scholarly journals Regulation of P-TEFb Elongation Complex Activity by CDK9 Acetylation

2007 ◽  
Vol 27 (13) ◽  
pp. 4641-4651 ◽  
Author(s):  
Junjiang Fu ◽  
Ho-Geun Yoon ◽  
Jun Qin ◽  
Jiemin Wong
Keyword(s):  
Rna Polymerase Ii ◽  
Elongation Factor ◽  
Transcriptional Elongation ◽  
Elongation Complex ◽  
Complex Activity ◽  
Interacting Protein ◽  
Pol Ii ◽  
Carboxy Terminal

ABSTRACT P-TEFb, comprised of CDK9 and a cyclin T subunit, is a global transcriptional elongation factor important for most RNA polymerase II (pol II) transcription. P-TEFb facilitates transcription elongation in part by phosphorylating Ser2 of the heptapeptide repeat of the carboxy-terminal domain (CTD) of the largest subunit of pol II. Previous studies have shown that P-TEFb is subjected to negative regulation by forming an inactive complex with 7SK small RNA and HEXIM1. In an effort to investigate the molecular mechanism by which corepressor N-CoR mediates transcription repression, we identified HEXIM1 as an N-CoR-interacting protein. This finding led us to test whether the P-TEFb complex is regulated by acetylation. We demonstrate that CDK9 is an acetylated protein in cells and can be acetylated by p300 in vitro. Through both in vitro and in vivo assays, we identified lysine 44 of CDK9 as a major acetylation site. We present evidence that CDK9 is regulated by N-CoR and its associated HDAC3 and that acetylation of CDK9 affects its ability to phosphorylate the CTD of pol II. These results suggest that acetylation of CDK9 is an important posttranslational modification that is involved in regulating P-TEFb transcriptional elongation function.

scholarly journals Cyclin-Dependent Kinase 9 (Cdk9) of Fission Yeast Is Activated by the CDK-Activating Kinase Csk1, Overlaps Functionally with the TFIIH-Associated Kinase Mcs6, and Associates with the mRNA Cap Methyltransferase Pcm1 In Vivo

2006 ◽  
Vol 26 (3) ◽  
pp. 777-788 ◽  
Author(s):  
Yi Pei ◽  
Hongyan Du ◽  
Juliet Singer ◽  
Courtney St. Amour ◽  
Selena Granitto ◽  
...  
Keyword(s):  
Fission Yeast ◽  
Rna Polymerase Ii ◽  
Elongation Factor ◽  
Growth Defect ◽  
Transcriptional Elongation ◽  
Cyclin Dependent Kinase ◽  
Elongation Complex ◽  
Pol Ii

ABSTRACT Cyclin-dependent kinase 9 (Cdk9) of fission yeast is an essential ortholog of metazoan positive transcription elongation factor b (P-TEFb), which is proposed to coordinate capping and elongation of RNA polymerase II (Pol II) transcripts. Here we show that Cdk9 is activated to phosphorylate Pol II and the elongation factor Spt5 by Csk1, one of two fission yeast CDK-activating kinases (CAKs). Activation depends on Cdk9 T-loop residue Thr-212. The other CAK—Mcs6, the kinase component of transcription factor IIH (TFIIH)—cannot activate Cdk9. Consistent with the specificities of the two CAKs in vitro, the kinase activity of Cdk9 is reduced ∼10-fold by csk1 deletion, and Cdk9 complexes from csk1Δ but not csk1 + cells can be activated by Csk1 in vitro. A cdk9 T212A mutant is viable but phenocopies conditional growth defects of csk1Δ strains, indicating a role for Csk1-dependent activation of Cdk9 in vivo. A cdk9 T212A mcs6 S165A strain, in which neither Cdk9 nor Mcs6 can be activated by CAK, has a synthetic growth defect, implying functional overlap between the two CDKs, which have distinct but overlapping substrate specificities. Cdk9 forms complexes in vivo with the essential cyclin Pch1 and with Pcm1, the mRNA cap methyltransferase. The carboxyl-terminal region of Cdk9, through which it interacts with another capping enzyme, the RNA triphosphatase Pct1, is essential. Together, the data support a proposed model whereby Cdk9/Pch1—the third essential CDK-cyclin complex described in fission yeast—helps to target the capping apparatus to the transcriptional elongation complex.

