scholarly journals Subregions of the adenovirus E1A transactivation domain target multiple components of the TFIID complex.

1995 ◽  
Vol 15 (11) ◽  
pp. 6283-6290 ◽  
Author(s):  
J V Geisberg ◽  
J L Chen ◽  
R P Ricciardi
Keyword(s):  
Transcriptional Activation ◽  
Terminal Region ◽  
Transactivation Domain ◽  
Adenovirus E1a ◽  
Critical Requirement ◽  
Tata Box Binding Protein ◽  
Carboxy Terminal ◽  
Tfiid Complex

Transcriptional activation by the adenovirus E1A 289R protein requires direct contacts with the TATA box-binding protein (TBP) and also displays a critical requirement for TBP-associated factors (TAFs) (T.G. Boyer and A. J. Berk, Genes Dev. 7:1810-1823, 1993; J. V. Geisberg, W. S. Lee, A. J. Berk, and R. P. Ricciardi, Proc. Natl. Acad. Sci. USA 91:2488-2492, 1994; W. S. Lee, C. C. Kao, G. O. Bryant, X. Liu, and A. J. Berk, Cell 67:365-376, 1991; and Q. Zhou, P. M. Lieberman, T. G. Boyer, and A. J. Berk, Genes Dev. 6:1964-1974, 1992). In this report, we demonstrate that the activation domain of E1A (CR3) specifically binds to two TAFs, human TAFII250 (hTAFII250) and Drosophila TAFII110 (dTAFII110). These interactions can take place both in vivo and in vitro and require the carboxy-terminal region of CR3; the zinc finger region of CR3, which binds TBP, is not needed to bind these TAFs. We mapped the E1A-binding sites on hTAFII250 to an internal region that contains a number of structural motifs, including an HMG box, a bromodomain, and direct repeats. This represents the first demonstration that hTAFII250 may serve as a target of a transcriptional activator. We also mapped the E1A binding on dTAFII110 to its C-terminal region. This is of significance since, by contrast, Sp1-mediated activation requires binding to the N-terminal domain of dTAFII110. Thus, distinct surfaces of dTAFII110 can serve as target sites for different activators. Our results indicate that E1A may activate transcription, in part, through direct contacts of the CR3 subdomains with selected components of the TFIID complex.

c-Fos-induced activation of a TATA-box-containing promoter involves direct contact with TATA-box-binding protein

1994 ◽  
Vol 14 (9) ◽  
pp. 6021-6029
Author(s):  
R Metz ◽  
A J Bannister ◽  
J A Sutherland ◽  
C Hagemeier ◽  
E C O'Rourke ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Transcriptional Activation ◽  
Binding Protein ◽  
Tata Box ◽  
Binding Motif ◽  
Protein Protein Interactions ◽  
High Concentrations ◽  
Tata Box Binding Protein

Transcriptional activation in eukaryotes involves protein-protein interactions between regulatory transcription factors and components of the basal transcription machinery. Here we show that c-Fos, but not a related protein, Fra-1, can bind the TATA-box-binding protein (TBP) both in vitro and in vivo and that c-Fos can also interact with the transcription factor IID complex. High-affinity binding to TBP requires c-Fos activation modules which cooperate to activate transcription. One of these activation modules contains a TBP-binding motif (TBM) which was identified through its homology to TBP-binding viral activators. This motif is required for transcriptional activation, as well as TBP binding. Domain swap experiments indicate that a domain containing the TBM can confer TBP binding on Fra-1 both in vitro and in vivo. In vivo activation experiments indicate that a GAL4-Fos fusion can activate a promoter bearing a GAL4 site linked to a TATA box but that this activity does not occur at high concentrations of GAL4-Fos. This inhibition (squelching) of c-Fos activity is relieved by the presence of excess TBP, indicating that TBP is a direct functional target of c-Fos. Removing the TBM from c-Fos severely abrogates activation of a promoter containing a TATA box but does not affect activation of a promoter driven only by an initiator element. Collectively, these results suggest that c-Fos is able to activate via two distinct mechanisms, only one of which requires contact with TBP. Since TBP binding is not exhibited by Fra-1, TBP-mediated activation may be one characteristic that discriminates the function of Fos-related proteins.

scholarly journals ADR1-Mediated Transcriptional Activation Requires the Presence of an Intact TFIID Complex

1998 ◽  
Vol 18 (10) ◽  
pp. 5861-5867 ◽  
Author(s):  
Philip B. Komarnitsky ◽  
Edward R. Klebanow ◽  
P. Anthony Weil ◽  
Clyde L. Denis
Keyword(s):  
Transcriptional Activation ◽  
Yeast Cells ◽  
Binding Studies ◽  
Whole Cell ◽  
Transactivation Domains ◽  
Cell Extracts ◽  
Genes Expression ◽  
Tfiid Complex

