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.

项目摘要 RNA聚合酶II（RNAPII）催化mRNA的合成，由细胞周期蛋白依赖性激酶共同调节 （CDK）和磷酸酶来控制转录周期进程，并确保延伸和 处理. RNAPII循环的紊乱是癌症和其他疾病的基础; CDK和磷酸酶 是潜在的药物靶点，但它们如何合作调节基因表达在很大程度上是未知的。我们寻求 以确定特定的磷酸化是如何以精确的顺序放置和移除的， 转录阶段之间。在过去的五年里，我们发现了两个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是如何工作的， 协调转录周期，以及它们如何被小分子破坏。 剖析CDK-PPP开关：我们确定了磷蛋白磷酸酶（PPP）家族的两个成员 作为Cdk 9（P-TEFb）抑制性磷酸化的靶标。PP 4和PP 1使Cdk 9底物去磷酸化 Spt 5分别调节快速伸长的开始和结束。为了定义Cdk 9-PPP串扰在 转录，我们将引入突变，锁定PP 4或PP 1在组成型活性（非磷酸化）或 非活性（磷酸化）状态，并分析全基因组对RNAPII动力学和基因表达的影响。 PPP家族磷酸酶：PP 4和相关PP 2A对RNAPII延伸的解开控制， 另一种磷酸酶被募集到转录复合物中，以类似的位点特异性使Spt 5去磷酸化， 并且可以协同调节RNAPII从启动子近端暂停释放。我们计划整合基因组 分析PP 4和PP 2A是否独立工作，组合或响应不同的信号。 了解磷酸化事件在RNAPII循环中的顺序：通过与细胞的类比 分裂周期--也受CDK调节--转录过程中磷酸化的顺序很可能是由 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Cdk 9的这一假设，它有几十个底物，并在整个延伸过程中是活跃的。 一个保守的延伸调节因子Spt 5的功能研究 因子DSIF，是Cdk 9-PPP开关控制延伸的关键; Cdk 9在 Spt 5中的不同位点在不同的时间被PP 4或PP 1去除，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Spt 5磷酸化，并阐明其差异调节CDK和磷酸酶。 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Cdk 7。随着人类细胞重新进入分裂 从静止循环，染色质结合Cdk 7被激活，潜在地增强其在G1特异性 基因转录我们将测试这个模型，并确定导致Cdk 7在细胞周期进入激活的途径。