One of the most misunderstood aspects of human Mediator function relates to the CDK8 submodule, a 600 kDa assembly that can reversibly associate with Mediator and contains the proteins CDK8, cyclin C, MED12, and MED13. One reason for confusion is that early preparations of human Mediator (when Mediator was provisionally called TRAP, ARC, or DRIP) actually contained a mixture of Mediator and CDK8-Mediator [3
]. Due to the presence of Mediator, these preparations exhibited co-activator function in reconstituted transcription assays. Only upon biochemical separation of CDK8-Mediator and Mediator itself was it established that Mediator was responsible for activator-dependent transcription, whereas CDK8-Mediator was unable to activate transcription [31
]. Subsequent studies indicated the CDK8 submodule represses activated transcription by binding to Mediator and inhibiting its ability to recruit and activate pol II [29
]. The Boyer lab identified another means by which the CDK8 submodule can repress transcription: through interactions with the G9a methyltransferase, which catalyzes histone H3K9 methylation, a repressive chromatin mark. G9a-dependent repression is particularly sensitive to the CDK8 submodule component MED12, which controls the recruitment of G9a to a subset of silenced neuronal genes in HeLa cells [32
]. Notably, MED12 knockdown does not appear to impact global levels of H3K9 methylation, suggesting a gene-selective role for MED12 in these cells.
The results linking MED12 to the G9a methyltransferase provided a key link between CDK8-Mediator and histone modifications; another link was revealed upon isolation and biochemical characterization of a distinct CDK8-Mediator complex that contained the TRRAP and GCN5L polypeptides [33
]. This “T/G-Mediator” complex directly modified chromatin templates, yet its chromatin-modifying activity does not simply result from the GCN5L acetyltransferase. Rather, CDK8 itself possesses intrinsic chromatin-modifying activity as a histone H3S10 kinase. In fact, CDK8 and GCN5L function synergistically within T/G-Mediator to phosphorylate and acetylate histone H3 (H3S10P/K14Ac) in vitro
and in cells; CDK8 knockdown resulted in global reduction in H3 S10P/K14Ac levels, suggesting T/G-Mediator is a widespread regulator of this H3 mark [33
]. Coupled H3 S10/K14 phosphorylation and acetylation correlates with activation of at least a subset of genes [34
]. Thus, these data identified a biochemical role for CDK8 in the activation of transcription: as an H3S10 kinase, CDK8 likely helps to establish a chromatin environment favorable for transcription. Potentially, the chromatin-modifying activity of T/G-Mediator might function together with chromatin remodelers, such as SWI/SNF, to help remodel the promoter prior to transcription initiation. Given the existence of multiple H3S10 kinases in mammalian cells [38
], it is currently unclear how many genes might rely upon CDK8-dependent H3S10 phosphorylation for their expression.
CDK8 has also been shown to function as a positive regulator at specific p53-regulated genes such as p21
]. Upon p53 activation by UVC, Mediator subunits occupy, but do not activate, the p21
promoter. By contrast, stimulation of p53 with the drug nutlin 3 strongly induces p21
expression. Mediator occupancy at p21
remains constant under these different conditions (UVC or nutlin 3), yet a significant increase in CDK8 submodule occupancy is observed only after nutlin 3 treatment [44
]. Interestingly, changes in CDK8 occupancy at p21
(UVC vs. nutlin 3 treatment) also correlate with changes in occupancy of other PEC factors. These results suggest the CDK8 submodule might promote PEC assembly under some conditions, perhaps through an indirect mechanism involving its kinase activity.
Additional evidence that CDK8 can serve as a co-activator derives from studies that focused on SMAD-dependent transcriptional activation in response to bone morphogenetic protein (SMAD1/5) or transforming growth factor beta (SMAD2/3) signaling. CDK8-dependent phosphorylation of the linker region within SMAD1/5 or SMAD2/3 complexes appears to activate these transcription factor assemblies [45
]. However, CDK8-dependent phosphorylation also targets SMAD complexes for proteasomal degradation, thereby also restraining transcriptional activation [45
]. A similar mechanism appears to operate with transcriptional activation by the Notch intracellular domain (ICD) enhancer complex. CDK8 phosphorylation of the Notch ICD activates this assembly while concomitantly targeting it for degradation [47
]. CDK8 also phosphorylates the E2F1 transcription factor; unlike the SMADs or the Notch enhancer complex, however, CDK8-dependent modification is linked to repression of E2F1 activity [48
]. As E2F1, in turn, represses β-catenin activity, this observation helped to identify CDK8 as a key colorectal cancer oncoprotein that regulates the β-catenin pathway [48
]. Importantly, oncogenesis is linked specifically to CDK8 kinase activity, as kinase-dead CDK8 mutants cannot transform cells or activate β-catenin target genes. These examples (SMAD1/5, SMAD2/3, Notch ICD, E2F1) reveal that the kinase activity of CDK8 is capable of positive or negative regulation of transcription factor activity (Box 2
). The number of functionally diverse targets for CDK8 suggests that regulation of its kinase activity must be strictly enforced. Recent insights regarding CDK8 kinase regulation are described in Box 2
Finally, a role for the CDK8 submodule in regulating transcription elongation was recently reported [50
]. Interestingly, this novel regulatory function for CDK8 requires positive transcription elongation factor b (P-TEFb). The CDK8 submodule appears to coordinate P-TEFb loading and activity at a number of serum-response genes, including FOS
, and EGR3
. At these genes, CDK8 helps control pol II CTD phosphorylation and elongation, most likely by regulating P-TEFb kinase activity. Significantly, these results provide the first clear evidence that CDK8 can impact pol II CTD phosphorylation (albeit perhaps indirectly, through P-TEFb) in human cells. Indeed, cellular knockdown of CDK8 yielded no detectable change in global pol II CTD phosphorylation levels (S5P or S2P), suggesting that the CDK8-dependent effect on pol II CTD phosphorylation is gene-specific [50
]. Precisely how CDK8 affects P-TEFb-directed pol II CTD phosphorylation at serum response genes remains unknown; regulation might occur via CDK8-dependent phosphorylation of P-TEFb or simply by CDK8 subcomplex–P-TEFb association. CDK8 and P-TEFb might also cooperatively regulate SMAD1/5 or SMAD 2/3 activity and stability, as both CDK8 and P-TEFb can modify the same regulatory sites within SMAD transcription factor complexes [45
Given the multiple and functionally diverse roles for the CDK8 submodule in gene regulation, a model consistent with the existing data involves the CDK8 submodule serving as a checkpoint for transcription (). A general feature of this model is that CDK8-Mediator provides a platform from which a variety of functionally divergent outcomes can be initiated. The reversible association of the CDK8 submodule with Mediator provides a facile means to regulate pol II initiation events, perhaps even resulting in long-term silencing of transcription. The ability of CDK8 to regulate transcription factor stability also implies a checkpoint role in that it would limit the duration that an activator remains stably bound at the promoter. These two distinct regulatory mechanisms result in similar functional outcomes in that each serves to check and restrain activated transcription. In each circumstance, re-activation of transcription might involve i) dissociation of the CDK8 submodule from Mediator, enabling subsequent recruitment of another pol II enzyme, or ii) subsequent binding of a newly-translated transcription factor to the promoter.
Figure 3 A framework for understanding how Mediator might function to regulate transcription. A) CDK8-Mediator provides a platform for multiple, functionally divergent events. Upon binding the TRRAP and GCN5L polypeptides, this assembly (provisionally called T/G-Mediator) (more ...)