Publications by authors named "Robert P Fisher"

56 Publications

New connections between ubiquitylation and methylation in the co-transcriptional histone modification network.

Curr Genet 2021 Jun 5. Epub 2021 Jun 5.

Department of Pharmacology and Therapeutics, McGill University, Montreal, Canada.

Co-transcriptional histone modifications are a ubiquitous feature of RNA polymerase II (RNAPII) transcription, with profound but incompletely understood effects on gene expression. Unlike the covalent marks found at promoters, which are thought to be instructive for transcriptional activation, these modifications occur in gene bodies as a result of transcription, which has made elucidation of their functions challenging. Here we review recent insights into the regulation and roles of two such modifications: monoubiquitylation of histone H2B at lysine 120 (H2Bub1) and methylation of histone H3 at lysine 36 (H3K36me). Both H2Bub1 and H3K36me are enriched in the coding regions of transcribed genes, with highly overlapping distributions, but they were thought to work largely independently. We highlight our recent demonstration that, as was previously shown for H3K36me, H2Bub1 signals to the histone deacetylase (HDAC) complex Rpd3S/Clr6-CII, and that Rpd3S/Clr6-CII and H2Bub1 function in the same pathway to repress aberrant antisense transcription initiating within gene coding regions. Moreover, both of these histone modification pathways are influenced by protein phosphorylation catalyzed by the cyclin-dependent kinases (CDKs) that regulate RNAPII elongation, chiefly Cdk9. Therefore, H2Bub1 and H3K36me are more tightly linked than previously thought, sharing both upstream regulatory inputs and downstream effectors. Moreover, these newfound connections suggest extensive, bidirectional signaling between RNAPII elongation complexes and chromatin-modifying enzymes, which helps to determine transcriptional outputs and should be a focus for future investigation.
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http://dx.doi.org/10.1007/s00294-021-01196-xDOI Listing
June 2021

Protein phosphatases in the RNAPII transcription cycle: erasers, sculptors, gatekeepers, and potential drug targets.

Genes Dev 2021 May 22;35(9-10):658-676. Epub 2021 Apr 22.

Department of Oncological Sciences, Icahn School of Medicine at Mount Sinai, New York, New York 10029, USA.

The transcription cycle of RNA polymerase II (RNAPII) is governed at multiple points by opposing actions of cyclin-dependent kinases (CDKs) and protein phosphatases, in a process with similarities to the cell division cycle. While important roles of the kinases have been established, phosphatases have emerged more slowly as key players in transcription, and large gaps remain in understanding of their precise functions and targets. Much of the earlier work focused on the roles and regulation of sui generis and often atypical phosphatases-FCP1, Rtr1/RPAP2, and SSU72-with seemingly dedicated functions in RNAPII transcription. Decisive roles in the transcription cycle have now been uncovered for members of the major phosphoprotein phosphatase (PPP) family, including PP1, PP2A, and PP4-abundant enzymes with pleiotropic roles in cellular signaling pathways. These phosphatases appear to act principally at the transitions between transcription cycle phases, ensuring fine control of elongation and termination. Much is still unknown, however, about the division of labor among the PPP family members, and their possible regulation by or of the transcriptional kinases. CDKs active in transcription have recently drawn attention as potential therapeutic targets in cancer and other diseases, raising the prospect that the phosphatases might also present opportunities for new drug development. Here we review the current knowledge and outstanding questions about phosphatases in the context of the RNAPII transcription cycle.
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http://dx.doi.org/10.1101/gad.348315.121DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8091971PMC
May 2021

Distinct Cdk9-phosphatase switches act at the beginning and end of elongation by RNA polymerase II.

Nat Commun 2020 08 28;11(1):4338. Epub 2020 Aug 28.

Department of Oncological Sciences, Icahn School of Medicine at Mount Sinai, New York, NY, 10029-6574, USA.

Reversible phosphorylation of Pol II and accessory factors helps order the transcription cycle. Here, we define two kinase-phosphatase switches that operate at different points in human transcription. Cdk9/cyclin T1 (P-TEFb) catalyzes inhibitory phosphorylation of PP1 and PP4 complexes that localize to 3' and 5' ends of genes, respectively, and have overlapping but distinct specificities for Cdk9-dependent phosphorylations of Spt5, a factor instrumental in promoter-proximal pausing and elongation-rate control. PP1 dephosphorylates an Spt5 carboxy-terminal repeat (CTR), but not Spt5-Ser666, a site between Kyrpides-Ouzounis-Woese (KOW) motifs 4 and 5, whereas PP4 can target both sites. In vivo, Spt5-CTR phosphorylation decreases as transcription complexes pass the cleavage and polyadenylation signal (CPS) and increases upon PP1 depletion, consistent with a PP1 function in termination first uncovered in yeast. Depletion of PP4-complex subunits increases phosphorylation of both Ser666 and the CTR, and promotes redistribution of promoter-proximally paused Pol II into gene bodies. These results suggest that switches comprising Cdk9 and either PP4 or PP1 govern pause release and the elongation-termination transition, respectively.
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http://dx.doi.org/10.1038/s41467-020-18173-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7455706PMC
August 2020

Dissecting the Pol II transcription cycle and derailing cancer with CDK inhibitors.

