scholarly journals Acetylation of Conserved Lysines in the Catalytic Core of Cyclin-Dependent Kinase 9 Inhibits Kinase Activity and Regulates Transcription

2008 ◽  
Vol 28 (7) ◽  
pp. 2201-2212 ◽  
Author(s):  
Arianna Sabò ◽  
Marina Lusic ◽  
Anna Cereseto ◽  
Mauro Giacca
Keyword(s):  
Rna Polymerase Ii ◽  
Transcriptional Activation ◽  
Elongation Factor ◽  
Cyclin Dependent Kinase ◽  
Catalytic Core ◽  
Carboxy Terminal Domain ◽  
Immunodeficiency Virus ◽  
Matrix Compartment ◽  
Carboxy Terminal

ABSTRACT Promoter clearance and transcriptional processivity in eukaryotic cells are fundamentally regulated by the phosphorylation of the carboxy-terminal domain of RNA polymerase II (RNAPII). One of the kinases that essentially performs this function is P-TEFb (positive transcription elongation factor b), which is composed of cyclin-dependent kinase 9 (CDK9) associated with members of the cyclin T family. Here we show that cellular GCN5 and P/CAF, members of the GCN5-related N-acetyltransferase family of histone acetyltransferases, regulate CDK9 function by specifically acetylating the catalytic core of the enzyme and, in particular, a lysine that is essential for ATP coordination and the phosphotransfer reaction. Acetylation markedly reduces both the kinase function and transcriptional activity of P-TEFb. In contrast to unmodified CDK9, the acetylated fraction of the enzyme is specifically found in the insoluble nuclear matrix compartment. Acetylated CDK9 associates with the transcriptionally silent human immunodeficiency virus type 1 provirus; upon transcriptional activation, it is replaced by the unmodified form, which is involved in the elongating phase of transcription marked by Ser2-phosphorylated RNAPII. Given the conservation of the CDK9 acetylated residues in the catalytic task of virtually all CDK proteins, we anticipate that this mechanism of regulation might play a broader role in controlling the function of other members of this kinase family.

scholarly journals Ubiquitylation of Cdk9 by Skp2 Facilitates Optimal Tat Transactivation

2005 ◽  
Vol 79 (17) ◽  
pp. 11135-11141 ◽  
Author(s):  
Matjaz Barboric ◽  
Fan Zhang ◽  
Mojca Besenicar ◽  
Ana Plemenitas ◽  
B. Matija Peterlin
Keyword(s):  
Rna Polymerase Ii ◽  
Elongation Factor ◽  
Transcriptional Elongation ◽  
Cyclin Dependent Kinase ◽  
Primary Mouse ◽  
Immunodeficiency Virus ◽  
F Box Protein ◽  
Hiv 1

ABSTRACT By recruiting the positive transcriptional elongation factor b (P-TEFb) to paused RNA polymerase II, the transactivator Tat stimulates transcriptional elongation of the human immunodeficiency virus type 1 (HIV-1) genome. We found that cyclin-dependent kinase 9 (Cdk9), the catalytic subunit of P-TEFb, is ubiquitylated in vivo. This ubiquitylation depended on the Skp1/Cul1/F-box protein E3 ubiquitin ligase Skp2. Likewise, Tat required Skp2 since its transactivation of the HIV-1 long terminal repeat decreased in primary mouse embryonic fibroblasts, which lacked Skp2. The ubiquitylation of Cdk9 by Skp2 facilitated the formation of the ternary complex between P-TEFb, Tat, and transactivation response element. Thus, our findings underscore the requirement of ubiquitylation for the coactivator function in regulating HIV-1 transcriptional elongation.

scholarly journals Human and Rodent Transcription Elongation Factor P-TEFb: Interactions with Human Immunodeficiency Virus Type 1 Tat and Carboxy-Terminal Domain Substrate

1999 ◽  
Vol 73 (7) ◽  
pp. 5448-5458 ◽  
Author(s):  
Y. Ramanathan ◽  
Syed M. Reza ◽  
Tara M. Young ◽  
Michael B. Mathews ◽  
Tsafi Pe’ery
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Elongation Factor ◽  
Cyclin T1 ◽  
Transcription Elongation Factor ◽  
Carboxy Terminal Domain ◽  
Immunodeficiency Virus ◽  
Carboxy Terminal

