scholarly journals Cdk7 Is Required for Full Activation of Drosophila Heat Shock Genes and RNA Polymerase II Phosphorylation In Vivo

2003 ◽  
Vol 23 (19) ◽  
pp. 6876-6886 ◽  
Author(s):  
Brian E. Schwartz ◽  
Stephane Larochelle ◽  
Beat Suter ◽  
John T. Lis
Keyword(s):  
Heat Shock ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Null Alleles ◽  
Temperature Sensitive ◽  
Heat Shock Genes ◽  
Elongation Complex ◽  
Pol Ii ◽  
Full Activation

ABSTRACT TFIIH has been implicated in several fundamental cellular processes, including DNA repair, cell cycle progression, and transcription. In transcription, the helicase activity of TFIIH functions to melt promoter DNA; however, the in vivo function of the Cdk7 kinase subunit of TFIIH, which has been hypothesized to be involved in RNA polymerase II (Pol II) phosphorylation, is not clearly understood. Using temperature-sensitive and null alleles of cdk7, we have examined the role of Cdk7 in the activation of Drosophila heat shock genes. Several in vivo approaches, including polytene chromosome immunofluorescence, nuclear run-on assays, and, in particular, a protein-DNA cross-linking assay customized for adults, revealed that Cdk7 kinase activity is required for full activation of heat shock genes, promoter-proximal Pol II pausing, and Pol II-dependent chromatin decondensation. The requirement for Cdk7 occurs very early in the transcription cycle. Furthermore, we provide evidence that TFIIH associates with the elongation complex much longer than previously suspected.

scholarly journals The exon junction complex regulates the release and phosphorylation of paused RNA polymerase II

2018 ◽  
Author(s):  
Junaid Akhtar ◽  
Nastasja Kreim ◽  
Federico Marini ◽  
Giriram Kumar Mohana ◽  
Daniel Brune ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Exon Skipping ◽  
Exon Junction Complex ◽  
Elongation Complex ◽  
Exon Definition ◽  
Pol Ii ◽  
Exon Junction ◽  
Transcriptional Regulatory

SUMMARYPromoter proximal pausing of RNA polymerase II (Pol II) is a widespread transcriptional regulatory step across metazoans. Here we find that the nuclear exon junction complex (pre-EJC) plays a critical and conserved role in this process. Depletion of pre-EJC subunits leads to a global decrease in Pol II pausing and to premature entry into elongation. This effect occurs, at least in part, via non-canonical recruitment of pre-EJC components at promoters. Failure to recruit the pre-EJC at promoters results in increased binding of the positive transcription elongation complex (P-TEFb) and in enhanced Pol II release. Notably, restoring pausing is sufficient to rescue exon skipping and the photoreceptor differentiation defect associated with depletion of pre-EJC components in vivo. We propose that the pre-EJC serves as an early transcriptional checkpoint to prevent premature entry into elongation, ensuring proper recruitment of RNA processing components that are necessary for exon definition.

scholarly journals P-TEFb Is Critical for the Maturation of RNA Polymerase II into Productive Elongation In Vivo

2007 ◽  
Vol 28 (3) ◽  
pp. 1161-1170 ◽  
Author(s):  
Zhuoyu Ni ◽  
Abbie Saunders ◽  
Nicholas J. Fuda ◽  
Jie Yao ◽  
Jose-Ramon Suarez ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Rna Processing ◽  
Transcription Initiation ◽  
Initiation Site ◽  
The Body ◽  
Elongation Complex ◽  
Pol Ii ◽  
Productive Elongation

ABSTRACT Positive transcription elongation factor b (P-TEFb) is the major metazoan RNA polymerase II (Pol II) carboxyl-terminal domain (CTD) Ser2 kinase, and its activity is believed to promote productive elongation and coupled RNA processing. Here, we demonstrate that P-TEFb is critical for the transition of Pol II into a mature transcription elongation complex in vivo. Within 3 min following P-TEFb inhibition, most polymerases were restricted to within 150 bp of the transcription initiation site of the active Drosophila melanogaster Hsp70 gene, and live-cell imaging demonstrated that these polymerases were stably associated. Polymerases already productively elongating at the time of P-TEFb inhibition, however, proceeded with elongation in the absence of active P-TEFb and cleared from the Hsp70 gene. Strikingly, all transcription factors tested (P-TEFb, Spt5, Spt6, and TFIIS) and RNA-processing factor CstF50 exited the body of the gene with kinetics indistinguishable from that of Pol II. An analysis of the phosphorylation state of Pol II upon the inhibition of P-TEFb also revealed no detectable CTD Ser2 phosphatase activity upstream of the Hsp70 polyadenylation site. In the continued presence of P-TEFb inhibitor, Pol II levels across the gene eventually recovered.