Nat Chem Biol 2020 07 22;16(7):716-724. Epub 2020 Jun 22.

Department of Oncological Sciences, Icahn School of Medicine at Mount Sinai, New York, NY, USA.

Largely non-overlapping sets of cyclin-dependent kinases (CDKs) regulate cell division and RNA polymerase II (Pol II)-dependent transcription. Here we review the molecular mechanisms by which specific CDKs are thought to act at discrete steps in the transcription cycle and describe the recent emergence of transcriptional CDKs as promising drug targets in cancer. We emphasize recent advances in understanding the transcriptional CDK network that were facilitated by development and deployment of small-molecule inhibitors with increased selectivity for individual CDKs. Unexpectedly, several of these compounds have also shown selectivity in killing cancer cells, despite the seemingly universal involvement of their target CDKs during transcription in all cells. Finally, we describe remaining and emerging challenges in defining functions of individual CDKs in transcription and co-transcriptional processes and in leveraging CDK inhibition for therapeutic purposes.
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http://dx.doi.org/10.1038/s41589-020-0563-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7914107PMC
July 2020

Cdk9 and H2Bub1 signal to Clr6-CII/Rpd3S to suppress aberrant antisense transcription.

Nucleic Acids Res 2020 07;48(13):7154-7168

Department of Oncological Sciences, Icahn School of Medicine at Mount Sinai, New York, NY, USA.

Mono-ubiquitylation of histone H2B (H2Bub1) and phosphorylation of elongation factor Spt5 by cyclin-dependent kinase 9 (Cdk9) occur during transcription by RNA polymerase II (RNAPII), and are mutually dependent in fission yeast. It remained unclear whether Cdk9 and H2Bub1 cooperate to regulate the expression of individual genes. Here, we show that Cdk9 inhibition or H2Bub1 loss induces intragenic antisense transcription of ∼10% of fission yeast genes, with each perturbation affecting largely distinct subsets; ablation of both pathways de-represses antisense transcription of over half the genome. H2Bub1 and phospho-Spt5 have similar genome-wide distributions; both modifications are enriched, and directly proportional to each other, in coding regions, and decrease abruptly around the cleavage and polyadenylation signal (CPS). Cdk9-dependence of antisense suppression at specific genes correlates with high H2Bub1 occupancy, and with promoter-proximal RNAPII pausing. Genetic interactions link Cdk9, H2Bub1 and the histone deacetylase Clr6-CII, while combined Cdk9 inhibition and H2Bub1 loss impair Clr6-CII recruitment to chromatin and lead to decreased occupancy and increased acetylation of histones within gene coding regions. These results uncover novel interactions between co-transcriptional histone modification pathways, which link regulation of RNAPII transcription elongation to suppression of aberrant initiation.
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http://dx.doi.org/10.1093/nar/gkaa474DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7367204PMC
July 2020

Splice or Die: When MYC Is Driving, Transcription Needs NUAK1 to Avoid Fatal Pileups.

Authors:
Robert P Fisher

Mol Cell 2020 03;77(6):1157-1158

Department of Oncological Sciences, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA. Electronic address:

In this issue of Molecular Cell, Cossa et al. (2020) uncover the basis for a dependency of tumor cells with deregulated MYC on the kinase NUAK1, which acts through PP1 and PNUTS to ensure that splicing keeps up with MYC-driven transcription.
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http://dx.doi.org/10.1016/j.molcel.2020.02.025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7910809PMC
March 2020

Histone H2B Ubiquitylation Regulates Histone Gene Expression by Suppressing Antisense Transcription in Fission Yeast.

Genetics 2019 09 25;213(1):161-172. Epub 2019 Jul 25.

Department of Pharmacology and Therapeutics, McGill University, Montreal, Quebec H3G 1Y6, Canada

Histone H2B monoubiquitylation (H2Bub1) is tightly linked to RNA polymerase II transcription elongation, and is also directly implicated in DNA replication and repair. Loss of H2Bub1 is associated with defects in cell cycle progression, but how these are related to its various functions, and the underlying mechanisms involved, is not understood. Here we describe a role for H2Bub1 in the regulation of replication-dependent histone genes in the fission yeast H2Bub1 activates histone genes indirectly by suppressing antisense transcription of -a gene encoding a GATA-type transcription factor that activates histone genes and is required for assembly of centromeric chromatin. Mutants lacking the ubiquitylation site in H2B or the H2B-specific E3 ubiquitin ligase Brl2 had elevated levels of antisense transcripts and reduced Ams2 protein levels. These defects were reversed upon inhibition of Cdk9-an ortholog of the kinase component of positive transcription elongation factor b (P-TEFb)-indicating that they likely resulted from aberrant transcription elongation. Reduced Cdk9 activity also partially rescued chromosome segregation phenotypes of H2Bub1 mutants. In a genome-wide analysis, loss of H2Bub1 led to increased antisense transcripts at over 500 protein-coding genes in H2Bub1 mutants; for a subset of these, including several genes involved in chromosome segregation and chromatin assembly, antisense derepression was Cdk9-dependent. Our results highlight antisense suppression as a key feature of cell cycle-dependent gene regulation by H2Bub1, and suggest that aberrant transcription elongation may underlie the effects of H2Bub1 loss on cell cycle progression.
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http://dx.doi.org/10.1534/genetics.119.302499DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6727805PMC
September 2019

Cdk7: a kinase at the core of transcription and in the crosshairs of cancer drug discovery.