Identification of rpo30, a vaccinia virus RNA polymerase gene with structural similarity to a eucaryotic transcription elongation factor

1990 ◽  
Vol 10 (10) ◽  
pp. 5433-5441
Author(s):  
B Y Ahn ◽  
P D Gershon ◽  
E V Jones ◽  
B Moss
Keyword(s):  
Rna Polymerase ◽  
Vaccinia Virus ◽  
Rna Polymerase Ii ◽  
Elongation Factor ◽  
Viral Rna ◽  
In Vitro Translation ◽  
Virus Protein ◽  
Transcriptional Elongation

Eucaryotic transcription factors that stimulate RNA polymerase II by increasing the efficiency of elongation of specifically or randomly initiated RNA chains have been isolated and characterized. We have identified a 30-kilodalton (kDa) vaccinia virus-encoded protein with apparent homology to SII, a 34-kDa mammalian transcriptional elongation factor. In addition to amino acid sequence similarities, both proteins contain C-terminal putative zinc finger domains. Identification of the gene, rpo30, encoding the vaccinia virus protein was achieved by using antibody to the purified viral RNA polymerase for immunoprecipitation of the in vitro translation products of in vivo-synthesized early mRNA selected by hybridization to cloned DNA fragments of the viral genome. Western immunoblot analysis using antiserum made to the vaccinia rpo30 protein expressed in bacteria indicated that the 30-kDa protein remains associated with highly purified viral RNA polymerase. Thus, the vaccinia virus protein, unlike its eucaryotic homolog, is an integral RNA polymerase subunit rather than a readily separable transcription factor. Further studies showed that the expression of rpo30 is regulated by dual early and later promoters.

scholarly journals Control of transcriptional elongation and cotranscriptional histone modification by the yeast BUR kinase substrate Spt5

2009 ◽  
Vol 106 (17) ◽  
pp. 6956-6961 ◽  
Author(s):  
Karen Zhou ◽  
Wei Hung William Kuo ◽  
Jeffrey Fillingham ◽  
Jack F. Greenblatt
Keyword(s):  
Histone Modification ◽  
Rna Polymerase Ii ◽  
Transcription Elongation ◽  
Consensus Sequence ◽  
Elongation Factor ◽  
Transcriptional Elongation ◽  
Histone H2b ◽  
Paf Complex

Elongation by RNA polymerase II (RNAPII) is a finely regulated process in which many elongation factors contribute to gene regulation. Among these factors are the polymerase-associated factor (PAF) complex, which associates with RNAPII, and several cyclin-dependent kinases, including positive transcription elongation factor b (P-TEFb) in humans and BUR kinase (Bur1–Bur2) and C-terminal domain (CTD) kinase 1 (CTDK1) in Saccharomyces cerevisiae. An important target of P-TEFb and CTDK1, but not BUR kinase, is the CTD of the Rpb1 subunit of RNAPII. Although the essential BUR kinase phosphorylates Rad6, which is required for histone H2B ubiquitination on K123, Rad6 is not essential, leaving a critical substrate(s) of BUR kinase unidentified. Here we show that BUR kinase is important for the phosphorylation in vivo of Spt5, a subunit of the essential yeast RNAPII elongation factor Spt4/Spt5, whose human orthologue is DRB sensitivity-inducing factor. BUR kinase can also phosphorylate the C-terminal region (CTR) of Spt5 in vitro. Like BUR kinase, the Spt5 CTR is important for promoting elongation by RNAPII and recruiting the PAF complex to transcribed regions. Also like BUR kinase and the PAF complex, the Spt5 CTR is important for histone H2B K123 monoubiquitination and histone H3 K4 and K36 trimethylation during transcription elongation. Our results suggest that the Spt5 CTR, which contains 15 repeats of a hexapeptide whose consensus sequence is S[T/A]WGG[A/Q], is a substrate of BUR kinase and a platform for the association of proteins that promote both transcription elongation and histone modification in transcribed regions.

scholarly journals Identification of rpo30, a vaccinia virus RNA polymerase gene with structural similarity to a eucaryotic transcription elongation factor.