scholarly journals Direct Interaction between the Subunit RAP30 of Transcription Factor IIF (TFIIF) and RNA Polymerase Subunit 5, Which Contributes to the Association between TFIIF and RNA Polymerase II

2001 ◽  
Vol 276 (15) ◽  
pp. 12266-12273 ◽  
Author(s):  
Wenxiang Wei ◽  
Dorjbal Dorjsuren ◽  
Yong Lin ◽  
Weiping Qin ◽  
Takahiro Nomura ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Middle Part ◽  
Wild Type ◽  
Binding Region ◽  
Pol Ii ◽  
Transcription Factor Iif ◽  
B Virus

The general transcription factor IIF (TFIIF) assembled in the initiation complex, and RAP30 of TFIIF, have been shown to associate with RNA polymerase II (pol II), although it remains unclear which pol II subunit is responsible for the interaction. We examined whether TFIIF interacts with RNA polymerase II subunit 5 (RPB5), the exposed domain of which binds transcriptional regulatory factors such as hepatitis B virus X protein and a novel regulatory protein, RPB5-mediating protein. The results demonstrated that RPB5 directly binds RAP30in vitrousing purified recombinant proteins andin vivoin COS1 cells transiently expressing recombinant RAP30 and RPB5. The RAP30-binding region was mapped to the central region (amino acids (aa) 47–120) of RPB5, which partly overlaps the hepatitis B virus X protein-binding region. Although the middle part (aa 101–170) and the N-terminus (aa 1–100) of RAP30 independently bound RPB5, the latter was not involved in the RPB5 binding when RAP30 was present in TFIIF complex. Scanning of the middle part of RAP30 by clustered alanine substitutions and then point alanine substitutions pinpointed two residues critical for the RPB5 binding inin vitroandin vivoassays. Wild type but not mutants Y124A and Q131A of RAP30 coexpressed with FLAG-RAP74 efficiently recovered endogenous RPB5 to the FLAG-RAP74-bound anti-FLAG M2 resin. The recovered endogenous RPB5 is assembled in pol II as demonstrated immunologically. Interestingly, coexpression of the central region of RPB5 and wild type RAP30 inhibited recovery of endogenous pol II to the FLAG-RAP74-bound M2 resin, strongly suggesting that the RAP30-binding region of RPB5 inhibited the association of TFIIF and pol II. The exposed domain of RPB5 interacts with RAP30 of TFIIF and is important for the association between pol II and TFIIF.

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 CTCF Interacts with and Recruits the Largest Subunit of RNA Polymerase II to CTCF Target Sites Genome-Wide

2007 ◽  
Vol 27 (5) ◽  
pp. 1631-1648 ◽  
Author(s):  
Igor Chernukhin ◽  
Shaharum Shamsuddin ◽  
Sung Yun Kang ◽  
Rosita Bergström ◽  
Yoo-Wook Kwon ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Gene Activation ◽  
Ctcf Binding ◽  
Pol Ii ◽  
Genome Wide ◽  
Target Sites ◽  
On Chip

ABSTRACT CTCF is a transcription factor with highly versatile functions ranging from gene activation and repression to the regulation of insulator function and imprinting. Although many of these functions rely on CTCF-DNA interactions, it is an emerging realization that CTCF-dependent molecular processes involve CTCF interactions with other proteins. In this study, we report the association of a subpopulation of CTCF with the RNA polymerase II (Pol II) protein complex. We identified the largest subunit of Pol II (LS Pol II) as a protein significantly colocalizing with CTCF in the nucleus and specifically interacting with CTCF in vivo and in vitro. The role of CTCF as a link between DNA and LS Pol II has been reinforced by the observation that the association of LS Pol II with CTCF target sites in vivo depends on intact CTCF binding sequences. “Serial” chromatin immunoprecipitation (ChIP) analysis revealed that both CTCF and LS Pol II were present at the β-globin insulator in proliferating HD3 cells but not in differentiated globin synthesizing HD3 cells. Further, a single wild-type CTCF target site (N-Myc-CTCF), but not the mutant site deficient for CTCF binding, was sufficient to activate the transcription from the promoterless reporter gene in stably transfected cells. Finally, a ChIP-on-ChIP hybridization assay using microarrays of a library of CTCF target sites revealed that many intergenic CTCF target sequences interacted with both CTCF and LS Pol II. We discuss the possible implications of our observations with respect to plausible mechanisms of transcriptional regulation via a CTCF-mediated direct link of LS Pol II to the DNA.

scholarly journals The Skn7 Response Regulator ofSaccharomyces cerevisiaeInteracts with Hsf1 In Vivo and Is Required for the Induction of Heat Shock Genes by Oxidative Stress