Authors:
Robert P Fisher

Transcription 2019 04 6;10(2):47-56. Epub 2018 Dec 6.

a Department of Oncological Sciences , Icahn School of Medicine at Mount Sinai , New York , NY , USA.

The transcription cycle of RNA polymerase II (Pol II) is regulated by a set of cyclin-dependent kinases (CDKs). Cdk7, associated with the transcription initiation factor TFIIH, is both an effector CDK that phosphorylates Pol II and other targets within the transcriptional machinery, and a CDK-activating kinase (CAK) for at least one other essential CDK involved in transcription. Recent studies have illuminated Cdk7 functions that are executed throughout the Pol II transcription cycle, from promoter clearance and promoter-proximal pausing, to co-transcriptional chromatin modification in gene bodies, to mRNA 3´-end formation and termination. Cdk7 has also emerged as a target of small-molecule inhibitors that show promise in the treatment of cancer and inflammation. The challenges now are to identify the relevant targets of Cdk7 at each step of the transcription cycle, and to understand how heightened dependence on an essential CDK emerges in cancer, and might be exploited therapeutically.
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http://dx.doi.org/10.1080/21541264.2018.1553483DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6602562PMC
April 2019

A Cdk9-PP1 switch regulates the elongation-termination transition of RNA polymerase II.

Nature 2018 06 13;558(7710):460-464. Epub 2018 Jun 13.

Department of Oncological Sciences, Icahn School of Medicine at Mount Sinai, New York, NY, USA.

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 .
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http://dx.doi.org/10.1038/s41586-018-0214-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6021199PMC
June 2018

Taking Aim at Glycolysis with CDK8 Inhibitors.

Authors:
Robert P Fisher

Trends Endocrinol Metab 2018 05 20;29(5):281-282. Epub 2018 Feb 20.

Department of Oncological Sciences, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA. Electronic address:

Dependence on glycolysis under aerobic conditions, a frequent metabolic derangement in cancer cells, suggests a therapeutic opportunity. Now, through chemical genetics, CDK8, a kinase associated with the Mediator transcriptional coactivator complex, has emerged as an upstream inducer of glycolysis and a possible target for anticancer drug discovery.
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http://dx.doi.org/10.1016/j.tem.2018.02.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5911231PMC
May 2018

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

Nat Commun 2018 02 7;9(1):543. Epub 2018 Feb 7.

Department of Molecular Biology and Genetics, Cornell University, 107 Biotechnology Building, 526 Campus Road, Ithaca, NY, 14853-2703, USA.

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.
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http://dx.doi.org/10.1038/s41467-018-03006-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5803247PMC
February 2018

Glucocorticoid-induced phosphorylation by CDK9 modulates the coactivator functions of transcriptional cofactor GRIP1 in macrophages.

Nat Commun 2017 11 23;8(1):1739. Epub 2017 Nov 23.

Graduate Program in Immunology and Microbial Pathogenesis, Weill Cornell Graduate School of Medical Sciences, 1300 York Avenue, New York, NY, 10021, USA.

The glucocorticoid (GC) receptor (GR) suppresses inflammation by activating anti-inflammatory and repressing pro-inflammatory genes. GR-interacting protein-1 (GRIP1) is a GR corepressor in macrophages, however, whether GRIP1 mediates GR-activated transcription, and what dictates its coactivator versus corepressor properties is unknown. Here we report that GRIP1 loss in macrophages attenuates glucocorticoid induction of several anti-inflammatory targets, and that GC treatment of quiescent macrophages globally directs GRIP1 toward GR binding sites dominated by palindromic GC response elements (GRE), suggesting a non-redundant GRIP1 function as a GR coactivator. Interestingly, GRIP1 is phosphorylated at an N-terminal serine cluster by cyclin-dependent kinase-9 (CDK9), which is recruited into GC-induced GR:GRIP1:CDK9 hetero-complexes, producing distinct GRE-specific GRIP1 phospho-isoforms. Phosphorylation potentiates GRIP1 coactivator but, remarkably, not its corepressor properties. Consistently, phospho-GRIP1 and CDK9 are not detected at GR transrepression sites near pro-inflammatory genes. Thus, GR restricts actions of its own coregulator via CDK9-mediated phosphorylation to a subset of anti-inflammatory genes.
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http://dx.doi.org/10.1038/s41467-017-01569-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5700924PMC
November 2017

Activation of the p53 Transcriptional Program Sensitizes Cancer Cells to Cdk7 Inhibitors.