ABSTRACT The yeast transcriptional activator ADR1, which is required forADH2 and other genes’ expression, contains four transactivation domains (TADs). While previous studies have shown that these TADs act through GCN5 and ADA2, and presumably TFIIB, other factors are likely to be involved in ADR1 function. In this study, we addressed the question of whether TFIID is also required for ADR1 action. In vitro binding studies indicated that TADI of ADR1 was able to retain TAFII90 from yeast extracts and TADII could retain TBP and TAFII130/145. TADIV, however, was capable of retaining multiple TAFIIs, suggesting that TADIV was binding TFIID from yeast whole-cell extracts. The ability of TADIV truncation derivatives to interact with TFIID correlated with their transcription activation potential in vivo. In addition, the ability of LexA-ADR1-TADIV to activate transcription in vivo was compromised by a mutation in TAFII130/145. ADR1 was found to associate in vivo with TFIID in that immunoprecipitation of either TAFII90 or TBP from yeast whole-cell extracts specifically coimmunoprecipitated ADR1. Most importantly, depletion of TAFII90 from yeast cells dramatically reducedADH2 derepression. These results indicate that ADR1 physically associates with TFIID and that its ability to activate transcription requires an intact TFIID complex.

scholarly journals The Gcn4p Activation Domain Interacts Specifically In Vitro with RNA Polymerase II Holoenzyme, TFIID, and the Adap-Gcn5p Coactivator Complex

1998 ◽  
Vol 18 (3) ◽  
pp. 1711-1724 ◽  
Author(s):  
Connie M. Drysdale ◽  
Belinda M. Jackson ◽  
Richard McVeigh ◽  
Edward R. Klebanow ◽  
Yu Bai ◽  
...  
Keyword(s):  
Rna Polymerase Ii ◽  
Transcriptional Activation ◽  
Adapter Proteins ◽  
Activation Domain ◽  
Hydrophobic Residues ◽  
Cell Extracts ◽  
Tata Box Binding Protein ◽  
Rna Polymerase Ii Holoenzyme

ABSTRACT The Gcn4p activation domain contains seven clusters of hydrophobic residues that make additive contributions to transcriptional activation in vivo. We observed efficient binding of a glutathioneS-transferase (GST)–Gcn4p fusion protein to components of three different coactivator complexes in Saccharomyces cerevisiae cell extracts, including subunits of transcription factor IID (TFIID) (yeast TAFII20 [yTAFII20], yTAFII60, and yTAFII90), the holoenzyme mediator (Srb2p, Srb4p, and Srb7p), and the Adap-Gcn5p complex (Ada2p and Ada3p). The binding to these coactivator subunits was completely dependent on the hydrophobic clusters in the Gcn4p activation domain. Alanine substitutions in single clusters led to moderate reductions in binding, double-cluster substitutions generally led to greater reductions in binding than the corresponding single-cluster mutations, and mutations in four or more clusters reduced binding to all of the coactivator proteins to background levels. The additive effects of these mutations on binding of coactivator proteins correlated with their cumulative effects on transcriptional activation by Gcn4p in vivo, particularly with Ada3p, suggesting that recruitment of these coactivator complexes to the promoter is a cardinal function of the Gcn4p activation domain. As judged by immunoprecipitation analysis, components of the mediator were not associated with constituents of TFIID and Adap-Gcn5p in the extracts, implying that GST-Gcn4p interacted with the mediator independently of these other coactivators. Unexpectedly, a proportion of Ada2p coimmunoprecipitated with yTAFII90, and the yTAFII20, -60, and -90 proteins were coimmunoprecipitated with Ada3p, revealing a stable interaction between components of TFIID and the Adap-Gcn5p complex. Because GST-Gcn4p did not bind specifically to highly purified TFIID, Gcn4p may interact with TFIID via the Adap-Gcn5p complex or some other adapter proteins. The ability of Gcn4p to interact with several distinct coactivator complexes that are physically and genetically linked to TATA box-binding protein can provide an explanation for the observation that yTAFII proteins are dispensable for activation by Gcn4p in vivo.

scholarly journals The erythroid Krüppel-like factor transactivation domain is a critical component for cell-specific inducibility of a beta-globin promoter.

