A Cdk9-PP1 switch regulates the elongation-termination transition of RNA polymerase II.
Nature
The end of the RNA polymerase II (Pol II) transcription cycle is strictly regulated to prevent interference between neighbouring genes and to safeguard transcriptome integrity . The accumulation of Pol II downstream of the cleavage and polyadenylation signal can facilitate the recruitment of factors involved in mRNA 3'-end formation and termination , but how this sequence is initiated remains unclear. In a chemical-genetic screen, human protein phosphatase 1 (PP1) isoforms were identified as substrates of positive transcription elongation factor b (P-TEFb), also known as the cyclin-dependent kinase 9 (Cdk9)-cyclin T1 (CycT1) complex . 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 Dis2 . Sites targeted by Cdk9 in the Spt5 carboxy-terminal domain can be dephosphorylated by Dis2 in vitro, and dis2 mutations retard Spt5 dephosphorylation after inhibition of Cdk9 in vivo. Chromatin immunoprecipitation and sequencing analysis indicates that Spt5 is dephosphorylated as transcription complexes traverse the cleavage and polyadenylation signal, concomitant with the accumulation of Pol II phosphorylated at residue Ser2 of the carboxy-terminal domain consensus heptad repeat . A conditionally lethal Dis2-inactivating mutation attenuates the drop in Spt5 phosphorylation on chromatin, promotes transcription beyond the normal termination zone (as detected by precision run-on transcription and sequencing ) and is genetically suppressed by the ablation of Cdk9 target sites in Spt5. These results suggest that the transition of Pol II from elongation to termination coincides with a Dis2-dependent reversal of Cdk9 signalling-a switch that is analogous to a Cdk1-PP1 circuit that controls mitotic progression .
10.1038/s41586-018-0214-z
Phase-separation mechanism for C-terminal hyperphosphorylation of RNA polymerase II.
Nature
Hyperphosphorylation of the C-terminal domain (CTD) of the RPB1 subunit of human RNA polymerase (Pol) II is essential for transcriptional elongation and mRNA processing. The CTD contains 52 heptapeptide repeats of the consensus sequence YSPTSPS. The highly repetitive nature and abundant possible phosphorylation sites of the CTD exert special constraints on the kinases that catalyse its hyperphosphorylation. Positive transcription elongation factor b (P-TEFb)-which consists of CDK9 and cyclin T1-is known to hyperphosphorylate the CTD and negative elongation factors to stimulate Pol II elongation. The sequence determinant on P-TEFb that facilitates this action is currently unknown. Here we identify a histidine-rich domain in cyclin T1 that promotes the hyperphosphorylation of the CTD and stimulation of transcription by CDK9. The histidine-rich domain markedly enhances the binding of P-TEFb to the CTD and functional engagement with target genes in cells. In addition to cyclin T1, at least one other kinase-DYRK1A -also uses a histidine-rich domain to target and hyperphosphorylate the CTD. As a low-complexity domain, the histidine-rich domain also promotes the formation of phase-separated liquid droplets in vitro, and the localization of P-TEFb to nuclear speckles that display dynamic liquid properties and are sensitive to the disruption of weak hydrophobic interactions. The CTD-which in isolation does not phase separate, despite being a low-complexity domain-is trapped within the cyclin T1 droplets, and this process is enhanced upon pre-phosphorylation by CDK7 of transcription initiation factor TFIIH. By using multivalent interactions to create a phase-separated functional compartment, the histidine-rich domain in kinases targets the CTD into this environment to ensure hyperphosphorylation and efficient elongation of Pol II.
10.1038/s41586-018-0174-3
SUMO suppresses and MYC amplifies transcription globally by regulating CDK9 sumoylation.