ABSTRACT The human immunodeficiency virus type 1 transcriptional regulator Tat increases the efficiency of elongation, and complexes containing the cellular kinase CDK9 have been implicated in this process. CDK9 is part of the Tat-associated kinase TAK and of the elongation factor P-TEFb (positive transcription elongation factor-b), which consists minimally of CDK9 and cyclin T. TAK and P-TEFb are both able to phosphorylate the carboxy-terminal domain (CTD) of RNA polymerase II, but their relationships to one another and to the stimulation of elongation by Tat are not well characterized. Here we demonstrate that human cyclin T1 (but not cyclin T2) interacts with the activation domain of Tat and is a component of TAK as well as of P-TEFb. Rodent (mouse and Chinese hamster) cyclin T1 is defective in Tat binding and transactivation, but hamster CDK9 interacts with human cyclin T1 to give active TAK in hybrid cells containing human chromosome 12. Although TAK is phosphorylated on both serine and threonine residues, it specifically phosphorylates serine 5 in the CTD heptamer. TAK is found in the nuclear and cytoplasmic fractions of human cells as a large complex (∼950 kDa). Magnesium or zinc ions are required for the association of Tat with the kinase. We suggest a model in which Tat first interacts with P-TEFb to form the TAK complex that engages with TAR RNA and the elongating transcription complex, resulting in hyperphosphorylation of the CTD on serine 5 residues.

scholarly journals Phosphorylation of the RNA Polymerase II Carboxy-Terminal Domain by the Bur1 Cyclin-Dependent Kinase

2001 ◽  
Vol 21 (13) ◽  
pp. 4089-4096 ◽  
Author(s):  
Stuart Murray ◽  
Rajesh Udupa ◽  
Sheng Yao ◽  
Grant Hartzog ◽  
Gregory Prelich
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcriptional Elongation ◽  
Cyclin Dependent Kinase ◽  
Terminal Domain ◽  
Carboxy Terminal Domain ◽  
Substrate Phosphorylation ◽  
Biochemical Approach ◽  
Selection For ◽  
Carboxy Terminal

ABSTRACT BUR1, which was previously identified by a selection for mutations that have general effects on transcription inSaccharomyces cerevisiae, encodes a cyclin-dependent kinase that is essential for viability, but none of its substrates have been identified to date. Using an unbiased biochemical approach, we have identified the carboxy-terminal domain (CTD) of Rpb1, the largest subunit of RNA polymerase II, as a Bur1 substrate. Phosphorylation of Rpb1 by Bur1 is likely to be physiologically relevant, sincebur1 mutations interact genetically with rpb1CTD truncations and with mutations in other genes involved in CTD function. Several genetic interactions are presented, implying a role for Bur1 during transcriptional elongation. These results identify Bur1 as a fourth S. cerevisiae CTD kinase and provide striking functional similarities between Bur1 and metazoan P-TEFb.

scholarly journals The Yin and Yang of P-TEFb Regulation: Implications for Human Immunodeficiency Virus Gene Expression and Global Control of Cell Growth and Differentiation

2006 ◽  
Vol 70 (3) ◽  
pp. 646-659 ◽  
Author(s):  
Qiang Zhou ◽  
Jasper H. N. Yik
Keyword(s):  
Human Immunodeficiency Virus ◽  
Rna Polymerase Ii ◽  
Elongation Factor ◽  
Transcriptional Elongation ◽  
Small Nuclear Rna ◽  
Cellular Genes ◽  
Immunodeficiency Virus ◽  
Cellular Cofactor ◽  
Cell Growth And Differentiation ◽  
Carboxy Terminal

