Genome-Wide Location of the Coactivator Mediator: Binding without Activation and Transient Cdk8 Interaction on DNA

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.

Section: Cdk8supporting

confidence: 89%

mentioning

confidence: 99%

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

González

Hamidi

Sol

et al. 2014

“…However, recent results suggest that the kinase module can play a role in transcriptional activation as well as repression (20,21). An exclusively repressive function would be difficult to reconcile with the observation that the genome-wide occupancy profiles of Cdk8 and Med13 characterized by ChIP match that of the core mediator complex (69,70). Our results support an essential and direct function for the Med12 and Med13 subunits in the activation of Wg target genes.…”

Section: Discussionmentioning

confidence: 51%

Pygopus activates Wingless target gene transcription through the mediator complex subunits Med12 and Med13

Carrera

Janody

Leeds

et al. 2008

120

112

Wnt target gene transcription is mediated by nuclear translocation of stabilized ␤-catenin, which binds to TCF and recruits Pygopus, a cofactor with an unknown mechanism of action. The mediator complex is essential for the transcription of RNA polymerase II-dependent genes; it associates with an accessory subcomplex consisting of the Med12, Med13, Cdk8, and Cyclin C subunits. We show here that the Med12 and Med13 subunits of the Drosophila mediator complex, encoded by kohtalo and skuld, are essential for the transcription of Wingless target genes. kohtalo and skuld act downstream of ␤-catenin stabilization both in vivo and in cell culture. They are required for transcriptional activation by the N-terminal domain of Pygopus, and their physical interaction with Pygopus depends on this domain. We propose that Pygopus promotes Wnt target gene transcription by recruiting the mediator complex through interactions with Med12 and Med13.Drosophila ͉ kinase module ͉ kohtalo ͉ skuld ͉ Wnt

“…Moreover, genomewide profiling indicates that Mediator can associate with gene-coding regions, suggesting that a portion of copurifying Mediator may even be in elongating pol II complexes (36,37). Purification of a tagged Mediator subunit under cross-linking conditions might distinguish between some of these possibilities.…”

Section: Discussionmentioning

confidence: 99%

Protein characterization of Saccharomyces cerevisiae RNA polymerase II after in vivo cross-linking

Tardiff

Abruzzi

Rosbash

2007

To characterize proteins associated with active transcription complexes, we purified RNA polymerase II (pol II) from Saccharomyces cerevisiae after fixing live cells with formaldehyde. The approach mimics ChIP and requires solubilizing cross-linked complexes with sonication. Pol II was affinity-purified, and associated proteins were identified by MS. Several classes of proteins depended on cross-linking, including Mediator, general transcription factors, elongation factors, ribonucleoprotein particle (RNP) proteins, and histones. A tagged RNP protein reciprocally purified pol II under identical cross-linking conditions, and the association between RNP proteins and pol II was largely RNase-sensitive. The data indicate that the cross-linked Pol II purification contains elongating pol II with associated nascent RNP. Consistent with this view, some elongation factors no longer associate with pol II after inactivation of transcription in the temperature-sensitive pol II mutant, rpb1-1. Taken together, our data suggest that the cross-linked pol II purification contains a mixed population of pol II, including initiating pol II and elongating pol II.mass spectrometry ͉ nascent RNP ͉ RNA processing ͉ transcription ͉ affinity purification P urification and mass spectrometric analysis of protein complexes is a ubiquitous approach in modern molecular biology (1). The identification of interacting proteins and their subsequent characterization provides valuable insight into the complexes that carry out essential cellular functions. Purifications can use multiple biochemical fractionations (e.g., ion exchange, gradients, gel filtration, and affinity purification) or a streamlined tandem affinity purification (TAP) approach (2). Conventional purification methods, however, often preclude the accurate identification of in vivo-relevant complexes. Major complications include dissociation of protein complexes, as a function of off-rates and extract dilution. Moreover, strong affinities can promote in vitro associations that do not exist in vivo. These can occur as a consequence of protein exchange and the absence of subcellular localization. Finally, major complexes may be insoluble, or harsh methods required to effect solubility can perturb complex composition. Ribonucleoprotein particles (RNPs) and especially transcription complexes are particularly subject to these caveats.The synthesis of protein-encoding mRNAs is directed by RNA polymerase II (pol II), an extensively studied macromolecular machine. The composition of pol II is dynamic and changes with the stage of transcription, namely, initiation, elongation, and termination (3, 4). Difficulties with complex purification underlie controversies, for example, to what extent initiation occurs by stepwise assembly of a preinitiation complex on DNA. Moreover, elongating pol II is tightly associated with DNA, which makes its characterization challenging (5).ChIP is a major tool for characterizing DNA-associated proteins and complexes, including transcription and cotranscriptional ...