Cell Rep 2017 Oct;21(2):467-481

Department of Oncological Sciences, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA. Electronic address:

Cdk7, the CDK-activating kinase and transcription factor IIH component, is a target of inhibitors that kill cancer cells by exploiting tumor-specific transcriptional dependencies. However, whereas selective inhibition of analog-sensitive (AS) Cdk7 in colon cancer-derived cells arrests division and disrupts transcription, it does not by itself trigger apoptosis efficiently. Here, we show that p53 activation by 5-fluorouracil or nutlin-3 synergizes with a reversible Cdk7 inhibitor to induce cell death. Synthetic lethality was recapitulated with covalent inhibitors of wild-type Cdk7, THZ1, or the more selective YKL-1-116. The effects were allele specific; a CDK7 mutation conferred both sensitivity to bulky adenine analogs and resistance to covalent inhibitors. Non-transformed colon epithelial cells were resistant to these combinations, as were cancer-derived cells with p53-inactivating mutations. Apoptosis was dependent on death receptor DR5, a p53 transcriptional target whose expression was refractory to Cdk7 inhibition. Therefore, p53 activation induces transcriptional dependency to sensitize cancer cells to Cdk7 inhibition.
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http://dx.doi.org/10.1016/j.celrep.2017.09.056DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5687273PMC
October 2017

Neonatal expression of RNA-binding protein IGF2BP3 regulates the human fetal-adult megakaryocyte transition.

J Clin Invest 2017 Jun 8;127(6):2365-2377. Epub 2017 May 8.

Department of Pathology, University of Virginia School of Medicine, Charlottesville, Virginia, USA.

Hematopoietic transitions that accompany fetal development, such as erythroid globin chain switching, play important roles in normal physiology and disease development. In the megakaryocyte lineage, human fetal progenitors do not execute the adult morphogenesis program of enlargement, polyploidization, and proplatelet formation. Although these defects decline with gestational stage, they remain sufficiently severe at birth to predispose newborns to thrombocytopenia. These defects may also contribute to inferior platelet recovery after cord blood stem cell transplantation and may underlie inefficient platelet production by megakaryocytes derived from pluripotent stem cells. In this study, comparison of neonatal versus adult human progenitors has identified a blockade in the specialized positive transcription elongation factor b (P-TEFb) activation mechanism that is known to drive adult megakaryocyte morphogenesis. This blockade resulted from neonatal-specific expression of an oncofetal RNA-binding protein, IGF2BP3, which prevented the destabilization of the nuclear RNA 7SK, a process normally associated with adult megakaryocytic P-TEFb activation. Knockdown of IGF2BP3 sufficed to confer both phenotypic and molecular features of adult-type cells on neonatal megakaryocytes. Pharmacologic inhibition of IGF2BP3 expression via bromodomain and extraterminal domain (BET) inhibition also elicited adult features in neonatal megakaryocytes. These results identify IGF2BP3 as a human ontogenic master switch that restricts megakaryocyte development by modulating a lineage-specific P-TEFb activation mechanism, revealing potential strategies toward enhancing platelet production.
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http://dx.doi.org/10.1172/JCI88936DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5451240PMC
June 2017

CDK regulation of transcription by RNAP II: Not over 'til it's over?

Authors:
Robert P Fisher

Transcription 2017 03 22;8(2):81-90. Epub 2016 Dec 22.

a Department of Oncological Sciences , Icahn School of Medicine at Mount Sinai , New York , NY , USA.

Transcription by RNA polymerase (RNAP) II is regulated at multiple steps by phosphorylation, catalyzed mainly by members of the cyclin-dependent kinase (CDK) family. The CDKs involved in transcription have overlapping substrate specificities, but play largely non-redundant roles in coordinating gene expression. Novel functions and targets of CDKs have recently emerged at the end of the transcription cycle, when the primary transcript is cleaved, and in most cases polyadenylated, and transcription is terminated by the action of the "torpedo" exonuclease Xrn2, which is a CDK substrate. Collectively, various functions have been ascribed to CDKs or CDK-mediated phosphorylation: recruiting cleavage and polyadenylation factors, preventing premature termination within gene bodies while promoting efficient termination of full-length transcripts, and preventing extensive readthrough transcription into intergenic regions or neighboring genes. The assignment of precise functions to specific CDKs is still in progress, but recent advances suggest ways in which the CDK network and RNAP II machinery might cooperate to ensure timely exit from the transcription cycle.
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http://dx.doi.org/10.1080/21541264.2016.1268244DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5423476PMC
March 2017

Getting to S: CDK functions and targets on the path to cell-cycle commitment.

Authors:
Robert P Fisher

F1000Res 2016 26;5:2374. Epub 2016 Sep 26.

Department of Oncological Sciences, Icahn School of Medicine at Mount Sinai, New York, NY, USA.

How and when eukaryotic cells make the irrevocable commitment to divide remain central questions in the cell-cycle field. Parallel studies in yeast and mammalian cells seemed to suggest analogous control mechanisms operating during the G1 phase-at Start or the restriction (R) point, respectively-to integrate nutritional and developmental signals and decide between distinct cell fates: cell-cycle arrest or exit versus irreversible commitment to a round of division. Recent work has revealed molecular mechanisms underlying this decision-making process in both yeast and mammalian cells but also cast doubt on the nature and timing of cell-cycle commitment in multicellular organisms. These studies suggest an expanded temporal window of mitogen sensing under certain growth conditions, illuminate unexpected obstacles and exit ramps on the path to full cell-cycle commitment, and raise new questions regarding the functions of cyclin-dependent kinases (CDKs) that drive G1 progression and S-phase entry.
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http://dx.doi.org/10.12688/f1000research.9463.1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5040153PMC
September 2016

P-TEFb regulation of transcription termination factor Xrn2 revealed by a chemical genetic screen for Cdk9 substrates.

