The regulatory role for the ERCC3 helicase of general transcription factor TFIIH during promoter escape in transcriptional activation

Fukuda, Aya; Nogi, Yasuhisa; Hisatake, Koji

doi:10.1073/pnas.251674198

Cited by 18 publications

(24 citation statements)

References 47 publications

(58 reference statements)

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“…To gain a mechanistic insight into the coactivator function in transcriptional activation, we utilized a model in vitro transcription system that included GAL4-VP16 as an activator and PC4 as a coactivator. This in vitro transcription system contained well-defined components, including recombinant factors (TFIIA, TFIIB, TFIIE, TFIIF, TFIIH, PC4, and GAL4-VP16) as well as highly purified HeLa cell-derived FLAG-tagged TFIID and RNA polymerase II (10)(11)(12), and exhibited marked transcriptional activation in response to GAL4-VP16 in a highly PC4-dependent manner. These features of this system provided an excellent opportunity to analyze mechanistic aspects of the coactivator function of PC4 in a quantitative manner.…”

Section: Resultsmentioning

confidence: 99%

“…Purification of recombinant factors (TFIIA, TFIIB, TFIIE, TFIIF, TFIIH, and GAL4-VP16), epitope-tagged TFIID, and RNA polymerase II were performed as described previously (10,12). Recombinant PC4 was purified as described previously (11).…”

Section: Methodsmentioning

confidence: 99%

“…Recombinant PC4 was purified as described previously (11). For 390-and 20-nucleotide (nt) transcripts, in vitro transcription reaction mixtures (25 l) contained 50 ng of negatively supercoiled pG5HMC2AT or its derivative, 12 mM HEPES-KOH (pH 7.9), 6% glycerol, 60 mM KCl, 0.6 mM EDTA, 8 (10), and the derived pppApC was treated with calf intestinal phosphatase to form ApC before electrophoresis (29). After electrophoresis, the transcripts were quantified by using a Fujix Bas2000 bioimaging analyzer.…”

Section: Methodsmentioning

confidence: 99%

“…However, given that transcription is a multistep process consisting of PIC assembly, promoter opening, initiation, promoter escape, elongation, and reinitiation, steps other than PIC assembly are potential targets for regulation as well. Indeed, despite predominant effects of activators-presumably in conjunction with coactivators-on PIC assembly, the effects on the subsequent steps (1,28,31,37) have also been demonstrated in various systems, including promoter opening (60), promoter escape (10,29), elongation (66), and reinitiation (26,67). However, the relative contributions of activators and coactivators in stimulating individual steps of transcription remain to be more clearly defined.…”

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confidence: 99%

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Transcriptional Coactivator PC4 Stimulates Promoter Escape and Facilitates Transcriptional Synergy by GAL4-VP16

Fukuda

Nakadai

Shimada

et al. 2004

Molecular and Cellular Biology

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Positive cofactor 4 (PC4) is a coactivator that strongly augments transcription by various activators, presumably by facilitating the assembly of the preinitiation complex (PIC). However, our previous observation of stimulation of promoter escape in GAL4-VP16-dependent transcription in the presence of PC4 suggested a possible role for PC4 in this step. Here, we performed quantitative analyses of the stimulatory effects of PC4 on initiation, promoter escape, and elongation in GAL4-VP16-dependent transcription and found that PC4 possesses the ability to stimulate promoter escape in response to GAL4-VP16 in addition to its previously demonstrated effect on PIC assembly. This stimulatory effect of PC4 on promoter escape required TFIIA and the TATA box binding protein-associated factor subunits of TFIID. Furthermore, PC4 displayed physical interactions with both TFIIH and GAL4-VP16 through its coactivator domain, and these interactions were regulated distinctly by PC4 phosphorylation. Finally, GAL4-VP16 and PC4 stimulated both initiation and promoter escape to similar extents on the promoters with three and five GAL4 sites; however, they stimulated promoter escape preferentially on the promoter with a single GAL4 site. These results provide insight into the mechanism by which PC4 permits multiply bound GAL4-VP16 to attain synergy to achieve robust transcriptional activation.Transcription of mRNA-coding genes involves RNA polymerase II and six general transcription factors (TFIIA, TFIIB, TFIID, TFIIE, TFIIF, and TFIIH) which comprise the basal transcription machinery that recognizes the core promoter elements and elicits the basal level of transcription (50). Activated transcription requires the binding of activators to the regulatory DNA sequences typically present upstream of the core promoter and their interactions with the general transcription machinery (32,49). Despite the well-documented direct interactions of activators with the general transcription factors and RNA polymerase II, activated transcription requires yet another group of transcription factors, termed mediators or coactivators, that confer on the general transcription machinery a markedly enhanced responsiveness to activators (2,18,20,36,41).A wide array of coactivators may be grouped into two broad categories according to the requirement of chromatin for their action in biochemical assays. The coactivators which function on the templates without chromatin include the TATA box binding protein-associated factors (TAFs) present in TFIID (58), positive cofactors (PCs) (PC1, PC2, PC3, and PC4) derived from the upstream factor stimulatory activity (USA) cofactor fraction (20), and metazoan multiprotein complexes that are structurally related to the yeast mediator (40) (TRAP/ SMCC, ARC, DRIP, NAT, murine mediator, human mediator, CRSP, and PC2) (36, 41). The coactivators which require chromatin templates for their functions include CBP/p300, PCAF and its related GCN5 proteins, and p160 family proteins that display histone acetyltransferase activities (4,...