SUMMARY The positive transcription elongation factor b (P-TEFb) stimulates transcriptional elongation by phosphorylating the carboxy-terminal domain of RNA polymerase II and antagonizing the effects of negative elongation factors. Not only is P-TEFb essential for transcription of the vast majority of cellular genes, but it is also a critical host cellular cofactor for the expression of the human immunodeficiency virus (HIV) type 1 genome. Given its important role in globally affecting transcription, P-TEFb's activity is dynamically controlled by both positive and negative regulators in order to achieve a functional equilibrium in sync with the overall transcriptional demand as well as the proliferative state of cells. Notably, this equilibrium can be shifted toward either the active or inactive state in response to diverse physiological stimuli that can ultimately affect the cellular decision between growth and differentiation. In this review, we examine the mechanisms by which the recently identified positive (the bromodomain protein Brd4) and negative (the noncoding 7SK small nuclear RNA and the HEXIM1 protein) regulators of P-TEFb affect the P-TEFb-dependent transcriptional elongation. We also discuss the consequences of perturbations of the dynamic associations of these regulators with P-TEFb in relation to the pathogenesis and progression of several major human diseases, such as cardiac hypertrophy, breast cancer, and HIV infection.

scholarly journals Spt5 Cooperates with Human Immunodeficiency Virus Type 1 Tat by Preventing Premature RNA Release at Terminator Sequences

2002 ◽  
Vol 22 (4) ◽  
pp. 1079-1093 ◽  
Author(s):  
Cyril F. Bourgeois ◽  
Young Kyeung Kim ◽  
Mark J. Churcher ◽  
Michelle J. West ◽  
Jonathan Karn
Keyword(s):  
Human Immunodeficiency Virus ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Dna Sequences ◽  
Transcription Elongation ◽  
Elongation Factor ◽  
Immunodeficiency Virus

ABSTRACT The human immunodeficiency virus type 1 (HIV-1) Tat protein activates transcription elongation by stimulating the Tat-activated kinase (TAK/p-TEFb), a protein kinase composed of CDK9 and its cyclin partner, cyclin T1. CDK9 is able to hyperphosphorylate the carboxyl-terminal domain (CTD) of the large subunit of RNA polymerase during elongation. In addition to TAK, the transcription elongation factor Spt5 is required for the efficient activation of transcriptional elongation by Tat. To study the role of Spt5 in HIV transcription in more detail, we have developed a three-stage Tat-dependent transcription assay that permits the isolation of active preinitiation complexes, early-stage elongation complexes, and Tat-activated elongation complexes. Spt5 is recruited in the transcription complex shortly after initiation. After recruitment of Tat during elongation through the transactivation response element RNA, CDK9 is activated and induces hyperphosphorylation of Spt5 in parallel to the hyperphosphorylation of the CTD of RNA polymerase II. However, immunodepletion experiments demonstrate that Spt5 is not required for Tat-dependent activation of the kinase. Chase experiments using the Spt5-depleted extracts demonstrate that Spt5 is not required for early elongation. However, Spt5 plays an important role in late elongation by preventing the premature dissociation of RNA from the transcription complex at terminator sequences and reducing the amount of polymerase pausing at arrest sites, including bent DNA sequences. This novel biochemical function of Spt5 is analogous to the function of NusG, an elongation factor found in Escherichia coli that enhances RNA polymerase stability on templates and shows sequence similarity to Spt5.

scholarly journals CDK12 loss in cancer cells affects DNA damage response genes through premature cleavage and polyadenylation

2018 ◽  
Author(s):  
Malgorzata Krajewska ◽  
Ruben Dries ◽  
Andrew V. Grassetti ◽  
Sofia Dust ◽  
Yang Gao ◽  
...  
Keyword(s):  
Dna Damage ◽  
Rna Polymerase Ii ◽  
Dna Damage Response ◽  
Mrna Processing ◽  
Cyclin Dependent Kinase ◽  
Carboxy Terminal Domain ◽  
Damage Response ◽  
Cleavage And Polyadenylation ◽  
Polyadenylation Sites ◽  
Carboxy Terminal

