Drosophila contains one (dCDK12) and humans contain two (hCDK12 and hCDK13) proteins that are the closest evolutionary relatives of yeast Ctk1, the catalytic subunit of the major elongation-phase C-terminal repeat domain (CTD) kinase in Saccharomyces cerevisiae, CTDK-I. However, until now, neither CDK12 nor CDK13 has been demonstrated to be a bona fide CTD kinase. Using Drosophila, we demonstrate that dCDK12 (CG7597) is a transcription-associated CTD kinase, the ortholog of yCtk1. Fluorescence microscopy reveals that the distribution of dCDK12 on formaldehyde-fixed polytene chromosomes is virtually identical to that of hyperphosphorylated RNA polymerase II (RNAPII), but is distinct from that of P-TEFb (dCDK9 + dCyclin T). Chromatin immunoprecipitation (ChIP) experiments confirm that dCDK12 is present on the transcribed regions of active Drosophila genes. Compared with P-TEFb, dCDK12 amounts are lower at the 5' end and higher in the middle and at the 3' end of genes (both normalized to RNAPII). Appropriately, Drosophila dCDK12 purified from nuclear extracts manifests CTD kinase activity in vitro. Intriguingly, we find that cyclin K is associated with purified dCDK12, implicating it as the cyclin subunit of this CTD kinase. Most importantly, we demonstrate that RNAi knockdown of dCDK12 in S2 cells alters the phosphorylation state of the CTD, reducing its Ser2 phosphorylation levels. Similarly, in human HeLa cells, we show that hCDK13 purified from nuclear extracts displays CTD kinase activity in vitro, as anticipated. Also, we find that chimeric (yeast/human) versions of Ctk1 containing the kinase homology domains of hCDK12/13 (or hCDK9) are functional in yeast cells (and also in vitro); using this system, we show that a bur1(ts) mutant is rescued more efficiently by a hCDK9 chimera than by a hCDK13 chimera, suggesting the following orthology relationships: Bur1 ↔ CDK9 and Ctk1 ↔ CDK12/13. Finally, we show that siRNA knockdown of hCDK12 in HeLa cells results in alterations in the CTD phosphorylation state. Our findings demonstrate that metazoan CDK12 and CDK13 are CTD kinases, and that CDK12 is orthologous to yeast Ctk1.
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"Next, to ask whether Xrn2 is a specific target of Cdk9 or a general CDK substrate, we measured the activity of purified Cdk7, Cdk12, or Cdk13 (Supplemental Fig. 2B) toward Flag-Xrn2. Cdk12 and Cdk13 form complexes with cyclin K and are thought to be Ser2 kinases (Bartkowiak et al. 2010) with roles in elongation and 3 ′ end formation (Davidson et al. 2014). The amount of each CDK was Figure 2. Xrn2 is a CDK substrate. "
[Show abstract][Hide abstract] ABSTRACT: The transcription cycle of RNA polymerase II (Pol II) is regulated at discrete transition points by cyclin-dependent
kinases (CDKs). Positive transcription elongation factor b (P-TEFb), a complex of Cdk9 and cyclin T1, promotes
release of paused Pol II into elongation, but the precise mechanisms and targets of Cdk9 action remain largely
unknown. Here, by a chemical genetic strategy, we identified
100 putative substrates of human P-TEFb, which
were enriched for proteins implicated in transcription and RNA catabolism. Among the RNA processing factors
phosphorylated by Cdk9 was the 5
exoribonuclease Xrn2, required in transcription termination by
Pol II, which we validated as a bona fide P-TEFb substrate in vivo and in vitro. Phosphorylation by Cdk9 or phos-
phomimetic substitution of its target residue, Thr439, enhanced enzymatic activity of Xrn2 on synthetic substrates
in vitro. Conversely, inhibition or depletion of Cdk9 or mutation of Xrn2-Thr439 to a nonphosphorylatable Ala
residue caused phenotypes consistent with inefficient termination in human cells: impaired Xrn2 chromatin
localization and increased readthrough transcription of endogenous genes. Therefore, in addition to its role in
elongation, P-TEFb regulates termination by promoting chromatin recruitment and activation of a
cotranscriptional RNA processing enzyme, Xrn2.