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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Transcriptional Coactivator PC4 Stimulates Promoter Escape and Facilitates Transcriptional Synergy by GAL4-VP16

Fukuda

Nakadai

Shimada

et al. 2004

Molecular and Cellular Biology

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“…The XPB helicase is a multi-function protein, being required to unwind DNA before all RNA Pol II-dependent transcription 64 , Table 10 for quantification and number of biological replicates). The black asterisks show significance points compared with control; red asterisks compared with Hfp hypomorph alone and green asterisks compared with Hay nc2RV1 in the Hfp hypomorph background.…”

Section: Discussionmentioning

confidence: 99%

Defective Hfp-dependent transcriptional repression of dMYC is fundamental to tissue overgrowth in Drosophila XPB models

et al. 2015

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Nucleotide excision DNA repair (NER) pathway mutations cause neurodegenerative and progeroid disorders (xeroderma pigmentosum (XP), Cockayne syndrome (CS) and trichothiodystrophy (TTD)), which are inexplicably associated with (XP) or without (CS/TTD) cancer. Moreover, cancer progression occurs in certain patients, but not others, with similar C-terminal mutations in the XPB helicase subunit of transcription and NER factor TFIIH. Mechanisms driving overproliferation and, therefore, cancer associated with XPB mutations are currently unknown. Here using Drosophila models, we provide evidence that C-terminally truncated Hay/XPB alleles enhance overgrowth dependent on reduced abundance of RNA recognition motif protein Hfp/FIR, which transcriptionally represses the MYC oncogene homologue, dMYC. The data demonstrate that dMYC repression and dMYC-dependent overgrowth in the Hfp hypomorph is further impaired in the C-terminal Hay/XPB mutant background. Thus, we predict defective transcriptional repression of MYC by the Hfp orthologue, FIR, might provide one mechanism for cancer progression in XP/CS.

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ABT1‐associated protein (ABTAP), a novel nuclear protein conserved from yeast to mammals, represses transcriptional activation by ABT1

Oda

Fukuda

Hagiwara

et al. 2004

J of Cellular Biochemistry

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Various TATA-binding protein (TBP)-associated proteins are involved in the regulation of gene expression through control of basal transcription directed by RNA polymerase (Pol) II. We recently identified a novel nuclear protein, activator of basal transcription 1 (ABT1), which binds TBP and DNA, and enhances Pol II-directed basal transcription. To better understand regulatory mechanisms for ABT1, we searched for ABT1-binding proteins using a yeast two-hybrid screening and isolated a cDNA clone encoding a novel protein termed ABT1-associated protein (ABTAP). ABTAP formed a complex with ABT1 and suppressed the ABT1-induced activation of Pol II-directed transcription in mammalian cells. Furthermore, ABTAP directly bound to ABT1, disrupted the interaction between ABT1 and TBP, and suppressed the ABT1-induced activation of Pol II-directed basal transcription in vitro. These two proteins colocalized in the nucleolus and nucleoplasm and were concomitantly relocalized into discrete nuclear bodies at higher expression of ABTAP. Taken together, these results suggest that ABTAP binds and negatively regulates ABT1. The ABT1/ABTAP complex is evolutionarily conserved and may constitute a novel regulatory system for basal transcription.

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The regulatory role for the ERCC3 helicase of general transcription factor TFIIH during promoter escape in transcriptional activation

Cited by 18 publications

References 47 publications

Transcriptional Coactivator PC4 Stimulates Promoter Escape and Facilitates Transcriptional Synergy by GAL4-VP16

Transcriptional Coactivator PC4 Stimulates Promoter Escape and Facilitates Transcriptional Synergy by GAL4-VP16

Defective Hfp-dependent transcriptional repression of dMYC is fundamental to tissue overgrowth in Drosophila XPB models

ABT1‐associated protein (ABTAP), a novel nuclear protein conserved from yeast to mammals, represses transcriptional activation by ABT1

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