Yu Fang,Shi Guang,Cheng Shimeng,Chen Jiwei,Wu Shwu-Yuan,Wang Zhiqiang,Xia Nansong,Zhai Yunhao,Wang Zhenxing,Peng Yu,Wang Dong,Du James X,Liao Lujian,Duan Sheng-Zhong,Shi Tieliu,Cheng Jinke,Chiang Cheng-Ming,Li Jiwen,Wong Jiemin
Cell research
Regulation of transcription is fundamental to the control of cellular gene expression and function. Although recent studies have revealed a role for the oncoprotein MYC in amplifying global transcription, little is known as to how the global transcription is suppressed. Here we report that SUMO and MYC mediate opposite effects upon global transcription by controlling the level of CDK9 sumoylation. On one hand, SUMO suppresses global transcription via sumoylation of CDK9, the catalytic subunit of P-TEFb kinase essential for productive transcriptional elongation. On the other hand, MYC amplifies global transcription by antagonizing CDK9 sumoylation. Sumoylation of CDK9 blocks its interaction with Cyclin T1 and thus the formation of active P-TEFb complex. Transcription profiling analyses reveal that SUMO represses global transcription, particularly of moderately to highly expressed genes and by generating a sumoylation-resistant CDK9 mutant, we confirm that sumoylation of CDK9 inhibits global transcription. Together, our data reveal that SUMO and MYC oppositely control global gene expression by regulating the dynamic sumoylation and desumoylation of CDK9.
10.1038/s41422-018-0023-9
RNA polymerase II-associated factor 1 regulates the release and phosphorylation of paused RNA polymerase II.
Yu Ming,Yang Wenjing,Ni Ting,Tang Zhanyun,Nakadai Tomoyoshi,Zhu Jun,Roeder Robert G
Science (New York, N.Y.)
Release of promoter-proximal paused RNA polymerase II (Pol II) during early elongation is a critical step in transcriptional regulation in metazoan cells. Paused Pol II release is thought to require the kinase activity of cyclin-dependent kinase 9 (CDK9) for the phosphorylation of DRB sensitivity-inducing factor, negative elongation factor, and C-terminal domain (CTD) serine-2 of Pol II. We found that Pol II-associated factor 1 (PAF1) is a critical regulator of paused Pol II release, that positive transcription elongation factor b (P-TEFb) directly regulates the initial recruitment of PAF1 complex (PAF1C) to genes, and that the subsequent recruitment of CDK12 is dependent on PAF1C. These findings reveal cooperativity among P-TEFb, PAF1C, and CDK12 in pausing release and Pol II CTD phosphorylation.
10.1126/science.aad2338
Nuclear CDKs drive Smad transcriptional activation and turnover in BMP and TGF-beta pathways.
Alarcón Claudio,Zaromytidou Alexia-Ileana,Xi Qiaoran,Gao Sheng,Yu Jianzhong,Fujisawa Sho,Barlas Afsar,Miller Alexandria N,Manova-Todorova Katia,Macias Maria J,Sapkota Gopal,Pan Duojia,Massagué Joan
Cell
TGF-beta and BMP receptor kinases activate Smad transcription factors by C-terminal phosphorylation. We have identified a subsequent agonist-induced phosphorylation that plays a central dual role in Smad transcriptional activation and turnover. As receptor-activated Smads form transcriptional complexes, they are phosphorylated at an interdomain linker region by CDK8 and CDK9, which are components of transcriptional mediator and elongation complexes. These phosphorylations promote Smad transcriptional action, which in the case of Smad1 is mediated by the recruitment of YAP to the phosphorylated linker sites. An effector of the highly conserved Hippo organ size control pathway, YAP supports Smad1-dependent transcription and is required for BMP suppression of neural differentiation of mouse embryonic stem cells. The phosphorylated linker is ultimately recognized by specific ubiquitin ligases, leading to proteasome-mediated turnover of activated Smad proteins. Thus, nuclear CDK8/9 drive a cycle of Smad utilization and disposal that is an integral part of canonical BMP and TGF-beta pathways.
10.1016/j.cell.2009.09.035
RNAP II CTD phosphorylated on threonine-4 is required for histone mRNA 3' end processing.
Hsin Jing-Ping,Sheth Amit,Manley James L
Science (New York, N.Y.)
The RNA polymerase II (RNAP II) largest subunit contains a C-terminal domain (CTD) with up to 52 Tyr(1)-Ser(2)-Pro(3)-Thr(4)-Ser(5)-Pro(6)-Ser(7) consensus repeats. Serines 2, 5, and 7 are known to be phosphorylated, and these modifications help to orchestrate the interplay between transcription and processing of messenger RNA (mRNA) precursors. Here, we provide evidence that phosphorylation of CTD Thr(4) residues is required specifically for histone mRNA 3' end processing, functioning to facilitate recruitment of 3' processing factors to histone genes. Like Ser(2), Thr(4) phosphorylation requires the CTD kinase CDK9 and is evolutionarily conserved from yeast to human. Our data thus illustrate how a CTD modification can play a highly specific role in facilitating efficient gene expression.
10.1126/science.1206034