Suppression of Mediator is regulated by Cdk8-dependent Grr1 turnover of the Med3 coactivator

Abstract

Mediator, an evolutionary conserved large multisubunit protein complex with a central role in regulating RNA polymerase II-transcribed genes, serves as a molecular switchboard at the interface between DNA binding transcription factors and the general transcription machinery. Mediator subunits include the Cdk8 module, which has both positive and negative effects on activator-dependent transcription through the activity of the cyclin-dependent kinase Cdk8, and the tail module, which is required for positive and negative regulation of transcription, correct preinitiation complex formation in basal and activated transcription, and Mediator recruitment. Currently, the molecular mechanisms governing Mediator function remain largely undefined. Here we demonstrate an autoregulatory mechanism used by Mediator to repress transcription through the activity of distinct components of different modules. We show that the function of the tail module component Med3, which is required for transcription activation, is suppressed by the kinase activity of the Cdk8 module. Med3 interacts with, and is phosphorylated by, Cdk8; site-specific phosphorylation triggers interaction with and degradation by the Grr1 ubiquitin ligase, thereby preventing transcription activation. This active repression mechanism involving Grr1-dependent ubiquitination of Med3 offers a rationale for the substoichiometric levels of the tail module that are found in purified Mediator and the corresponding increase in tail components seen in cdk8 mutants.

Analysis of Polygenic Mutants Suggests a Role for Mediator in Regulating Transcriptional Activation Distance in Saccharomyces cerevisiae

“…Our isolation of a grr1 mutation also implicates Mediator based on recent evidence that Grr1 destabilizes Med3 in a Cdk8-dependent fashion (Gonzalez et al 2014). However, in contrast to that study, we did not detect elevated levels of Med3 in a grr1D background.…”

“…Our results, showing a modestly increased level of Med3 recruitment have not distinguished between recruitment of the tail domain or the entire complex. Tests of additional Mediator components will shed light on the role of the rest of the complex in the regulation of activation distance.Our isolation of a grr1 mutation also implicates Mediator based on recent evidence that Grr1 destabilizes Med3 in a Cdk8-dependent fashion (Gonzalez et al 2014). However, in contrast to that study, we did not detect elevated levels of Med3 in a grr1D background.…”

“…However, in contrast to that study, we did not detect elevated levels of Med3 in a grr1D background. This difference may be because we measured Med3 levels with the MED3 gene in single copy, while Gonzalez et al (2014) made their observations when MED3 was overexpressed from a plasmid. Since we failed to The CCW12 promoter is a previously characterized site where Mediator is known to associate.…”

The Mediator Kinase Module Restrains Epidermal Growth Factor Receptor Signaling and Represses Vulval Cell Fate Specification in Caenorhabditis elegans

“…As S. cerevisiae CDK8 inhibits the Mediator tail module triad composed of MED2, MED3, and MED15 (van de Peppel et al 2005;Gonzalez et al 2014), we hypothesized that their putative C. elegans orthologs MDT-29, MDT-27, and MDT-15 (Bourbon 2008), might be targets for CKM inhibition. mdt-15 and mdt-29 knockdown had no effect on vulva formation in wild-type animals (i.e., causing neither Muv nor Vul phenotypes and displaying normal VPC induction; Table 1), but significantly reduced the Muv penetrance of mdt-13/let-19 mutants (Figure 6A); mdt-27 RNAi caused a similar trend ( Figure 6A).…”

“…(Fryer et al 2004;Donner et al 2007;Firestein et al 2008;Zhou et al 2012). Furthermore, in Saccharomyces cerevisiae, the CKM regulates the activity of the Mediator tail module subunits MED2, MED3, and MED15 (van de Peppel et al 2005;Gonzalez et al 2014). However, whether such intra-Mediator signaling effects occur in metazoans and affect e.g., animal development has not yet been tested.…”