Genes Dev 2016 Jan;30(1):117-31

Department of Oncological Sciences, Icahn School of Medicine at Mount Sinai, New York, New York 10029, USA;

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'-to-3' "torpedo" 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 phosphomimetic 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.
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http://dx.doi.org/10.1101/gad.269589.115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4701974PMC
January 2016

Functional interaction of Rpb1 and Spt5 C-terminal domains in co-transcriptional histone modification.

Nucleic Acids Res 2015 Nov 14;43(20):9766-75. Epub 2015 Aug 14.

Department of Pharmacology and Therapeutics, McGill University, Montreal, Quebec, H3G 1Y6, Canada

Transcription by RNA polymerase II (RNAPII) is accompanied by a conserved pattern of histone modifications that plays important roles in regulating gene expression. The establishment of this pattern requires phosphorylation of both Rpb1 (the largest RNAPII subunit) and the elongation factor Spt5 on their respective C-terminal domains (CTDs). Here we interrogated the roles of individual Rpb1 and Spt5 CTD phospho-sites in directing co-transcriptional histone modifications in the fission yeast Schizosaccharomyces pombe. Steady-state levels of methylation at histone H3 lysines 4 (H3K4me) and 36 (H3K36me) were sensitive to multiple mutations of the Rpb1 CTD repeat motif (Y1S2P3T4S5P6S7). Ablation of the Spt5 CTD phospho-site Thr1 reduced H3K4me levels but had minimal effects on H3K36me. Nonetheless, Spt5 CTD mutations potentiated the effects of Rpb1 CTD mutations on H3K36me, suggesting overlapping functions. Phosphorylation of Rpb1 Ser2 by the Cdk12 orthologue Lsk1 positively regulated H3K36me but negatively regulated H3K4me. H3K36me and histone H2B monoubiquitylation required Rpb1 Ser5 but were maintained upon inactivation of Mcs6/Cdk7, the major kinase for Rpb1 Ser5 in vivo, implicating another Ser5 kinase in these regulatory pathways. Our results elaborate the CTD 'code' for co-transcriptional histone modifications.
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http://dx.doi.org/10.1093/nar/gkv837DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4787787PMC
November 2015

The structure and substrate specificity of human Cdk12/Cyclin K.

Nat Commun 2014 Mar 24;5:3505. Epub 2014 Mar 24.

1] Group Physical Biochemistry, Center of Advanced European Studies and Research, Ludwig-Erhard-Allee 2, Bonn 53175, Germany [2] Department of Physical Biochemistry, Max Planck Institute of Molecular Physiology, Otto-Hahn-Strasse 11, Dortmund 44227, Germany.

Phosphorylation of the RNA polymerase II C-terminal domain (CTD) by cyclin-dependent kinases is important for productive transcription. Here we determine the crystal structure of Cdk12/CycK and analyse its requirements for substrate recognition. Active Cdk12/CycK is arranged in an open conformation similar to that of Cdk9/CycT but different from those of cell cycle kinases. Cdk12 contains a C-terminal extension that folds onto the N- and C-terminal lobes thereby contacting the ATP ribose. The interaction is mediated by an HE motif followed by a polybasic cluster that is conserved in transcriptional CDKs. Cdk12/CycK showed the highest activity on a CTD substrate prephosphorylated at position Ser7, whereas the common Lys7 substitution was not recognized. Flavopiridol is most potent towards Cdk12 but was still 10-fold more potent towards Cdk9. T-loop phosphorylation of Cdk12 required coexpression with a Cdk-activating kinase. These results suggest the regulation of Pol II elongation by a relay of transcriptionally active CTD kinases.
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http://dx.doi.org/10.1038/ncomms4505DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3973122PMC
March 2014

The PAF complex and Prf1/Rtf1 delineate distinct Cdk9-dependent pathways regulating transcription elongation in fission yeast.

PLoS Genet 2013 26;9(12):e1004029. Epub 2013 Dec 26.

Department of Pharmacology and Therapeutics, McGill University, Montreal, Canada.

Cyclin-dependent kinase 9 (Cdk9) promotes elongation by RNA polymerase II (RNAPII), mRNA processing, and co-transcriptional histone modification. Cdk9 phosphorylates multiple targets, including the conserved RNAPII elongation factor Spt5 and RNAPII itself, but how these different modifications mediate Cdk9 functions is not known. Here we describe two Cdk9-dependent pathways in the fission yeast Schizosaccharomyces pombe that involve distinct targets and elicit distinct biological outcomes. Phosphorylation of Spt5 by Cdk9 creates a direct binding site for Prf1/Rtf1, a transcription regulator with functional and physical links to the Polymerase Associated Factor (PAF) complex. PAF association with chromatin is also dependent on Cdk9 but involves alternate phosphoacceptor targets. Prf1 and PAF are biochemically separate in cell extracts, and genetic analyses show that Prf1 and PAF are functionally distinct and exert opposing effects on the RNAPII elongation complex. We propose that this opposition constitutes a Cdk9 auto-regulatory mechanism, such that a positive effect on elongation, driven by the PAF pathway, is kept in check by a negative effect of Prf1/Rtf1 and downstream mono-ubiquitylation of histone H2B. Thus, optimal RNAPII elongation may require balanced action of functionally distinct Cdk9 pathways.
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http://dx.doi.org/10.1371/journal.pgen.1004029DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3873232PMC
August 2014

Modelling the CDK-dependent transcription cycle in fission yeast.

