Characterizing the roles of the CDK8 module in human Mediator structure and function

The human Mediator complex is a multi-subunit co-regulator that serves as a transcriptional hub, relaying signals from DNA binding activators to the general transcription machinery to regulate RNA polymerase II (RNAPII) function. Despite its importance in the regulation of mRNA production, little is known about the mechanisms by which Mediator operates. One mechanism known to dictate Mediator function is its interaction with a four-protein "CDK8 subcomplex" made up of the kinase CDK8, cyclin C, and two larger proteins of unknown function named MED12 and MED13. Whereas core Mediator is a potent co-activator, CDK8-Mediator strongly represses RNAPII transcription. To better define the regulatory roles of the CDK8 subcomplex, we express and purify a recombinant human CDK8 subcomplex, as well as isolate an endogenous CDK8 subcomplex (eK8) from human cells. We identify a kinase activating function within MED12, distinguishing it biochemically from MED13 within the subcomplex. We also uncover three novel CDK8 kinase substrates; MED13, histone H3, and CDK8 itself. Moreover, mass spectrometry analysis of eK8 reveals associated factors, including the TRiC chaperonin, which may help control its biological function. In support of this, electron microscopy analysis suggests TRiC sequesters the CDK8 subcomplex and kinase assays reveal eK8 has altered kinase substrate specificity compared to the recombinant subcomplex.;Past studies suggested CDK8 kinase activity is required for its repressive function. Using a reconstituted transcription system, we demonstrate that in fact MED12 and MED13 are critical for subcomplex-dependent repression whereas CDK8 kinase activity is not. Structural and biochemical studies confirm the CDK8 module binds the Mediator leg domain via the MED13 subunit, and this module-Mediator association precludes RNAPII. Collectively, these results reveal the CDK8 subcomplex functions as a switch that controls the Mediator-RNAPII interaction to regulate transcription initiation and re-initiation events. As Mediator is required for expression of virtually all protein-coding genes, this may reflect a common mechanism by which activated transcription is shut down in human cells.