Mediator is a large complex containing up to 30 subunits that consist of four modules each: head, middle, tail and CDK/Cyclin. Recent studies have shown that CDK8, a subunit of the CDK/Cyclin module, is one of the key subunits of Mediator that mediates its pivotal roles in transcriptional regulation. In addition to CDK8, CDK19 was identified in human Mediator with a great deal of similarity to CDK8 but was conserved only in vertebrates. Previously, we reported that human CDK19 could form the Mediator complexes independent of CDK8. To further investigate the in vivo transcriptional activities of the complexes, we used a luciferase assay in combined with siRNA‐mediated knockdown to show that CDK8 and CDK19 possess opposing functions in viral activator VP16‐dependent transcriptional regulation. CDK8 supported transcriptional activation, whereas CDK19, however, counteracted it. In this study, we further characterized CDK19. We used microarrays to identify target genes for each CDK, and we selected six genes: two target genes of CDK8, two target genes of CDK19 and two genes that were targets for both. Surprisingly, it turned out that both CDKs bound to all six target genes, regardless of their effects in transcription upon binding, suggesting Mediator as a context‐specific transcriptional regulator.

Background: Two CDK subunits of the Mediator complex play pivotal roles in transcription by a mechanism that has not yet been elucidated. Results: The histone arginine methyltransferase PRMT5 is a Mediator CDK-interacting protein. Conclusion:Mediator-associated PRMT5 symmetrically dimethylates histone H4 arginine 3, and this might cause transcriptional repression. Significance: This work enables further exploration of Mediator functions in transcriptional repression.

The Mediator complex (Mediator) plays key roles in transcription and functions as the nexus for integration of various transcriptional signals. Previously, we screened for Mediator cyclin-dependent kinase (CDK)-interacting factors and identified three proteins related to chromatin regulation. One of them, SUZ12 is required for both stability and activity of Polycomb Repressive Complex 2 (PRC2). PRC2 primarily suppresses gene expression through histone H3 lysine 27 trimethylation, resulting in stem cell maintenance and differentiation; perturbation of this process leads to oncogenesis. Recent work showed that Mediator contributes to the embryonic stem cell state through DNA loop formation, which is strongly associated with chromatin architecture; however, it remains unclear how Mediator regulates gene expression in cooperation with chromatin regulators (i.e. writers, readers and remodelers). We found that Mediator CDKs interact directly with the PRC2 subunit EZH2, as well as SUZ12. Known PRC2 target genes were deregulated by Mediator CDK knockdown during neuronal differentiation, and both Mediator and PRC2 complexes co-occupied the promoters of developmental genes regulated by retinoic acid. Our results provide a mechanistic link between Mediator and PRC2 during neuronal differentiation.

In eukaryotes, holo-Mediator consists of four modules: head, middle, tail, and CDK/Cyclin. The head module performs an essential function involved in regulation of RNA polymerase II (Pol II). We studied the human head module subunit MED17 (hMED17). Recent structural studies showed that yeast MED17 may function as a hinge connecting the neck and movable jaw regions of the head module to the fixed jaw region. Luciferase assays in hMED17-knockdown cells showed that hMED17 supports transcriptional activation, and pulldown assays showed that hMED17 interacted with Pol II and the general transcription factors TFIIB, TBP, TFIIE, and TFIIH. In addition, hMED17 bound to a DNA helicase subunit of TFIIH, XPB, which is essential for both transcription and nucleotide excision repair (NER). Because hMED17 associates with p53 upon UV-C irradiation, we treated human MCF-7 cells with either UV-C or the MDM2 inhibitor Nutlin-3. Both treatments resulted in accumulation of p53 in the nucleus, but hMED17 remained concentrated in the nucleus in response to UV-C. hMED17 colocalized with the NER factors XPB and XPG following UV-C irradiation, and XPG and XPB bound to hMED17 in vitro. These findings suggest that hMED17 may play essential roles in switching between transcription and NER.

The Mediator complex (Mediator) is conserved among eukaryotes and is comprised of head, middle, tail and CDK/cyclin modules. The head module has received the most attention because its interaction with RNA polymerase II (Pol II) and the general transcription factors TFIIH and TBP facilitates phosphorylation of the carboxy-terminal domain (CTD) of the largest subunit of Pol II. We studied the human head module subunit hMED18 to elucidate how Mediator is involved in both transcriptional activation and repression. siRNA-mediated hMED18 depletion augmented transcription, indicating that hMED18 functions in transcriptional repression. Treatment of cells with two histone deacetylase (HDAC) inhibitors, the HDAC inhibitor trichostatin A (TSA) and the SIRT inhibitor nicotinamide showed that this repression was not caused by those HDAC activities. A screen for hMED18-target genes showed that the promoters for cap RNA methyltransferase RNMT-activating mini protein (RAM/FAM103A1) and divalent metal transporter 1 (DMT1/SLC11A2) genes were bound by hMED18. Depletion of hMED18 showed hMED18 and the middle module subunit hMED1 were lost from the promoters of those genes, whereas the CDK/cyclin module subunit hCDK8 remained bound. This indicates a novel transcriptional repression mechanism of hMED18 mediated by hCDK8 and further a novel positive role of free CDK/cyclin module in transcriptional activation. [Correction added on 12 June 2014, after first online publication: SLC11A2 amended from SCL11A2.]

The Mediator complex consists of more than 20 subunits. This is composed of four modules: head, middle, tail and CDK/Cyclin. Importantly, Mediator complex is known to play pivotal roles in transcriptional regulation, but its molecular mechanisms are still elusive. Many studies, including our own, have revealed that CDK8, a kinase subunit of the CDK/Cyclin module, is one of the key subunits involved in these roles. Additionally, we previously demonstrated that a novel CDK component, CDK19, played similar roles. It is assumed that various factors that directly affect transcriptional regulation target these two CDKs; thus, we conducted yeast two-hybrid screenings to isolate the CDK19-interacting proteins. From a screening of 40 million colonies, we obtained 287 clones that provided positive results encoded mRNAs, and it turned out that 59 clones of them encoded nuclear proteins. We checked the reading frames of the candidate clones and obtained three positive clones, all of which encoded the transcriptional cofactors, Brahma-related gene 1, B-cell CLL/lymphoma 6 and suppressor of zeste 12 homolog. Intriguingly, these three cofactors are also related to chromatin regulation. Further studies demonstrated that those could bind not only to CDK19 but also to CDK8. These results help elucidate the functional mechanism for the mutual regulations between transcription and chromatin.