The end of the RNA polymerase II (Pol II) transcription cycle is strictly regulated to prevent interference between neighbouring genes and safeguard transcriptome integrity1. Pol II accumulation downstream of the cleavage and polyadenylation signal (CPS) can facilitate recruitment of factors involved in mRNA 3’-end formation and termination2, but how this sequence is initiated remains unclear. In a chemical-genetic screen, we identified human protein phosphatase 1 (PP1) isoforms as substrates of positive transcription elongation factor b (P-TEFb), the cyclin-dependent kinase 9 (Cdk9)-cyclin T1 complex3. Here we show that Cdk9 and PP1 govern phosphorylation of the conserved elongation factor Spt5 in the fission yeast Schizosaccharomyces pombe. Cdk9 phosphorylates both Spt5 and a negative regulatory site on the PP1 isoform Dis24. Sites targeted by Cdk9 in the Spt5 carboxy-terminal domain (CTD) can be dephosphorylated by Dis2 in vitro, and dis2 mutations retard Spt5 dephosphorylation after Cdk9 inhibition in vivo. Chromatin immunoprecipitation and sequencing (ChIP-seq) analysis indicates that Spt5 is dephosphorylated as transcription complexes traverse the CPS, concomitant with accumulation of Pol II phosphorylated at CTD repeat residue Ser25. A conditionally lethal Dis2-inactivating mutation attenuates the drop in Spt5 phosphorylation (pSpt5) on chromatin, promotes transcription beyond the normal termination zone detected by precision run-on transcription and sequencing (PRO-seq)6, and is genetically suppressed by ablation of Cdk9 target sites in Spt5. These results suggest that the transition from elongation to termination by Pol II coincides with Dis2-dependent reversal of Cdk9 signaling—a switch analogous to a Cdk1-PP1 circuit that controls mitotic progression4.

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.

SUMMARY The zinc-finger transcription factor, Mxr1 activates methanol utilization and peroxisome biogenesis genes in the methylotrophic yeast, P. pastoris. Expression of Mxr1-dependent genes is regulated in response to various carbon sources by an unknown mechanism. We show here that this mechanism involves the highly conserved 14-3-3 proteins. 14-3-3 proteins participate in many biological processes in different eukaryotes. We have characterized a putative 14-3-3-binding region at Mxr1 residues 212–225 and mapped the major activation domain of Mxr1 to residues 246–280, and showed that phenylalanine residues in this region are critical for its function. Furthermore, we report that an unique and previously uncharacterized 14-3-3 family protein in P. pastoris complements S. cerevisiae 14-3-3 functions and interacts with Mxr1 through its 14-3-3-binding region via phosphorylation of Ser215 in a carbon source-dependent manner. Indeed, our in vivo results suggest a carbon source-dependent regulation of expression of Mxr1-activated genes by 14-3-3 in P. pastoris. Interestingly, we observed 14-3-3-independent binding of Mxr1 to the promoters, suggesting a post-DNA binding function of 14-3-3 in regulating transcription. We provide the first molecular explanation of carbon source-mediated regulation of Mxr1 activity, whose mechanism involves a post-DNA binding role of 14-3-3.

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