The cyclin-dependent kinase 12 (CDK12) modulates transcription elongation by phosphorylating the carboxy-terminal domain of RNA polymerase II and appears to selectively affect the expression of DNA damage response (DDR) and mRNA processing genes. Yet, the mechanism(s) by which it achieves this selectivity remains unclear. Using a highly selective CDK12/13 inhibitor, THZ531, and nascent RNA sequencing, we show that CDK12 inhibition results in gene length-dependent elongation defects, leading to premature cleavage and polyadenylation (PCPA) as well as loss of expression of long (>45 kb) genes, a substantial proportion of which participate in the DDR. This early termination phenotype correlated with an increased proportion of intronic polyadenylation sites, a feature that was especially prominent among DDR genes. Finally, phosphoproteomic analysis indicated that pre-mRNA processing factors, including those involved in PCPA, are direct phosphotargets of CDKs 12 and 13. These results support a model in which DDR genes are uniquely susceptible to CDK12 inhibition due primarily to their relatively longer lengths and lower ratios of U1 snRNP binding to intronic polyadenylation sites.

scholarly journals NF-κB-Repressing Factor Inhibits Elongation of Human Immunodeficiency Virus Type 1 Transcription by DRB Sensitivity-Inducing Factor

2005 ◽  
Vol 25 (17) ◽  
pp. 7473-7483 ◽  
Author(s):  
Ursula Dreikhausen ◽  
Kirsten Hiebenthal-Millow ◽  
Myriam Bartels ◽  
Klaus Resch ◽  
Mahtab Nourbakhsh
Keyword(s):  
Human Immunodeficiency Virus ◽  
Rna Polymerase Ii ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Transcriptional Activation ◽  
Specific Binding ◽  
Transcription Elongation ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) is able to establish a latent infection during which the integrated provirus remains transcriptionally silent. In response to specific stimuli, the HIV-1 long terminal repeat (LTR) is highly activated, enhancing both transcriptional initiation and elongation. Here, we have identified a specific binding sequence of the nuclear NF-κB-repressing factor (NRF) within the HIV-1 LTR. The aim of this work was to define the role of NRF in regulating the LTR. Our data show that the endogenous NRF is required for transcriptional activation of the HIV-1 LTR in stimulated cells. In unstimulated cells, however, NRF inhibits HIV-1 LTR activity at the level of transcription elongation. Binding of NRF to the LTR in unstimulated cells prevents recruitment of elongation factor DRB sensitivity-inducing factor and formation of processive elongation complexes by hyperphosphorylated RNA polymerase II. Our data suggest that NRF interrupts the regulatory coupling of LTR binding factors and transcription elongation events. This inhibitory mechanism might contribute to transcriptional quiescence of integrated HIV-1 provirus.

scholarly journals CA150, a nuclear protein associated with the RNA polymerase II holoenzyme, is involved in Tat-activated human immunodeficiency virus type 1 transcription.

1997 ◽  
Vol 17 (10) ◽  
pp. 6029-6039 ◽  
Author(s):  
C Suñé ◽  
T Hayashi ◽  
Y Liu ◽  
W S Lane ◽  
R A Young ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcriptional Activation ◽  
Nuclear Protein ◽  
Cellular Factors ◽  
Immunodeficiency Virus ◽  
Rna Polymerase Ii Holoenzyme ◽  
Hiv 1

Maximal human immunodeficiency virus type 1 (HIV-1) gene expression requires specific cellular factors in addition to the virus-encoded trans-activator protein Tat and the RNA element TAR. We developed a functional assay, based on transcriptional activation in vitro, to identify these cellular factors. Here, we describe the purification and molecular cloning of CA150, a nuclear protein that is associated with the human RNA polymerase II holoenzyme and is involved in Tat-dependent HIV-1 transcriptional activation. The sequence of CA150 contains an extensive glutamine- and alanine-rich repeat that is found in transcriptional modulators such as GAL11 and SSN6 in Saccharomyces cerevisiae and Zeste in Drosophila melanogaster. Immunodepletion of CA150 abolished Tat trans activation in vitro. Moreover, overexpression of a mutant CA150 protein specifically and dramatically decreased Tat-mediated activation of the HIV-1 promoter in vivo, strongly suggesting a role for CA150 in HIV-1 gene regulation. Immunoprecipitation experiments demonstrated that both CA150 and Tat associate with the RNA polymerase II holoenzyme. Furthermore, we found that functional Tat associates with the holoenzyme whereas activation-deficient Tat mutants do not. Thus, we propose that Tat action is transduced via an RNA polymerase II holoenzyme that contains CA150.

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