1990 ◽  
Vol 10 (10) ◽  
pp. 5433-5441 ◽  
Author(s):  
B Y Ahn ◽  
P D Gershon ◽  
E V Jones ◽  
B Moss
Keyword(s):  
Rna Polymerase ◽  
Vaccinia Virus ◽  
Rna Polymerase Ii ◽  
Elongation Factor ◽  
Viral Rna ◽  
In Vitro Translation ◽  
Virus Protein ◽  
Transcriptional Elongation

Eucaryotic transcription factors that stimulate RNA polymerase II by increasing the efficiency of elongation of specifically or randomly initiated RNA chains have been isolated and characterized. We have identified a 30-kilodalton (kDa) vaccinia virus-encoded protein with apparent homology to SII, a 34-kDa mammalian transcriptional elongation factor. In addition to amino acid sequence similarities, both proteins contain C-terminal putative zinc finger domains. Identification of the gene, rpo30, encoding the vaccinia virus protein was achieved by using antibody to the purified viral RNA polymerase for immunoprecipitation of the in vitro translation products of in vivo-synthesized early mRNA selected by hybridization to cloned DNA fragments of the viral genome. Western immunoblot analysis using antiserum made to the vaccinia rpo30 protein expressed in bacteria indicated that the 30-kDa protein remains associated with highly purified viral RNA polymerase. Thus, the vaccinia virus protein, unlike its eucaryotic homolog, is an integral RNA polymerase subunit rather than a readily separable transcription factor. Further studies showed that the expression of rpo30 is regulated by dual early and later promoters.

scholarly journals JMJD6 cleaves MePCE to release positive transcription elongation factor b (P-TEFb) in higher eukaryotes

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Schuyler Lee ◽  
Haolin Liu ◽  
Ryan Hill ◽  
Chunjing Chen ◽  
Xia Hong ◽  
...  
Keyword(s):  
Rna Polymerase Ii ◽  
Transcription Elongation ◽  
Elongation Factor ◽  
Factor B ◽  
Pol Ii ◽  
Positive Transcription Elongation Factor ◽  
Transcription Elongation Factor ◽  
Higher Eukaryotes

More than 30% of genes in higher eukaryotes are regulated by promoter-proximal pausing of RNA polymerase II (Pol II). Phosphorylation of Pol II CTD by positive transcription elongation factor b (P-TEFb) is a necessary precursor event that enables productive transcription elongation. The exact mechanism on how the sequestered P-TEFb is released from the 7SK snRNP complex and recruited to Pol II CTD remains unknown. In this report, we utilize mouse and human models to reveal methylphosphate capping enzyme (MePCE), a core component of the 7SK snRNP complex, as the cognate substrate for Jumonji domain-containing 6 (JMJD6)’s novel proteolytic function. Our evidences consist of a crystal structure of JMJD6 bound to methyl-arginine, enzymatic assays of JMJD6 cleaving MePCE in vivo and in vitro, binding assays, and downstream effects of Jmjd6 knockout and overexpression on Pol II CTD phosphorylation. We propose that JMJD6 assists bromodomain containing 4 (BRD4) to recruit P-TEFb to Pol II CTD by disrupting the 7SK snRNP complex.

scholarly journals JMJD6 Cleaves MePCE to Release P-TEFb

2019 ◽  
Author(s):  
Schuyler Lee ◽  
Haolin Liu ◽  
Ryan Hill ◽  
Xia Hong ◽  
Xinjian Liu ◽  
...  
Keyword(s):  
Rna Polymerase Ii ◽  
Transcription Elongation ◽  
Elongation Factor ◽  
Binding Assays ◽  
Enzymatic Assays ◽  
Pol Ii ◽  
Downstream Effects ◽  
Capping Enzyme

