CDKN1C negatively regulates RNA polymerase II C-terminal domain phosphorylation in an E2F1-dependent manner.
CDKN1C is a cyclin-dependent kinase inhibitor and is a candidate tumor suppressor gene. We previously found that the CDKN1C protein represses E2F1-driven transcription in an apparent negative feedback loop. Herein, we explore the mechanism by which CDKN1C represses transcription. We find that adenoviral-mediated overexpression of CDKN1C leads to a dramatic reduction in phosphorylation of the RNA polymerase II (pol II) C-terminal domain (CTD). RNA interference studies demonstrate that this activity is not an artifact of CDKN1C overexpression, because endogenous CDKN1C mediates an inhibition of RNA pol II CTD phosphorylation in HeLa cells upon treatment with dexamethasone. Surprisingly, we find that CDKN1C-mediated repression of RNA pol II phosphorylation is E2F1-dependent, suggesting that E2F1 may direct CDKN1C to chromatin. Chromatin immunoprecipitation assays demonstrate that CDKN1C is associated with E2F1-regulated promoters in vivo and that this association can dramatically reduce the level of RNA pol II CTD phosphorylation at both Ser-2 and Ser-5 of the C-terminal domain repeat. In addition, we show that CDKN1C interacts with both CDK7 and CDK9 (putative RNA pol II CTD kinases) and that CDKN1C blocks their ability to phosphorylate a glutathione S-transferase-CTD fusion protein in vitro. E2F1 and CDKN1C are found to form stable complexes both in vivo and in vitro. Molecular studies demonstrate that the E2F1-CDKN1C interaction is mediated by two E2F domains. A central E2F1 domain interacts directly with CDKN1C, whereas a C-terminal E2F1 domain interacts with CDKN1C via interaction with Rb. The results presented in this report highlight a novel mechanism of tumor suppression by CDKN1C.
Ma
Yihong
Y
Molecular Oncology Program, H. Lee Moffitt Cancer Center and Research Institute, Tampa, Florida 33612.
Chen
Lu
L
Molecular Oncology Program, H. Lee Moffitt Cancer Center and Research Institute, Tampa, Florida 33612.
Wright
Gabriela M
GM
Molecular Oncology Program, H. Lee Moffitt Cancer Center and Research Institute, Tampa, Florida 33612.
Pillai
Smitha R
SR
Molecular Oncology Program, H. Lee Moffitt Cancer Center and Research Institute, Tampa, Florida 33612.
Chellappan
Srikumar P
SP
Molecular Oncology Program, H. Lee Moffitt Cancer Center and Research Institute, Tampa, Florida 33612.
Cress
W Douglas
WD
Molecular Oncology Program, H. Lee Moffitt Cancer Center and Research Institute, Tampa, Florida 33612. Electronic address: douglas.cress@moffitt.org.
D002453:Cell Cycle; D045744:Cell Line, Tumor; D047369:Chromatin Immunoprecipitation; D050761:Cyclin-Dependent Kinase Inhibitor p57 / Q000378:metabolism*; D050687:E2F1 Transcription Factor / Q000378:metabolism*; D015971:Gene Expression Regulation, Enzymologic; D006367:HeLa Cells; D006801:Humans; D008954:Models, Biological; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D017434:Protein Structure, Tertiary; D012319:RNA Polymerase II / Q000737:chemistry / Q000502:physiology*; D014158:Transcription, Genetic
C493964:CDKN1C protein, human; D050761:Cyclin-Dependent Kinase Inhibitor p57; D050687:E2F1 Transcription Factor; D012319:RNA Polymerase II