Biochem Soc Trans 2013 Dec;41(6):1660-5

*Department of Structural and Chemical Biology, Icahn School of Medicine at Mount Sinai, One Gustave L. Levy Place, Box 1677, New York, NY 10029-6574, U.S.A.

CDKs (cyclin-dependent kinases) ensure directionality and fidelity of the eukaryotic cell division cycle. In a similar fashion, the transcription cycle is governed by a conserved subfamily of CDKs that phosphorylate Pol II (RNA polymerase II) and other substrates. A genetic model organism, the fission yeast Schizosaccharomyces pombe, has yielded robust models of cell-cycle control, applicable to higher eukaryotes. From a similar approach combining classical and chemical genetics, fundamental principles of transcriptional regulation by CDKs are now emerging. In the present paper, we review the current knowledge of each transcriptional CDK with respect to its substrate specificity, function in transcription and effects on chromatin modifications, highlighting the important roles of CDKs in ensuring quantity and quality control over gene expression in eukaryotes.
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http://dx.doi.org/10.1042/BST20130238DOI Listing
December 2013

The CDK-activating kinase Cdk7: taking yes for an answer.

Cell Cycle 2013 Oct 13;12(20):3239-40. Epub 2013 Sep 13.

Department of Structural and Chemical Biology; Graduate School of Biomedical Sciences; Icahn School of Medicine at Mount Sinai; New York, NY USA.

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http://dx.doi.org/10.4161/cc.26355DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3885630PMC
October 2013

Pause, play, repeat: CDKs push RNAP II's buttons.

Transcription 2013 Jul-Aug;4(4):146-52. Epub 2013 Jun 11.

Department of Structural and Chemical Biology; Icahn School of Medicine at Mount Sinai; New York, NY USA.

Cyclin-dependent kinases (CDKs) play a central role in governing eukaryotic cell division. It is becoming clear that the transcription cycle of RNA polymerase II (RNAP II) is also regulated by CDKs; in metazoans, the cell cycle and transcriptional CDK networks even share an upstream activating kinase, which is itself a CDK. From recent chemical-genetic analyses we know that CDKs and their substrates control events both early in transcription (the transition from initiation to elongation) and late (3' end formation and transcription termination). Moreover, mutual dependence on CDK activity might couple the "beginning" and "end" of the cycle, to ensure the fidelity of mRNA maturation and the efficient recycling of RNAP II from sites of termination to the transcription start site (TSS). As is the case for CDKs involved in cell cycle regulation, different transcriptional CDKs act in defined sequence on multiple substrates. These phosphorylations are likely to influence gene expression by several mechanisms, including direct, allosteric effects on the transcription machinery, co-transcriptional recruitment of proteins needed for mRNA-capping, splicing and 3' end maturation, dependent on multisite phosphorylation of the RNAP II C-terminal domain (CTD) and, perhaps, direct regulation of RNA-processing or histone-modifying machinery. Here we review these recent advances, and preview the emerging challenges for transcription-cycle research.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3977912PMC
http://dx.doi.org/10.4161/trns.25146DOI Listing
March 2015

The CDK Network: Linking Cycles of Cell Division and Gene Expression.

Authors:
Robert P Fisher

Genes Cancer 2012 Nov;3(11-12):731-8

Mount Sinai School of Medicine, New York, NY, USA.

Cyclin-dependent kinases (CDKs) play essential roles in cell proliferation and gene expression. Although distinct sets of CDKs work in cell division and transcription by RNA polymerase II (Pol II), they share a CDK-activating kinase (CAK), which is itself a CDK-Cdk7-in metazoans. Thus a unitary CDK network controls and may coordinate cycles of cell division and gene expression. Recent work reveals decisive roles for Cdk7 in both pathways. The CAK function of Cdk7 helps determine timing of activation and cyclin-binding preferences of different CDKs during the cell cycle. In the transcription cycle, Cdk7 is both an effector kinase, which phosphorylates Pol II and other proteins and helps establish promoter-proximal pausing; and a CAK for Cdk9 (P-TEFb), which releases Pol II from the pause. By governing the transition from initiation to elongation, Cdk7, Cdk9 and their substrates influence expression of genes important for developmental and cell-cycle decisions, and ensure co-transcriptional maturation of Pol II transcripts. Cdk7 engaged in transcription also appears to be regulated by phosphorylation within its own activation (T) loop. Here I review recent studies of CDK regulation in cell division and gene expression, and propose a model whereby mitogenic signals trigger a cascade of CDK T-loop phosphorylation that drives cells past the restriction (R) point, when continued cell-cycle progression becomes growth factor-independent. Because R-point control is frequently deregulated in cancer, the CAK-CDK pathway is an attractive target for chemical inhibition aimed at impeding the inappropriate commitment to cell division.
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http://dx.doi.org/10.1177/1947601912473308DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3636752PMC
November 2012

A Cdk7-Cdk4 T-loop phosphorylation cascade promotes G1 progression.

Mol Cell 2013 Apr;50(2):250-60

Department of Structural and Chemical Biology, Graduate School of Biomedical Sciences, Icahn School of Medicine at Mount Sinai, New York, NY 10029-6574, USA.

