The CAK Network in Metazoan Cell Cycle Regulation

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DESCRIPTION (provided by applicant): Cyclin-dependent kinases (CDKs) control cell division and transcription by RNA polymerase II (Pol II), and are themselves stringently regulated. An activator common to both cell-cycle and transcription pathways in metazoans is the CDK-activating kinase (CAK): a complex of Cdk7, cyclin H and Mat1. The Cdk7 complex has essential functions in cell division and gene expression: as a CAK, to activate CDKs that drive S phase and mitosis; and, as a component of the transcription factor TFIIH, to phosphorylate Pol II (and other proteins). Cdk7 and its targets form a network that links gene expression with genome duplication and segregation in dividing cells. Our long-term goal is to understand how Cdk7 accomplishes its dual functions, and whether it serves to coordinate patterns of gene expression with cell division. We have defined requirements for Cdk7 in cell-cycle control and transcription in mammalian cells, by a novel, chemical-genetic approach: the creation of a human cancer cell line in which Cdk7 can be specifically inhibited with small molecules. Cdk7 is required for the activation of Cdk2, the major CDK active in S phase; and for the assembly and activation of Cdk1/cyclin B, the enzymatic trigger of mitosis. Inhibition of Cdk7 represses expression of a specific subset of Pol ll-dependent genes. The ability to manipulate Cdk7 activity in vivo will allow us to evaluate the CAK-CDK pathway as a therapeutic target in human disease. We will take a chemical genetic approach, to address our Specific Aims: 1) To determine the targets and timing of Cdk7 function in G1. 2) To understand the mechanism(s) enforcing dependency of Cdk1/cyclin B assembly on CAK in vivo. 3) To ask if Cdk7 regulates Cdk1/cyclin B assembly and activity during a mitotic arrest. 4) To define precise requirements for Cdk7 activity in gene expression, through functional genomics to identify Cdk7-responsive genes, and through biochemistry to identify its critical protein targets and collaborators. Cell proliferation and the regulated expression of genetic information are both disturbed in cancer. A network of regulatory enzymes called CDKs coordinates both processes. We take a novel approach, with engineered human cancer cells sensitive to specifically designed CDK inhibitors, to probe the functions of CDKs, and to test whether these enzymes are potential targets for inhibition in the treatment of cancer.