AbstractMore than 30% of genes in higher eukaryotes are regulated by promoter-proximal pausing of RNA polymerase II (Pol II). Phosphorylation of Pol II-CTD by positive transcription elongation factor (P-TEFb) is a necessary precursor event that enables productive transcription elongation. The exact mechanism on how the sequestered P-TEFb is released from the 7SK snRNP complex and recruited to Pol II-CTD remains unknown. In this report, we reveal methylphosphate capping enzyme (MePCE), a core component of the 7SK snRNP complex, as the cognate substrate for Jumonji domain-containing 6 (JMJD6)’s novel proteolytic function. Our evidences consist of a crystal structure of JMJD6 bound to methyl-arginine, enzymatic assays of JMJD6 cleaving MePCE in vivo and in vitro, binding assays, and downstream effects of Jmjd6 knockout and overexpression on Pol II-CTD phosphorylation. We propose that JMJD6 assists bromodomain containing 4 (BRD4) to recruit P-TEFb to Pol II-CTD by disrupting the 7SK snRNP complex.

scholarly journals TFIIH-Associated Cdk7 Kinase Functions in Phosphorylation of C-Terminal Domain Ser7 Residues, Promoter-Proximal Pausing, and Termination by RNA Polymerase II

2009 ◽  
Vol 29 (20) ◽  
pp. 5455-5464 ◽  
Author(s):  
Kira Glover-Cutter ◽  
Stéphane Larochelle ◽  
Benjamin Erickson ◽  
Chao Zhang ◽  
Kevan Shokat ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Elongation Factor ◽  
Histone H4 ◽  
Pol Ii ◽  
Terminal Domain ◽  
Histone H4 Acetylation ◽  
Flanking Regions

ABSTRACT The function of human TFIIH-associated Cdk7 in RNA polymerase II (Pol II) transcription and C-terminal domain (CTD) phosphorylation was investigated in analogue-sensitive Cdk7 as/as mutant cells where the kinase can be inhibited without disrupting TFIIH. We show that both Cdk7 and Cdk9/PTEFb contribute to phosphorylation of Pol II CTD Ser5 residues on transcribed genes. Cdk7 is also a major kinase of CTD Ser7 on Pol II at the c-fos and U snRNA genes. Furthermore, TFIIH and recombinant Cdk7-CycH-Mat1 as well as recombinant Cdk9-CycT1 phosphorylated CTD Ser7 and Ser5 residues in vitro. Inhibition of Cdk7 in vivo suppressed the amount of Pol II accumulated at 5′ ends on several genes including c-myc, p21, and glyceraldehyde-3-phosphate dehydrogenase genes, indicating reduced promoter-proximal pausing or polymerase “leaking” into the gene. Consistent with a 5′ pausing defect, Cdk7 inhibition reduced recruitment of the negative elongation factor NELF at start sites. A role of Cdk7 in regulating elongation is further suggested by enhanced histone H4 acetylation and diminished histone H4 trimethylation on lysine 36—two marks of elongation—within genes when the kinase was inhibited. Consistent with a new role for TFIIH at 3′ ends, it was detected within genes and 3′-flanking regions, and Cdk7 inhibition delayed pausing and transcription termination.

scholarly journals The Growth Factor Granulin Interacts with Cyclin T1 and Modulates P-TEFb-Dependent Transcription

2003 ◽  
Vol 23 (5) ◽  
pp. 1688-1702 ◽  
Author(s):  
Mainul Hoque ◽  
Tara M. Young ◽  
Chee-Gun Lee ◽  
Ginette Serrero ◽  
Michael B. Mathews ◽  
...  
Keyword(s):  
Rna Polymerase Ii ◽  
Direct Action ◽  
Elongation Factor ◽  
Cellular Protein ◽  
Interacting Protein ◽  
Cyclin T1 ◽  
Rich Domain ◽  
Hiv 1

ABSTRACT Cyclin T1, together with the kinase CDK9, is a component of the transcription elongation factor P-TEFb which binds the human immunodeficiency virus type 1 (HIV-1) transactivator Tat. P-TEFb facilitates transcription by phosphorylating the carboxy-terminal domain (CTD) of RNA polymerase II. Cyclin T1 is an exceptionally large cyclin and is therefore a candidate for interactions with regulatory proteins. We identified granulin as a cyclin T1-interacting protein that represses expression from the HIV-1 promoter in transfected cells. The granulins, mitogenic growth factors containing repeats of a cysteine-rich motif, were reported previously to interact with Tat. We show that granulin formed stable complexes in vivo and in vitro with cyclin T1 and Tat. Granulin bound to the histidine-rich domain of cyclin T1, which was recently found to bind to the CTD, but not to cyclin T2. Binding of granulin to P-TEFb inhibited the phosphorylation of a CTD peptide. Granulin expression inhibited Tat transactivation, and tethering experiments showed that this effect was due, at least in part, to a direct action on cyclin T1 in the absence of Tat. In addition, granulin was a substrate for CDK9 but not for the other transcription-related kinases CDK7 and CDK8. Thus, granulin is a cellular protein that interacts with cyclin T1 to inhibit transcription.

