We have shown here that Cdk7 can act to establish the promoter-proximal pause through its control of the TFIIE-DSIF switch, and to release Pol II from the pause through its ability to activate Cdk9. Therefore, within the context of the Pol II transcription machinery, Cdk7 is both an effector CDK, which phosphorylates Pol II and other proteins directly involved in transcription; and an upstream regulator of Cdk9 (and, possibly, other transcriptional CDKs). The identification of Cdk7 as a Cdk9-activating kinase in human cells resolves a long-standing puzzle, and suggests that mammals might have only a single CAK for all CDKs that depend on T-loop phosphorylation, regardless of their function. In contrast, transcriptional CDKs in fungi are activated by a non-cyclin-dependent, single-subunit CAK, even in fission yeast, where the Cdk7 ortholog is a physiologic activator of cell-cycle CDKs22,40,41
To drive the cell cycle, CDKs work in sequences defined by the timed expression of different cyclins, and by checkpoints that ensure dependence of later events on completion of earlier ones42
. Although transcription by Pol II likewise involves multiple CDKs, mechanisms enforcing their order of action have been slow to emerge. Recent work in budding and fission yeast revealed one basis for sequential CDK function: recruitment of P-TEFb depends on activity of the TFIIH-associated kinase33,34
. In fission yeast, moreover, Ser7 phosphorylation of the Pol II CTD by the Cdk7 ortholog Mcs6 can promote subsequent phosphorylation by Cdk934,43
. Although the latter mechanism—CTD “priming” by an early-acting CDK—appears to be conserved in human cells44
, data presented here indicate that the former is unlikely to operate in metazoans; inhibition of Cdk7 in human cells did not diminish recruitment of Cdk9 to chromatin.
Instead, we uncovered a third way in which distinct CDK functions can be ordered in the Pol II transcription cycle—direct activation of one CDK by another—that is likely to be unique to metazoans. In human cells, P-TEFb is activated by Cdk7, as are CDKs involved in cell division4,45
. Therefore, cell proliferation and gene expression are controlled by CDK cascades with a common upstream activating kinase. During cell-cycle progression, different CDK—cyclin complexes are active at different times, and phosphorylate substrates in a temporal order determined by properties of both enzyme (distinct recognition motifs on different cyclins) and substrate (relative affinity for given CDK—cyclin pairs)46
. The individual subunits of TFIIH and P-TEFb are expressed constitutively, and apparently recruited to chromatin en bloc
. Our ChIP analyses of Cdk7 and Cdk9 suggest that a degree of temporal regulation might instead be achieved by T-loop phosphorylation during the transcription cycle: the ratios of phosphorylated-to-unphosphorylated isoforms at different positions on transcribed chromatin predict that the specific activity of both CDKs would increase along genes in a 5′ to 3′ direction. Consistent with this idea, a read-out of chromatin-associated CDK activity—Ser2 phosphorylation of the Rpb1 CTD—also increases towards the 3′ end, and is diminished when Cdk7 is inhibited. The decrease in the Ser2P: total Pol II ratio was modest relative to the drop in Cdk9-Thr186P: total Cdk9, perhaps because reduced Pol II density on transcribed chromatin lowered the effective concentration of the Cdk9 substrate (Rpb1 CTD), simultaneously with the reduction in specific activity of the CTD-modifying enzyme (Cdk9).
Cdk7 activity is required at two distinct execution points in G1 and G2 phases of the cell cycle4
. Cdk7 also influences the Pol II cycle at multiple points: promoting pausing by recruitment of DSIF and NELF to chromatin, and activating a downstream CDK to overcome the pause. Inefficient recruitment of DSIF and NELF would lower the threshold of P-TEFb activity required to overcome pausing, and thus might explain why pausing is attenuated by inhibition of Cdk7 even while Cdk9 is also indirectly inhibited. Moreover, that inhibition is not complete: wild-type Cdk9 had measurable activity in the absence of detectable Thr186 phosphorylation, and a Cdk9T186A
mutant also had basal activity towards Spt5 in vitro
(), which might suffice to support elongation when Cdk7 is inhibited. Although elongation is permitted under these circumstances, Pol II density in coding regions is generally diminished, steady-state levels of many transcripts are decreased, and RNA-processing is disrupted (5
, this report and unpublished observations). Antagonistic downstream effects elicited by the same kinase suggest incoherent feedforward ()—a network motif that produces biphasic response kinetics in cellular signaling pathways47
. In this case, the proposed delay between recruitment of elongation factors and activation of Cdk9 could ensure a transient pause, to promote processive transcription and proper loading of mRNA-processing machinery.
Our results raise the possibility that CDKs also act in similar fashion to enforce the stable pausing of Pol II in promoter-proximal regions of stringently regulated genes, together with known pause factors such as NELF1,2
. Like NELF depletion2,37
, selective inhibition of Cdk7 caused decreased Pol II crosslinking to chromatin at multiple genes, and deranged mRNA 3′-end formation (5
, this report). The model of elongation control we have proposed () appears “hard-wired” to produce a transient pause. The relative rates of the individual steps might be subject to differential regulation, however, which could shorten or lengthen the pause as needed. For example, T-loop phosphorylation of Cdk7—which appears to be a post-recruitment event at the genes we have analyzed—could accelerate phosphorylation of Pol II, Spt5 and TFIIE without affecting the rate of Cdk9 activation, and thereby increase pause duration. An intrinsically slow rate of Cdk9 T-loop phosphorylation might be further retarded, on the other hand, by negative regulatory factors or features of the chromatin landscape at specific genes, to generate a more durable pause. The chemical-genetic approach uncovered a CDK cascade at the core of the Pol II transcription machinery. It could now illuminate the regulatory capabilities of that module, and allow the identification of additional targets of both Cdk7 and Cdk9 within the transcription machinery.