Cdk9 regulates a promoter-proximal checkpoint to modulate RNA polymerase II elongation rate in fission yeast

Abstract

Post-translational modifications of the transcription elongation complex provide mechanisms to fine-tune gene expression, yet their specific impacts on RNA polymerase II regulation remain difficult to ascertain. Here, in Schizosaccharomyces pombe, we examine the role of Cdk9, and related Mcs6/Cdk7 and Lsk1/Cdk12 kinases, on transcription at base-pair resolution with Precision Run-On sequencing (PRO-seq). Within a minute of Cdk9 inhibition, phosphorylation of Pol II-associated factor, Spt5 is undetectable. The effects of Cdk9 inhibition are more severe than inhibition of Cdk7 and Cdk12, resulting in a shift of Pol II toward the transcription start site (TSS). A time course of Cdk9 inhibition reveals that early transcribing Pol II can escape promoter-proximal regions, but with a severely reduced elongation rate of only ~400 bp/min. Our results in fission yeast suggest the existence of a conserved global regulatory checkpoint that requires Cdk9 kinase activity.

Selective kinase inhibition shows that Bur1 (Cdk9) phosphorylates the Rpb1 linker in vivo

“…In contrast to these other reports, we observed a pronounced drop off in RNApII ChIP signal from 5' to 3' ends of genes in bur2Δ budding yeast (24). Similarly, recent experiments in S. pombe (17) showed that inhibition of a Cdk9-AS strain also caused a marked polar elongation defect. Finally, Cdk9 inhibition in mammalian cells blocks RNApII from escaping past the early pause site (38).…”

“…As seen for Ctk1-IS and Bur1-IS, Kin28-IS inhibition was also transient, with recovery apparent by 60 minutes after CMK addition. In contrast to Rodriguez-Molina et al (4) and multiple Kin28-AS studies (11,12,14,15,17), we found that Kin28-IS inhibition also strongly inhibited Ser2P formation, as assayed by either 3E10 or H5 antibodies (Figs 3A and 4).…”

“…Another important observation is that in vivo inhibition of Kin28-IS produces strong inhibition of Ser2P, as well as Ser5P and Ser7P. The Ser2P drop was unexpected, as this phosphorylation was reported to be unaffected in multiple earlier studies using AS alleles of Kin28/Cdk7 in budding or fission yeasts (11,12,14,15,17), as well as in a paper using the same Kin28-IS allele used by us (4). Mammalian Cdk7-AS cells showed a partial reduction in both Ser5P and Ser2P (19,20), while inhibition of mammalian Cdk7 with the covalent kinase inhibitor THZ1 strongly blocks Ser5P and Ser2P (21,30).…”

“…AS alleles have frequently been used to inhibit these transcription-related kinases in budding and fission yeast (8)(9)(10)(11)(12)(13)(14)(15)(16)(17) as well as mammalian cells (18)(19)(20). Chemical inhibitors are available that target the native mammalian kinases with varying levels of specificity (21)(22)(23).…”

CDK12 controls G1/S progression by regulating RNAPII processivity at core DNA replication genes

“…One possible explanation might be the presence of CDK13 and BRD4, redundant P-Ser2 kinases, in humans [12,20,74]. In Schizosaccharomyces pombe, short (5 min) inhibition of AS Lsk1, a non-essential CDK12 homolog, decreased Ser2 phosphorylation, but had only a subtle effect on RNAPII distribution and transcription [75]. Although we cannot completely rule out that very short (in minutes) CDK12 inhibition globally affects transcription in human cells, this seems unlikely, since bulk P-Ser2 and P-Ser5 levels in cells are either not affected or only subtly (Figs 1D and EV1D) [48].…”