A kinase subunit of the human mediator complex, CDK8, positively regulates transcriptional activation

Abstract

The human t hyroid hormone r eceptor-a ssociated p roteins (TRAP)/Mediator and related complexes mediate transcription through regulatory factors. To further understand the structural and functional diversity of these complexes we established three HeLa cell lines each expressing one of three epitope-tagged human TRAP/Mediator subunits, MED6, MED7, and CDK8 and isolated the complexes in which these subunits were contained by affinity and HPLC-gel filtration chromatography. The largest complexes from each cell line had a molecular mass of 1.5 MDa and possessed almost identical subunit compositions; we designated these complexes T RAP/ M ediatorl ike c omplex 1 (TMLC1). Two potential subcomplexes were additionally observed: a 1-MDa complex from the CDK8-cell line (TMLC2) and a 600-kDa complex from the MED6-cell line (TMLC3). All three complexes regulated transcription in vitro ; TMLC1 and TMLC3 augmented transcriptional activation, whereas TMLC2 repressed it. TMLC1 and TMLC2 phosphorylated RNA polymerase II (Pol II), but TMLC3 did not. Furthermore, TMLC1 predominantly interacted with the general transcription factors TFIIE, TFIIF, and TFIIH, which function during transcription initiation and the transition to elongation. In a final experiment, knockdown of CDK8 using RNA interference prevented transcriptional activation by Gal4-VP16 in a luciferase-assay. This, together with the effect of TMLC1 on transcription in vitro , suggests that CDK8 play positive roles in transcriptional activation.

Mediator Complex Recruits Epigenetic Regulators via Its Two Cyclin-dependent Kinase Subunits to Repress Transcription of Immune Response Genes

“…According to the current consensus, CDK8 generally functions as a negative regulatory component of Mediator (16,17). However, recent studies have shown that CDK8 also plays a positive role in transcriptional regulation (18,19). We observed that at least two Mediator subcomplexes con-tain human CDK8 yet exert opposite effects on transcriptional activation.…”

“…Previously, based on in vitro transcription studies, Mediator was thought to act as a negative transcriptional regulator, and S-Mediator was thought to act as a positive regulator (11). However, recent studies demonstrated positive transcriptional regulatory properties of Mediator, suggesting that it may require further classification into additional subtypes (3,19,23). The molecular mechanisms of Mediator's activities in transcriptional repression remain elusive.…”

“…These nuclear extracts were prepared from large cultures of previously prepared HA/FLAG-tagged CDK8 (HF-CDK8)-and CDK19 (HF-CDK19)-expressing stable cell lines previously established by our group (19,25). Each CDK-containing Mediator complex was purified using anti-FLAG M2 agarose beads (Fig.…”

“…DNA microarray analysis of the target genes of each CDK complex revealed extensive overlap in their target gene preferences (26). Therefore, we decided to explore the idea that Mediators play a pivotal role in transcriptional regulation (19,25).…”

RNA Polymerase II C-terminal Heptarepeat Domain Ser-7 Phosphorylation Is Established in a Mediator-dependent Fashion

“…CDK8 and the paralogue CDC2L6 are associated with the Mediator complex (13,14). CDK8 has mostly been associated with repression of transcription, yet there are also reports that specific genes are activated by it (15,16). CDK9 is thought to release RNAPII that is paused by negative inhibitory factors 5,6-dichloro-1-␤-D-ribofuranosylbenzimidazole (DRB) sensitivity-inducing factor and negative elongation factor some 30 -40 base pairs downstream of the transcription start sites.…”