Preview · Article · Jan 2016 · Genes & development
"Among all members of the CDK super family, CDK12 deserves special attention because of its reported phosphorylation of the Pol II CTD on Ser2, an ability that is shared with CDK9 (Bartkowiak et al., 2010). To determine whether i-CDK9 also affects CDK12 kinase activity, we examined the abilities of affinity-purified Flag-tagged CDK12 (CDK12-F), CDK9 (CDK9-F) and their associated cyclin partners to phosphorylate GST-CTD in the presence of increasing amounts of i-CDK9. "
[Show abstract][Hide abstract] ABSTRACT: CDK9 is the kinase subunit of positive transcription elongation factor b (P-TEFb) that enables RNA polymerase (Pol) II's transition from promoter-proximal pausing to productive elongation. Although considerable interest exists in CDK9 as a therapeutic target, little progress has been made due to lack of highly selective inhibitors. Here, we describe the development of i-CDK9 as such an inhibitor that potently suppresses CDK9 phosphorylation of substrates and causes genome-wide Pol II pausing. While most genes experience reduced expression, MYC and other primary response genes increase expression upon sustained i-CDK9 treatment. Essential for this increase, the bromodomain protein BRD4 captures P-TEFb from 7SK snRNP to deliver to target genes and also enhances CDK9's activity and resistance to inhibition. Because the i-CDK9-induced MYC expression and binding to P-TEFb compensate for P-TEFb's loss of activity, only simultaneously inhibiting CDK9 and MYC/BRD4 can efficiently induce growth arrest and apoptosis of cancer cells, suggesting the potential of a combinatorial treatment strategy.
"These conclusions are supported by ChIP analysis of the proviral genome in 2D10 cells, which shows that the virus-encoded Tat protein recruits Ssu72, P-TEFb, and AFF4/SEC to the HIV-1 promoter concomitant with the release of NELF and movement of RNAPII elongation complexes containing Spt4, Spt5, and Spt6 into the proviral coding region. Interestingly, Tat, Ssu72, P-TEFb, and AFF4/SEC did not travel with RNAPII to the 39 end of the provirus, consistent with previous studies showing that P-TEFb does not colocalize with the peak of S2P–RNAPII at the 39 end of cellular genes (Bartkowiak et al. 2010). In yeast, Ssu72 is commonly associated with the S2P–S5P transition at gene terminator regions, whereas Rtr1 (human RPAP2) controls promoter-proximal S5P levels for many active genes. "
[Show abstract][Hide abstract] ABSTRACT: HIV-1 Tat stimulates transcription elongation by recruiting the P-TEFb (positive transcription elongation factor-b) (CycT1:CDK9) C-terminal domain (CTD) kinase to the HIV-1 promoter. Here we show that Tat transactivation also requires the Ssu72 CTD Ser5P (S5P)-specific phosphatase, which mediates transcription termination and intragenic looping at eukaryotic genes. Importantly, HIV-1 Tat interacts directly with Ssu72 and strongly stimulates its CTD phosphatase activity. We found that Ssu72 is essential for Tat:P-TEFb-mediated phosphorylation of the S5P-CTD in vitro. Interestingly, Ssu72 also stimulates nascent HIV-1 transcription in a phosphatase-dependent manner in vivo. Chromatin immunoprecipitation (ChIP) experiments reveal that Ssu72, like P-TEFb and AFF4, is recruited by Tat to the integrated HIV-1 proviral promoter in TNF-α signaling 2D10 T cells and leaves the elongation complex prior to the termination site. ChIP-seq (ChIP combined with deep sequencing) and GRO-seq (genome-wide nuclear run-on [GRO] combined with deep sequencing) analysis further reveals that Ssu72 predominantly colocalizes with S5P-RNAPII (RNA polymerase II) at promoters in human embryonic stem cells, with a minor peak in the terminator region. A few genes, like NANOG, also have high Ssu72 at the terminator. Ssu72 is not required for transcription at most cellular genes but has a modest effect on cotranscriptional termination. We conclude that Tat alters the cellular function of Ssu72 to stimulate viral gene expression and facilitate the early S5P-S2P transition at the integrated HIV-1 promoter.
Full-text · Article · Oct 2014 · Genes & Development