Cyclin-Dependent Kinase 8 Positively Cooperates with Mediator To Promote Thyroid Hormone Receptor-Dependent Transcriptional Activation

Abstract

Mediator is a multisubunit assemblage of proteins originally identified in humans as a coactivator bound to thyroid hormone receptors (TRs) and essential for thyroid hormone (T3)-dependent transcription. Cyclin-dependent kinase 8 (CDK8), cyclin C, MED12, and MED13 form a variably associated Mediator subcomplex (termed the CDK8 module) whose functional role in TR-dependent transcription remains unclear. Using in vitro and cellular approaches, we show here that Mediator complexes containing the CDK8 module are specifically recruited into preinitiation complexes at the TR target gene type I deiodinase (DioI) together with RNA polymerase II (Pol II) in a TR- and T3-dependent manner. We found that CDK8 is essential for robust T3-dependent Dio1 transcription and that CDK8 knockdown via RNA interference decreased Pol II occupancy, and also the recruitment of the Pol II kinase CDK9, at the DioI promoter. Chromatin immunoprecipitation revealed CDK8 occupancy at the DioI promoter concurrent with active transcription, thus suggesting CDK8 involvement in transcriptional reinitiation. Mutagenesis assays showed that CDK8 kinase activity is necessary for full T3-dependent DioI activation, whereas in vitro kinase studies indicated that CDK8 may contribute to Pol II phosphorylation. Collectively, our data suggest CDK8 plays an important coactivator role in TR-dependent transcription by promoting Pol II recruitment and activation at TR target gene promoters.

We thank Krassimir Yankulov for providing the recombinant baculovirus expressing CDK8 and cyclin C, Yoshi Ohkuma for providing the CDK8 mutant expression vector, Pradeep Pandey for the pCIN4-MED17 vector, and Ravi Vijayvargia for generating Mediator siRNAs.

This work was funded by a grant from the National Institutes of Health (DK054030) awarded to J.D.F.