2000 ◽  
Vol 11 (7) ◽  
pp. 2335-2347 ◽  
Author(s):  
Desmond C. Raitt ◽  
Anthony L. Johnson ◽  
Alexander M. Erkine ◽  
Kozo Makino ◽  
Brian Morgan ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Heat Shock ◽  
Response Regulator ◽  
Coiled Coil ◽  
Normal Growth ◽  
Temperature Sensitive ◽  
Regulatory Sequences ◽  
Heat Shock Genes

The Skn7 response regulator has previously been shown to play a role in the induction of stress-responsive genes in yeast, e.g., in the induction of the thioredoxin gene in response to hydrogen peroxide. The yeast Heat Shock Factor, Hsf1, is central to the induction of another set of stress-inducible genes, namely the heat shock genes. These two regulatory trans-activators, Hsf1 and Skn7, share certain structural homologies, particularly in their DNA-binding domains and the presence of adjacent regions of coiled-coil structure, which are known to mediate protein–protein interactions. Here, we provide evidence that Hsf1 and Skn7 interact in vitro and in vivo and we show that Skn7 can bind to the same regulatory sequences as Hsf1, namely heat shock elements. Furthermore, we demonstrate that a strain deleted for the SKN7 gene and containing a temperature-sensitive mutation in Hsf1 is hypersensitive to oxidative stress. Our data suggest that Skn7 and Hsf1 cooperate to achieve maximal induction of heat shock genes in response specifically to oxidative stress. We further show that, like Hsf1, Skn7 can interact with itself and is localized to the nucleus under normal growth conditions as well as during oxidative stress.

scholarly journals Fcp1 Dephosphorylation of the RNA Polymerase II C-Terminal Domain Is Required for Efficient Transcription of Heat Shock Genes

2012 ◽  
Vol 32 (17) ◽  
pp. 3428-3437 ◽  
Author(s):  
N. J. Fuda ◽  
M. S. Buckley ◽  
W. Wei ◽  
L. J. Core ◽  
C. T. Waters ◽  
...  
Keyword(s):  
Heat Shock ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Heat Shock Genes ◽  
Terminal Domain

scholarly journals Yeast NC2 Associates with the RNA Polymerase II Preinitiation Complex and Selectively Affects Transcription In Vivo

2001 ◽  
Vol 21 (8) ◽  
pp. 2736-2742 ◽  
Author(s):  
Joseph V. Geisberg ◽  
Frank C. Holstege ◽  
Richard A. Young ◽  
Kevin Struhl
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcriptional Activation ◽  
Negative Regulator ◽  
Preinitiation Complex ◽  
Positive Role ◽  
Pol Ii ◽  
Basal Transcription Factors

ABSTRACT NC2 (Dr1-Drap1 or Bur6-Ydr1) has been characterized in vitro as a general negative regulator of RNA polymerase II (Pol II) transcription that interacts with TATA-binding protein (TBP) and inhibits its function. Here, we show that NC2 associates with promoters in vivo in a manner that correlates with transcriptional activity and with occupancy by basal transcription factors. NC2 rapidly associates with promoters in response to transcriptional activation, and it remains associated under conditions in which transcription is blocked after assembly of the Pol II preinitiation complex. NC2 positively and negatively affects approximately 17% of Saccharomyces cerevisiaegenes in a pattern that resembles the response to general environmental stress. Relative to TBP, NC2 occupancy is high at promoters where NC2 is positively required for normal levels of transcription. Thus, NC2 is associated with the Pol II preinitiation complex, and it can play a direct and positive role at certain promoters in vivo.

scholarly journals A Nonconserved Surface of the TFIIB Zinc Ribbon Domain Plays a Direct Role in RNA Polymerase II Recruitment

2004 ◽  
Vol 24 (7) ◽  
pp. 2863-2874 ◽  
Author(s):  
Thomas C. Tubon ◽  
William P. Tansey ◽  
Winship Herr
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
Terminal Region ◽  
General Role ◽  
Pol Ii ◽  
Amino Terminal ◽  
Zinc Ribbon

ABSTRACT The general transcription factor TFIIB is a highly conserved and essential component of the eukaryotic RNA polymerase II (pol II) transcription initiation machinery. It consists of a single polypeptide with two conserved structural domains: an amino-terminal zinc ribbon structure (TFIIBZR) and a carboxy-terminal core (TFIIBCORE). We have analyzed the role of the amino-terminal region of human TFIIB in transcription in vivo and in vitro. We identified a small nonconserved surface of the TFIIBZR that is required for pol II transcription in vivo and for different types of basal pol II transcription in vitro. Consistent with a general role in transcription, this TFIIBZR surface is directly involved in the recruitment of pol II to a TATA box-containing promoter. Curiously, although the amino-terminal human TFIIBZR domain can recruit both human pol II and yeast (Saccharomyces cerevisiae) pol II, the yeast TFIIB amino-terminal region recruits yeast pol II but not human pol II. Thus, a critical process in transcription from many different promoters—pol II recruitment—has changed in sequence specificity during eukaryotic evolution.

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