THZ1 Reveals Roles for Cdk7 in Co-transcriptional Capping and Pausing

Mol Cell. 2015 Aug 20;59(4):576-87. doi: 10.1016/j.molcel.2015.06.032. Epub 2015 Aug 6.

Abstract

The Cdk7 subunit of TFIIH phosphorylates RNA polymerase II (Pol II) during initiation, and, while recent studies show that inhibition of human Cdk7 negatively influences transcription, the mechanisms involved are unclear. Using in vitro transcription with nuclear extract, we demonstrate that THZ1, a covalent Cdk7 inhibitor, causes defects in Pol II phosphorylation, co-transcriptional capping, promoter proximal pausing, and productive elongation. THZ1 does not affect initiation but blocks essentially all Pol II large subunit C-terminal domain (CTD) phosphorylation. We found that guanylylation of nascent RNAs is length dependent and modulated by a THZ1-sensitive factor present in nuclear extract. THZ1 impacts pausing through a capping-independent block of DSIF and NELF loading. The P-TEFb-dependent transition into productive elongation was also inhibited by THZ1, likely due to loss of DSIF. Capping and pausing were also reduced in THZ1-treated cells. Our results provide mechanistic insights into THZ1 action and how Cdk7 broadly influences transcription and capping.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Antineoplastic Agents / chemistry*
  • Antineoplastic Agents / pharmacology
  • Cyclin-Dependent Kinases / antagonists & inhibitors
  • Cyclin-Dependent Kinases / chemistry*
  • Cyclin-Dependent Kinases / metabolism
  • HeLa Cells
  • Humans
  • Kinetics
  • Nuclear Proteins / chemistry
  • Nuclear Proteins / metabolism
  • Phenylenediamines / chemistry*
  • Phenylenediamines / pharmacology
  • Phosphorylation
  • Promoter Regions, Genetic
  • Protein Processing, Post-Translational
  • Protein Structure, Tertiary
  • Pyrimidines / chemistry*
  • Pyrimidines / pharmacology
  • RNA Polymerase II / chemistry
  • RNA Processing, Post-Transcriptional
  • Transcription Factors / chemistry
  • Transcription Factors / metabolism
  • Transcription Initiation, Genetic*
  • Transcriptional Elongation Factors / chemistry
  • Transcriptional Elongation Factors / metabolism

Substances

  • Antineoplastic Agents
  • NSMF protein, human
  • Nuclear Proteins
  • Phenylenediamines
  • Pyrimidines
  • SUPT5H protein, human
  • THZ1 compound
  • Transcription Factors
  • Transcriptional Elongation Factors
  • Cyclin-Dependent Kinases
  • cyclin-dependent kinase-activating kinase
  • RNA Polymerase II