The transcription cycle of RNA polymerase II (Pol II) depends on sequential functions of distinct cyclin-dependent kinases (CDKs), but the mechanisms that order those functions are still emerging. We have taken a chemical-genetic approach-replacement of wild- type with analog-sensitive (AS) mutant CDKs-to reveal both "early" and "late" functions of the CDK network in transcription. First, by selective inhibition of Cdk7-a component of transcription initiation factor TFIIH-in human cells, we uncovered two unexpected, and seemingly antagonistic functions. Cdk7 activity is required to recruit factors that establish a promoter-proximal pause by Pol II, and to activate Cdk9, catalytic subunit of positive transcription elongation factor b (P-TEFb), which releases the pause. Therefore the CDK network appears to depend on incoherent feedforward to raise a transient kinetic barrier to Pol II elongation, and thus create a temporal window to recruit mRNA- processing and chromatin-modifying machinery. Consistent with a requirement for CDK- directed pausing to ensure faithful RNA processing, inhibition of Cdk7 or Cdk9 leads to defects in termination and 3'-end maturation of Pol II transcripts. Second, in a chemical- genetic screen for Cdk9 substrates, we identified multiple proteins involved in RNA 5'- end decapping and the "torpedo" pathway of transcription termination, which has recently been suggested to influence pausing and divergent antisense transcription. We will dissect the initiation-elongation transition of Pol II, and elucidate regulation of transcription termination and polarity, to uncover novel modes of gene regulation by CDKs.
The specific aims are: 1. To identify functions and targets of CDKs at the initiation-elongation transition 2. To investigate possible regulation of the transcription termination pathway by P- TEFb, uncovered in a chemical genetic screen for Cdk9 targets 3. To dissect functions of Cdk7 and Cdk9 by chemical genetics in human cells Our studies reveal a CDK cascade at the core of the Pol II transcription cycle; completion of our aims will illuminate how Cdk7 and Cdk9 collaborate to ensure unidirectional transitions between phases of that cycle. By turning CDKs into chemical switches that can be manipulated with customized small molecules, we will distinguish their specific roles and substrates, and reveal new anti-cancer or anti-viral drug targets.

Public Health Relevance

Proper control of gene expression is essential to normal human development and is deranged in cancer cells. Genes important for cell proliferation and developmental decisions are often regulated during the elongation stage of transcription, which is in turn controlled by a network of cyclin-dependent kinases (CDKs). CDK regulation may be disturbed in cancer cells, making CDKs logical targets for chemotherapy. Chemical genetics allows selective inhibition of one CDK at a time in engineered human cells- derived from cancers or normal tissue-and enables evaluation of different CDKs, with distinct roles in gene expression, as potential targets for inhibition by anti-cancer drugs.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM104291-01A1
Application #
8630081
Study Section
Cellular Signaling and Regulatory Systems Study Section (CSRS)
Program Officer
Sledjeski, Darren D
Project Start
2014-02-15
Project End
2017-12-31
Budget Start
2014-02-15
Budget End
2014-12-31
Support Year
1
Fiscal Year
2014
Total Cost
$322,050
Indirect Cost
$132,050
Name
Icahn School of Medicine at Mount Sinai
Department
Biology
Type
Schools of Medicine
DUNS #
078861598
City
New York
State
NY
Country
United States
Zip Code
10029
Bösken, Christian A; Farnung, Lucas; Hintermair, Corinna et al. (2014) The structure and substrate specificity of human Cdk12/Cyclin K. Nat Commun 5:3505