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

项目摘要 细胞周期蛋白依赖性蛋白激酶(CDKs)调节细胞分裂和转录。要了解其作用机制 这种调控，或者是针对癌细胞中的CDK网络，我们需要确定其功能和底物 特定的CDK。为了实现这一目标，我们开创了一种化学遗传学方法，结合了 具有化学抑制的速度和可逆性的遗传学，通过用类似物取代野生型- 活细胞中敏感的(AS)突变的CDK。在未来五年，我们将把化学遗传学与 功能基因组学以新的方式，建立细胞周期和转录调控的新范式，并 发现与CDK网络相互作用的新途径，这可能是癌症的靶点。 细胞周期调控中的CAK-CDK网络：我们的研究揭示了CDK的不同激活途径 在细胞周期的不同阶段发挥作用；未来五年的目标是了解这些途径是如何 受上游信号调节，并与细胞周期调节的转录相关。我们将调查一个 由CDK激活激酶(CAK)CDK7和其靶标CDK4组成的级联在G1中被开启 静止的细胞重新进入分裂周期，以及当细胞退出周期时，CDK7是如何特别下调的。 CDK对转录周期的调节：抑制转录CDKs扰动RNA聚合酶(POL)II 动力学的方式让人想起细胞周期停滞和检查点故障，但确切的机制仍然 需要被定义。CDK7在从起始到启动的转变中需要建立启动子-近端的停顿 延长，并通过激活正转录延长因子b(P-TEFb，a)促进暂停释放 CDK9/细胞周期蛋白T1复合体)。我们发现正常的POLII延伸率依赖于分裂中的CDK9活性 酵母，以及定义的人和酵母CDK9底物组，这些底物富含与 RNA处理。在接下来的五年里，我们将测试CDK9作用于不同底物的想法 刺激转录延伸和RNA加工，以确保它们的动力学耦合。 定义转录退出网络：CDK调节持续到转录周期结束；我们 验证了终止酶Xrn2和蛋白磷酸酶1(PP1)，参与切割和 多聚腺苷基化)作为真正的CDK9底物。CDK9的磷酸化激活Xrn2但抑制PP1， 这是使延伸因子(和CDK9靶)Spt5去磷酸化所必需的。在未来五年，我们将 测试控制伸长-终止转变的双稳态CDK9-PP1开关的紧急模型。 合成-致死相互作用的化学-遗传发现CDK已成为药物的靶点 利用某些癌细胞特有的转录依赖性。我们在结肠中诱导了这种依赖 肿瘤细胞通过联合肿瘤抑制因子P53的激活剂和CDK7的抑制剂实现合成 杀伤力。在接下来的五年里，我们将发现与CDK网络相互作用的新途径--并可能 导致抗癌药物组合-通过在依赖于CDK的人类细胞中进行合成致死筛选。

Project Summary Cyclin-dependent kinases (CDKs) regulate cell division and transcription. To understand the mechanisms of this regulation, or to target the CDK network in cancer cells, we need to identify the functions and substrates of specific CDKs. To achieve this goal, we pioneered a chemical-genetic approach combining the specificity of genetics with the speed and reversibility of chemical inhibition, by replacement of wild-type with analog- sensitive (AS) mutant CDKs in living cells. In the next five years, we will combine chemical genetics with functional genomics in novel ways, to establish new paradigms of cell-cycle and transcriptional control, and to discover new pathways that interact with the CDK network, which might be targeted in cancer. The CAK-CDK network in cell-cycle control: Our studies revealed distinct activation pathways for CDKs that act in different phases of the cell cycle; a goal for the next five years is to understand how those pathways are regulated by upstream signaling and linked to cell cycle-regulated transcription. We will investigate how a cascade comprising the CDK-activating kinase (CAK) Cdk7 and its target Cdk4 is switched on in G1 when quiescent cells re-enter the division cycle, and how Cdk7 is specifically down-regulated as cells exit the cycle. CDK regulation of the transcription cycle: Inhibiting transcriptional CDKs perturbs RNA polymerase (Pol) II dynamics in ways reminiscent of cell-cycle arrests and checkpoint failures, but the precise mechanisms still need to be defined. Cdk7 is required to establish a promoter-proximal pause in the transition from initiation to elongation, and to promote pause release by activating positive transcription elongation factor b (P-TEFb, a Cdk9/cyclin T1 complex). We showed that normal Pol II elongation rates depend on Cdk9 activity in fission yeast, and defined sets of human and yeast Cdk9 substrates, which are enriched for proteins implicated in RNA processing. Over the next five years, we will test the idea that Cdk9 acts on different substrates to stimulate both transcription elongation and RNA processing, to ensure their kinetic coupling. Defining a transcription exit network: CDK regulation persists to the end of the transcription cycle; we validated the termination enzyme Xrn2 and protein phosphatase 1 (PP1, implicated in cleavage and polyadenylation) as bona fide Cdk9 substrates. Phosphorylation by Cdk9 activates Xrn2 but inhibits PP1, which is required to dephosphorylate the elongation factor (and Cdk9 target) Spt5. In the next five years we will test the emergent model of a bistable Cdk9-PP1 switch that controls the elongation-termination transition. Chemical-genetic discovery of synthetic-lethal interactions CDKs have emerged as targets of drugs that exploit transcriptional dependencies unique to certain cancer cells. We induced such a dependency in colon cancer cells by combining activators of the tumor suppressor p53 with inhibitors of Cdk7 to achieve synthetic lethality. In the next five years, we will uncover novel pathways that interact with the CDK network—and might lead to anti-cancer drug combinations—by synthetic-lethal screens in human cells dependent on AS CDKs.