The CDK8-submodule of Mediator is a pleiotropic regulator of gene expression. On one hand, this protein complex has been characterized as a transcriptional repressor that inhibits RNAPII by multiple mechanisms, including: 1) kinase-independent allosteric regulation of Mediator-RNAPII interactions29
; 2) kinase-dependent inactivation of TFIIH via phosphorylation of its cyclin H subunit52
; 3) gene silencing via recruitment of histone methyl-transferases33
. In contrast, several reports indicate that CDK8 is a positive regulator of gene activity in other scenarios. In yeast, SRB10 promotes RNAPII activity in vitro and in vivo34
. In human cells, CDK8 depletion impairs activation of genes responsive to p53 and β-catenin27,35
. Additionally, CDK8-Mediator complexes can perform histone modifications associated with gene activation36
. In this report, we provide evidence that CDK8 is a direct positive regulator of transcription within the serum response network. We found that CDK8 is dispensable for RNAPII recruitment to promoters carrying pre-loaded RNAPII (FOS, EGR1) as well as promoters displaying inducible RNAPII recruitment (EGR2, EGR3). Furthermore, CDK8 depletion did not produce a drastic impairment in RNAPII escape from promoter regions, as significant amounts of RNAPII were detected throughout the intragenic regions of these genes upon activation. Instead, decreased IEG expression in CDK8 knock-down cells is explained by the appearance of slower elongation complexes containing hypophosphorylated RNAPII. The findings in this report represent a marked advance in our understanding of how CDK8 promotes gene activity, but also prompt more general questions about the role of CTD phosphorylation, CTD kinases and Mediator in mammalian gene expression control.
Here, we establish a strong correlation between CDK8 status, CTD phosphorylation and RNAPII elongation rates. Although some effect of CDK8 depletion on IEG RNA processing can not be fully discarded, our results indicate that most of the negative effects of CDK8 knock-down on IEG expression are explained by decreased elongation rates. In nuclear run-on experiments, CDK8 depletion decreases elongation rates by ~2-3-fold at the FOS locus and >10-fold at the EGR loci. Importantly, these differences are attenuated when additional time is allowed for elongation complexes derived from CDK8-depleted cells to proceed, with the FOS locus showing equivalent RNA synthesis and the EGR loci reaching up to 30–60% of wild type levels by 60 minutes. Interestingly, the impact of CDK8 on CTD phosphorylation is more pronounced at the EGR loci, further supporting a link between CTD phosphorylation and elongation rates. The notion that decreased transcription, rather than impaired processing, causes lower IEG mRNA expression in CDK8-depleted cells is reinforced by the fact that these cells accumulate lower levels of pre-mRNAs.
Despite the fact that CDK8 is a CTD-kinase and that its depletion leads to hypophosphorylated CTD and slower elongation complexes, a simple interpretation of these results is complicated by the fact that recruitment of two other CTD-kinases, CDK7 and CDK9, is impaired by CDK8 depletion. Until recently, the prevalent view on CTD phosphorylation maintained that CDK7 was the major Ser5-kinase and CDK9 the predominant Ser2-kinase. This view originated with the early characterization of CDK7 and the CDK9-like kinases in yeast16,53
. However, recent chemical genetic experiments in yeast and human cells showed that inhibition of CDK7 kinase activity does not result in global defects in Ser5 phosphorylation24,54,55
; but CDK7 may instead be the major kinase for Ser7, an alternate phospho-acceptor site within the CTD24,56
. Interestingly, CDK9 may be a relevant Ser5-kinase in certain contexts57,58
. CDK8 is known to phosphorylate both Ser2 and Ser5 in vitro23
, but its in vivo contributions remain to be elucidated. Interplay between the CTD kinases is supported by the fact that Ser5 phosphorylation by CDK7 ‘primes’ the CTD for subsequent Ser2 phosphorylation by CDK959
. Yeast Mediator was shown to be required for TFIIH recruitment to specific promoters and our data suggest that a similar interaction between CDK8-Mediator and TFIIH may also take place at IEGs50
. Additionally, our data indicate that P-TEFb association with the RNAPII machinery is also facilitated by CDK8-Mediator and we provide evidence of a physical interaction between the two complexes.
Though much is known regarding the critical role of P-TEFb in elongation control, little is known about the mechanisms by which this complex is recruited to allow for signalling- and gene-specific regulation. Several modes of P-TEFb recruitment have been described, including direct interactions with DNA-binding proteins or with the bromodomain protein BRD413,38,48,49,60
. Interestingly, BRD4 has been shown repeatedly to interact with Mediator and our results confirm this interaction. Furthermore, our results agree with those of Wu and Chiang who found that BRD4 associates with both CDK8-Mediator and core Mediator48
. In contrast, we find that P-TEFb associates preferentially with CDK8-Mediator and the free CDK8-submodule. These observations suggest that BRD4 and P-TEFb interact with Mediator by different means.
Early evidence that Mediator regulates RNAPII activity at post-recruitment steps was demonstrated by Wang et al26
. Using MED23 null mice, they found that abolishing Mediator recruitment to the EGR1 locus drastically impairs transcriptional activation without affecting binding of TFIIA, TFIID, histone acetylation, methylation or association of the chromatin remodelling factor BRG1. Importantly, the defects in EGR1 transcription observed in MED23−/− cells could not be merely explained by reduced RNAPII association, as a significant fraction of RNAPII remained associated with the promoter in mutant cells. Thus, they concluded that Mediator affected both recruitment and post-recruitment steps. Given that RNAPII occupancy remains unaffected in CDK8 knock-down cells, we have been able to more clearly define the contribution of Mediator to post-recruitment steps. A significant fraction of MED23 remains associated with the promoter upon CDK8 depletion, which may explain why we do not observe any recruitment defects. Instead, most effects of CDK8 knock-down on IEG expression can be explained by impaired P-TEFb recruitment. This notion is supported by the fact that CDK9 inhibition by flavopiridol resembles (albeit with more penetrance) the effects of CDK8 knock-down. Given past results that demonstrate mutually exclusive association of CDK8-submodule and RNAPII with Mediator28,29
, our data further suggest dynamic association of the CDK8-submodule at the promoter of active serum response genes. Taken together, these results invoke a mechanism involving CDK8-submodule exchange at the promoter to regulate RNAPII and P-TEFb association within the transcriptional apparatus at post-recruitment steps.
CDK8 is a potent oncogene in colon cancer. CDK8 overexpression promotes cell proliferation, anchorage-independent growth, and tumor growth in xenografts35
. Part of these effects can be attributed to the fact that CDK8 promotes transcription mediated by the oncogenic transcription factor β-catenin/TCF35
. Here, we report that CDK8 is a potent positive regulator of the serum response network, which has been implicated in multiple tumorigenic phenotypes. The precise contributions of the β-catenin and serum response networks to the overall oncogenic effects of CDK8 overexpression await further experimentation. Whereas the transformation-inducing effects of β-catenin can be effectively blocked by overexpression of a dominant negative version of its binding partner TCF35
, the equivalent task for the serum response network is not straightforward. Upon serum stimulation, ERKs translocate to the nucleus and activate a myriad of transcription factors, including multiple members of the Ets family, MYC and C/EBP-β39
. Possible functional redundancy within the Ets family will impose a combinatorial genetic approach to determine their precise interaction with CDK8 during tumor development. Nonetheless, the fact that many MAPK-regulated transcription factors and their target genes have well demonstrated roles in cancer development will make this a worthy task that would lead to a detailed understanding of the oncogenic effects of CDK8 overexpression.