Genetic Analysis Of Mammalian Cdk7 Kinase Submodule Of Tfiih: Uncoupling Of Rnapii Ctd Serine 5 Phosphorylation And Transcription

The Cdk7 submodule has been suggested to function as a general activator of RNA Polymerase II (RNAPII) transcription by phosphorylating Serine 5 of the C‐terminal domain (CTD) and of cell cycle through T‐loop phosphorylation of Cdks. Genetic analyses in mammalian cells have suggested that Cdk7 is not essential in post‐mitotic cells, and indeed our recent results show that downregulation of Cdk7 is associated with adipogenesis, and suggest that Cdk7 acts as a roadblock to adipogenesis through inhibitory phosphorylation of PPARγ on Serine 112. We analyzed cell cycle and transcriptional changes following loss‐of‐function of Cdk7 kinase in mouse fibroblasts (MEFs) and human cancer cells. Our results show that in both exponentially growing MEFs and U2OS osteosarcoma cells loss of Cdk7 kinase, either through conditional knockout of Mat1 or Cdk7 knockdown, led to a G1 cell cycle arrest associated with decreased Cdk2 phosphorylation followed by cell death. These results are consistent with a role for Cdk7 in activating cell cycle Cdks. The adipogenic effects we previously noted following Mat1 loss in MEFs suggested that Cdk7 would not regulate general mRNA transcription. Comparison of expression profiles of Mat1‐/‐ and control MEFs using Affymetrix expression arrays demonstrated changes in less than 1% of mRNAs, indicating that Cdk7 activity is not crucial for transcription of most genes. However, analysis of RNAPII CTD phosphorylation demonstrated a dramatic drop in Ser‐5 phosphorylation and a concomitant decrease in Ser‐2 phosphorylation. The unexpected results demonstrate that the level of RNAPII CTD Ser‐5 phosphorylation is not correlated with general transcription. In addition, no significant defects were noted in RNA splicing or polyadenylation.