Eukaryotic cell division is controlled by cyclin-dependent kinases (CDKs), which require phosphorylation by a CDK-activating kinase (CAK) for full activity. Chemical genetics uncovered requirements for the metazoan CAK Cdk7 in determining cyclin specificity and activation order of Cdk2 and Cdk1 during S and G2 phases. It was unknown if Cdk7 also activates Cdk4 and Cdk6 to promote passage of the restriction (R) point, when continued cell-cycle progression becomes mitogen independent, or if CDK-activating phosphorylation regulates G1 progression. Here we show that Cdk7 is a Cdk4- and Cdk6-activating kinase in human cells, required to maintain activity, not just to establish the active state, as is the case for Cdk1 and Cdk2. Activating phosphorylation of Cdk7 rises concurrently with that of Cdk4 as cells exit quiescence and accelerates Cdk4 activation in vitro. Therefore, mitogen signaling drives a CDK-activation cascade during G1 progression, and CAK might be rate-limiting for R point passage.
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http://dx.doi.org/10.1016/j.molcel.2013.04.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3677717PMC
April 2013

Cyclin-dependent kinase control of the initiation-to-elongation switch of RNA polymerase II.

Nat Struct Mol Biol 2012 Nov 14;19(11):1108-15. Epub 2012 Oct 14.

Department of Structural and Chemical Biology, Mount Sinai School of Medicine, New York, New York, USA.

Promoter-proximal pausing by RNA polymerase II (Pol II) ensures gene-specific regulation and RNA quality control. Structural considerations suggested a requirement for initiation-factor eviction in elongation-factor engagement and pausing of transcription complexes. Here we show that selective inhibition of Cdk7--part of TFIIH--increases TFIIE retention, prevents DRB sensitivity-inducing factor (DSIF) recruitment and attenuates pausing in human cells. Pause release depends on Cdk9-cyclin T1 (P-TEFb); Cdk7 is also required for Cdk9-activating phosphorylation and Cdk9-dependent downstream events--Pol II C-terminal domain Ser2 phosphorylation and histone H2B ubiquitylation--in vivo. Cdk7 inhibition, moreover, impairs Pol II transcript 3'-end formation. Cdk7 thus acts through TFIIE and DSIF to establish, and through P-TEFb to relieve, barriers to elongation: incoherent feedforward that might create a window to recruit RNA-processing machinery. Therefore, cyclin-dependent kinases govern Pol II handoff from initiation to elongation factors and cotranscriptional RNA maturation.
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http://dx.doi.org/10.1038/nsmb.2399DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3746743PMC
November 2012

Chemical genetics reveals a specific requirement for Cdk2 activity in the DNA damage response and identifies Nbs1 as a Cdk2 substrate in human cells.

PLoS Genet 2012 Aug 23;8(8):e1002935. Epub 2012 Aug 23.

Department of Structural and Chemical Biology, Mount Sinai School of Medicine, New York, New York, USA.

The cyclin-dependent kinases (CDKs) that promote cell-cycle progression are targets for negative regulation by signals from damaged or unreplicated DNA, but also play active roles in response to DNA lesions. The requirement for activity in the face of DNA damage implies that there are mechanisms to insulate certain CDKs from checkpoint inhibition. It remains difficult, however, to assign precise functions to specific CDKs in protecting genomic integrity. In mammals, Cdk2 is active throughout S and G2 phases, but Cdk2 protein is dispensable for survival, owing to compensation by other CDKs. That plasticity obscured a requirement for Cdk2 activity in proliferation of human cells, which we uncovered by replacement of wild-type Cdk2 with a mutant version sensitized to inhibition by bulky adenine analogs. Here we show that transient, selective inhibition of analog-sensitive (AS) Cdk2 after exposure to ionizing radiation (IR) enhances cell-killing. In extracts supplemented with an ATP analog used preferentially by AS kinases, Cdk2(as) phosphorylated the Nijmegen Breakage Syndrome gene product Nbs1-a component of the conserved Mre11-Rad50-Nbs1 complex required for normal DNA damage repair and checkpoint signaling-dependent on a consensus CDK recognition site at Ser432. In vivo, selective inhibition of Cdk2 delayed and diminished Nbs1-Ser432 phosphorylation during S phase, and mutation of Ser432 to Ala or Asp increased IR-sensitivity. Therefore, by chemical genetics, we uncovered both a non-redundant requirement for Cdk2 activity in response to DNA damage and a specific target of Cdk2 within the DNA repair machinery.
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http://dx.doi.org/10.1371/journal.pgen.1002935DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3426557PMC
August 2012

A positive feedback loop links opposing functions of P-TEFb/Cdk9 and histone H2B ubiquitylation to regulate transcript elongation in fission yeast.

PLoS Genet 2012 2;8(8):e1002822. Epub 2012 Aug 2.

Department of Structural and Chemical Biology, Mount Sinai School of Medicine, New York, New York, United States of America.

