targeting; Elongation Factor; Ensure; Event; Family; Gene Expression; Gene Expression Regulation; Genetic Transcription; Genomics; Goals; Growth and Development function; Human; Link; Malignant Neoplasms; Messenger RNA; Modeling; Mutation; Pathway interactions; Phase; Phosphoric Monoester Hydrolases; Phosphorylation; Protein Dephosphorylation; Protein Phosphatase 2A Regulatory Subunit PR53; Protein phosphatase; RNA Polymerase II; Regulation; Role; Signal Transduction; Site; Specificity; Substrate Specificity; Testing; Therapeutic; Transcriptional Regulation; Work; cancer cell; cancer therapy; genome editing; genome-wide; member; phosphatase-1 kinase; promoter; protein expression; recruit; response; small molecule; transcription factor TFIIH

Project Summary RNA polymerase II (RNAPII), which catalyzes mRNA synthesis, is co-regulated by cyclin-dependent kinases (CDKs) and phosphatases to control transcription cycle progression and ensure coupling of elongation and processing. Derangements of the RNAPII cycle underlie cancer and other diseases; CDKs and phosphatases are potential drug targets, but how they collaborate to regulate gene expression is largely unknown. We seek to determine how specific phosphorylations are placed and removed in precise order to execute transitions between phases of transcription. In the past five years we uncovered two CDK-phosphatase switches that regulate RNAPII elongation. Our goals for the next five years are to understand how these circuits work to coordinate the transcription cycle, and how they might be disrupted with small molecules. Dissecting CDK-PPP switches: We identified two members of the phosphoprotein phosphatase (PPP) family as targets of inhibitory phosphorylation by Cdk9 (P-TEFb). PP4 and PP1 dephosphorylate the Cdk9 substrate Spt5 to regulate the onset and end of rapid elongation, respectively. To define roles of Cdk9-PPP crosstalk in transcription, we will introduce mutations that lock PP4 or PP1 in constitutively active (unphosphorylated) or inactive (phosphorylated) states and analyze genome-wide effects on RNAPII dynamics and gene expression. Unraveling control of RNAPII elongation by PPP family phosphatases: PP4 and the related PP2A, another phosphatase recruited to transcription complexes, dephosphorylate Spt5 with similar site specificity, and may cooperate to regulate RNAPII release from the promoter-proximal pause. We plan integrated genomic analyses to ask whether PP4 and PP2A work independently, in combination, or in response to different signals. Understanding how phosphorylation events are ordered in the RNAPII cycle: By analogy with the cell division cycle—also regulated by CDKs—the order of phosphorylations during transcription is likely determined by substrate specificities of CDKs and phosphatases, and by structural features of the substrates themselves. We will test this hypothesis for Cdk9, which has scores of substrates and is active throughout elongation. Functional dissection of a conserved elongation regulator: Spt5, large subunit of the conserved elongation factor DSIF, is the linchpin of elongation control by Cdk9-PPP switches; phosphorylations placed by Cdk9 at different sites in Spt5 are removed at different times by PP4 or PP1 and are likely to perform different functions in the transcription cycle. We will take a genome editing approach in human cells to dissect the functions of distinct Spt5 phosphorylations, and to elucidate their differential regulation by CDKs and phosphatases. Illuminating a link between transcription and cell cycle control: One CDK is a core component of both cell-division and transcriptional machineries: the TFIIH-associated Cdk7. As human cells re-enter the division cycle from quiescence, chromatin-bound Cdk7 is activated, potentially enhancing its function in G1-specific gene transcription. We will test this model and identify pathways that lead to Cdk7 activation at cell cycle entry.

项目概要 RNA 聚合酶 II (RNAPII) 催化 mRNA 合成，由细胞周期蛋白依赖性激酶共同调节 （CDK）和磷酸酶控制转录周期进程并确保延伸和转录的耦合 加工。 RNAPII 循环的紊乱是癌症和其他疾病的基础； CDK 和磷酸酶 是潜在的药物靶标，但它们如何协作调节基因表达很大程度上未知。我们寻求 确定如何以精确的顺​​序放置和去除特定磷酸化以执行转换 转录阶段之间。在过去五年中，我们发现了两种 CDK 磷酸酶开关 调节 RNAPII 延伸。我们未来五年的目标是了解这些电路如何工作 协调转录周期，以及如何用小分子破坏它们。 剖析 CDK-PPP 开关：我们鉴定了磷蛋白磷酸酶 (PPP) 家族的两个成员 作为 Cdk9 (P-TEFb) 抑制性磷酸化的靶标。 PP4 和 PP1 使 Cdk9 底物去磷酸化 Spt5分别调节快速伸长的开始和结束。定义 Cdk9-PPP 串扰的作用 转录时，我们将引入突变，将 PP4 或 PP1 锁定在组成型活性（非磷酸化）或 非活性（磷酸化）状态并分析全基因组范围内对 RNAPII 动态和基因表达的影响。 揭示 PPP 家族磷酸酶对 RNAPII 延伸的控制：PP4 和相关的 PP2A， 另一种磷酸酶被招募到转录复合物中，以相似的位点特异性使 Spt5 去磷酸化， 并可能合作调节 RNAPII 从启动子近端暂停的释放。我们计划整合基因组 分析PP4和PP2A是否独立工作、组合工作或响应不同的信号。 了解 RNAPII 循环中磷酸化事件的顺序：通过与细胞类比 分裂周期（也受 CDK 调节）转录过程中磷酸化的顺序可能是确定的 通过 CDK 和磷酸酶的底物特异性以及底物本身的结构特征。 我们将测试 Cdk9 的这一假设，它具有大量底物并且在整个延伸过程中都很活跃。 保守伸长调节因子的功能解剖：Spt5，保守伸长的大亚基 因子 DSIF，是 Cdk9-PPP 开关伸长控制的关键； Cdk9 磷酸化 Spt5 中的不同位点在不同时间被 PP4 或 PP1 移除，并且可能执行不同的功能 在转录周期中。我们将在人类细胞中采用基因组编辑方法来剖析 不同的 Spt5 磷酸化，并阐明 CDK 和磷酸酶对它们的差异调节。 阐明转录和细胞周期控制之间的联系：一个 CDK 是两者的核心组成部分 细胞分裂和转录机制：TFIIH 相关的 Cdk7。当人体细胞重新进入分裂状态时 从静止周期开始，染色质结合的 Cdk7 被激活，可能增强其在 G1 特异性中的功能 基因转录。我们将测试该模型并确定导致细胞周期进入时 Cdk7 激活的途径。