scholarly journals A Cdk9-PP1 kinase-phosphatase switch regulates the elongation-termination transition of RNA polymerase II

2017 ◽  
Author(s):  
Pabitra K. Parua ◽  
Gregory T. Booth ◽  
Miriam Sansó ◽  
Bradley Benjamin ◽  
Jason C. Tanny ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Elongation Factor ◽  
Bistable Switch ◽  
Pol Ii ◽  
Mrna Maturation ◽  
Cleavage And Polyadenylation ◽  
Transcription Complexes

The end of the RNA polymerase II (Pol II) transcription cycle is strictly regulated to ensure proper mRNA maturation and prevent interference between neighboring genes1. Pol II slowing downstream of the cleavage and polyadenylation signal (CPS) leads to recruitment of cleavage and polyadenylation factors and termination2, but how this chain of events is initiated remains unclear. In a chemical-genetic screen, we identified protein phosphatase 1 (PP1) isoforms as substrates of human 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 transcription factor Spt5 in the fission yeast Schizosaccharomyces pombe. Cdk9 phosphorylates both Spt5 and a negative regulatory site on the PP1 isoform Dis24. Sites phosphorylated by Cdk9 in the Spt5 carboxy-terminal domain (CTD) are dephosphorylated by Dis2 in vitro, and Cdk9 inhibition in vivo leads to rapid Spt5 dephosphorylation that is retarded by concurrent Dis2 inactivation. Chromatin immunoprecipitation and sequencing (ChIP-seq) analysis indicates that Spt5 is dephosphorylated as transcription complexes traverse the CPS, prior to or concomitant with slowing of Pol II5. A Dis2-inactivating mutation stabilizes 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 suppressed by ablation of Cdk9 target sites in Spt5. These results support a model whereby the transition of Pol II from elongation to termination is regulated by opposing activities of Cdk9 and Dis2 towards their common substrate Spt5—a bistable switch analogous to a Cdk1-PP1 module that controls mitotic progression4.

scholarly journals AFF1 acetylation by p300 temporally inhibits transcription during genotoxic stress response

2019 ◽  
Vol 116 (44) ◽  
pp. 22140-22151 ◽  
Author(s):  
Nidhi Kumari ◽  
M. Abul Hassan ◽  
Xiangdong Lu ◽  
Robert G. Roeder ◽  
Debabrata Biswas
Keyword(s):  
Rna Polymerase Ii ◽  
Mammalian Cells ◽  
Elongation Factor ◽  
Genotoxic Stress ◽  
Regulation Of Transcription ◽  
Strongly Correlated ◽  
Elongation Complex ◽  
Transcriptional Inhibition

Soon after exposure to genotoxic reagents, mammalian cells inhibit transcription to prevent collisions with repair machinery and to mount a proper DNA damage response. However, mechanisms underlying early transcriptional inhibition are poorly understood. In this report, we show that site-specific acetylation of super elongation complex (SEC) subunit AFF1 by p300 reduces its interaction with other SEC components and impairs P-TEFb−mediated C-terminal domain phosphorylation of RNA polymerase II both in vitro and in vivo. Reexpression of wild-type AFF1, but not an acetylation mimic mutant, restores SEC component recruitment and target gene expression in AFF1 knockdown cells. Physiologically, we show that, upon genotoxic exposure, p300-mediated AFF1 acetylation is dynamic and strongly correlated with concomitant global down-regulation of transcription—and that this can be reversed by overexpression of an acetylation-defective AFF1 mutant. Therefore, we describe a mechanism of dynamic transcriptional regulation involving p300-mediated acetylation of a key elongation factor during genotoxic stress.

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