Transcript elongation by RNA polymerase II (RNAPII) is accompanied by conserved patterns of histone modification. Whereas histone modifications have established roles in transcription initiation, their functions during elongation are not understood. Mono-ubiquitylation of histone H2B (H2Bub1) plays a key role in coordinating co-transcriptional histone modification by promoting site-specific methylation of histone H3. H2Bub1 also regulates gene expression through an unidentified, methylation-independent mechanism. Here we reveal bidirectional communication between H2Bub1 and Cdk9, the ortholog of metazoan positive transcription elongation factor b (P-TEFb), in the fission yeast Schizosaccharomyces pombe. Chemical and classical genetic analyses indicate that lowering Cdk9 activity or preventing phosphorylation of its substrate, the transcription processivity factor Spt5, reduces H2Bub1 in vivo. Conversely, mutations in the H2Bub1 pathway impair Cdk9 recruitment to chromatin and decrease Spt5 phosphorylation. Moreover, an Spt5 phosphorylation-site mutation, combined with deletion of the histone H3 Lys4 methyltransferase Set1, phenocopies morphologic and growth defects due to H2Bub1 loss, suggesting independent, partially redundant roles for Cdk9 and Set1 downstream of H2Bub1. Surprisingly, mutation of the histone H2B ubiquitin-acceptor residue relaxes the Cdk9 activity requirement in vivo, and cdk9 mutations suppress cell-morphology defects in H2Bub1-deficient strains. Genome-wide analyses by chromatin immunoprecipitation also demonstrate opposing effects of Cdk9 and H2Bub1 on distribution of transcribing RNAPII. Therefore, whereas mutual dependence of H2Bub1 and Spt5 phosphorylation indicates positive feedback, mutual suppression by cdk9 and H2Bub1-pathway mutations suggests antagonistic functions that must be kept in balance to regulate elongation. Loss of H2Bub1 disrupts that balance and leads to deranged gene expression and aberrant cell morphologies, revealing a novel function of a conserved, co-transcriptional histone modification.
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http://dx.doi.org/10.1371/journal.pgen.1002822DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3410854PMC
December 2012

Why minimal is not optimal: driving the mammalian cell cycle--and drug discovery--with a physiologic CDK control network.

Cell Cycle 2012 Jul 15;11(14):2600-5. Epub 2012 Jul 15.

Department of Structural and Chemical Biology, Mount Sinai School of Medicine, New York, NY, USA.

Progression through the eukaryotic cell division cycle is governed by the activity of cyclin-dependent kinases (CDKs). For a CDK to become active it must (1) bind a positive regulatory subunit (cyclin) and (2) be phosphorylated on its activation (T) loop. In metazoans, multiple CDK catalytic subunits, each with a distinct set of preferred cyclin partners, regulate the cell cycle, but it has been difficult to assign functions to individual CDKs in vivo. Biochemical analyses and experiments with dominant-negative alleles suggested that specific CDK/cyclin complexes regulate different events, but genetic loss of interphase CDKs (Cdk2, -4 and -6), alone or in combination, did not block proliferation of cells in culture. These knockout and knockdown studies suggested redundancy or plasticity built into the CDK network but did not address whether there was true redundancy in normal cells with a full complement of CDKs. Here, we discuss recent work that took a chemical-genetic approach to reveal that the activity of a genetically non-essential CDK, Cdk2, is required for cell proliferation when normal cyclin pairing is maintained. These results have implications for the systems-level organization of the cell cycle, for regulation of the restriction point and G 1/S transition and for efforts to target Cdk2 therapeutically in human cancers.
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http://dx.doi.org/10.4161/cc.20758DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3409006PMC
July 2012

Separate domains of fission yeast Cdk9 (P-TEFb) are required for capping enzyme recruitment and primed (Ser7-phosphorylated) Rpb1 carboxyl-terminal domain substrate recognition.

Mol Cell Biol 2012 Jul 16;32(13):2372-83. Epub 2012 Apr 16.

Department of Structural and Chemical Biology, Mount Sinai School of Medicine, New York, New York, USA.

In fission yeast, discrete steps in mRNA maturation and synthesis depend on a complex containing the 5'-cap methyltransferase Pcm1 and Cdk9, which phosphorylates the RNA polymerase II (Pol II) carboxyl-terminal domain (CTD) and the processivity factor Spt5 to promote transcript elongation. Here we show that a Cdk9 carboxyl-terminal extension, distinct from the catalytic domain, mediates binding to both Pcm1 and the Pol II CTD. Removal of this segment diminishes Cdk9/Pcm1 chromatin recruitment and Spt5 phosphorylation in vivo and leads to slow growth and hypersensitivity to cold temperature, nutrient limitation, and the IMP dehydrogenase inhibitor mycophenolic acid (MPA). These phenotypes, and the Spt5 phosphorylation defect, are suppressed by Pcm1 overproduction, suggesting that normal transcript elongation and gene expression depend on physical linkage between Cdk9 and Pcm1. The extension is dispensable, however, for recognition of CTD substrates "primed" by Mcs6 (Cdk7). On defined peptide substrates in vitro, Cdk9 prefers CTD repeats phosphorylated at Ser7 over unmodified repeats. In vivo, Ser7 phosphorylation depends on Mcs6 activity, suggesting a conserved mechanism, independent of chromatin recruitment, to order transcriptional CDK functions. Therefore, fission yeast Cdk9 comprises a catalytic domain sufficient for primed substrate recognition and a multivalent recruitment module that couples transcription with capping.
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http://dx.doi.org/10.1128/MCB.06657-11DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3434489PMC
July 2012