1995 ◽  
Vol 15 (2) ◽  
pp. 852-860 ◽  
Author(s):  
J J Bieker ◽  
C M Southwood
Keyword(s):  
Dna Binding ◽  
Transcriptional Activation ◽  
Erythroid Cell ◽  
Transactivation Domain ◽  
Beta Globin ◽  
Cell Type Specificity ◽  
Globin Promoter ◽  
Murine Erythroleukemia

Erythroid Krüppel-like factor (EKLF) is an erythroid cell-specific DNA-binding protein that activates transcription from the beta-globin CACCC element, a functionally important and evolutionarily conserved component of globin as well as other erythroid cell-specific promoters and enhancers. We have attempted to elucidate the molecular role of EKLF in erythrocyte-specific transcriptional activation. First, in vivo and in vitro analyses have been used to demonstrate that the level of activation by EKLF is dependent on the orientation and number of CACCC elements, that EKLF contains separable activation and DNA-binding domains, and that the EKLF proline-rich region is a potent activator in CV-1 cells when fused to a nonrelated DNA-binding module. Second, we have established a transient assay in murine erythroleukemia cells in which reproducible levels of a reporter can be induced when linked to a locus control region enhancer-beta-globin promoter and in which induction is abolished when the promoter CAC site is mutated to a GAL site. Third, we demonstrate that the EKLF transactivation region, when fused to the GAL DNA-binding domain, can restore inducibility to this mutated construct and that this inducibility exhibits activator-, promoter-, and cell-type specificity. These results demonstrate that EKLF provides a crucial transactivation function for globin expression and further reinforce the idea that EKLF is an important regulator of CACCC element-directed transcription in erythroid cells.

scholarly journals The hsp70 gene CCAAT-binding factor mediates transcriptional activation by the adenovirus E1a protein.

1992 ◽  
Vol 12 (6) ◽  
pp. 2599-2605 ◽  
Author(s):  
L S Lum ◽  
S Hsu ◽  
M Vaewhongs ◽  
B Wu
Keyword(s):  
Transcriptional Activation ◽  
Dependent Manner ◽  
Hsp70 Gene ◽  
Hsp70 Promoter ◽  
Adenovirus E1a ◽  
Cell Biol ◽  
Binding Factor ◽  
E1a Protein

Expression of the human hsp70 gene is cell cycle regulated and is inducible by both serum and the adenovirus E1a protein (K. Milarski and R. Morimoto, Proc. Natl. Acad. Sci. USA 83:9517-9521, 1986; M. C. Simon, K. Kitchener, H.-T. Kao, E. Hickey, L. Weber, R. Voellmy, N. Heintz, and J. R. Nevins, Mol. Cell. Biol. 7:2884-2890, 1987; B. Wu, H. Hurst, N. Jones, and R. Morimoto, Mol. Cell. Biol. 6:2994-2999, 1986; B. Wu and R. Morimoto, Proc. Natl. Acad. Sci. USA 82:6070-6074, 1985). This regulated expression is predominantly controlled by the CCAAT element at position -70 relative to the transcriptional initiation site (G. Williams, T. McClanahan, and R. Morimoto, Mol. Cell. Biol. 9:2574-2587, 1989; B. Wu, H. Hurst, N. Jones, and R. Morimoto, Mol. Cell. Biol. 6:2994-2999, 1986). A corresponding CCAAT-binding factor (CBF) of 999 amino acids has recently been cloned and shown to stimulate transcription selectively from the hsp70 promoter in a CCAAT element-dependent manner (L. Lum, L. Sultzman, R. Kaufman, D. Linzer, and B. Wu, Mol. Cell. Biol. 10:6709-6717, 1990). We report here that the first 192 residues of CBF, when fused to the DNA-binding domain of the heterologous activator GAL-4, are necessary and sufficient to mediate E1a-dependent transcriptional activation. E1a and CBF exhibit complex formation in vitro, suggesting that an in vivo interaction between these proteins may be relevant to the well-characterized E1a-induced transcriptional activation of the hsp70 promoter.

scholarly journals AR1 Is an Integral Part of the Adenovirus Type 2 E1A-CR3 Transactivation Domain

1998 ◽  
Vol 72 (7) ◽  
pp. 5978-5983 ◽  
Author(s):  
Anne-Christine Ström ◽  
Petra Ohlsson ◽  
Göran Akusjärvi
Keyword(s):  
Amino Acid ◽  
Transcriptional Activation ◽  
Adenovirus Type ◽  
Genetic Dissection ◽  
Transactivation Domain ◽  
Sequence Elements ◽  
Carboxy Terminal ◽  
Adenovirus Type 2

ABSTRACT We have previously shown that the nonconserved carboxy-terminal exon of the adenovirus type 2 E1A-289R protein contains two interchangeable sequence elements, auxiliary region (AR) 1 and AR2, that are required for efficient CR3-mediated transcriptional activation of the viral E4 promoter (M. Bondesson, C. Svensson, S. Linder, and G. Akusjärvi, EMBO J. 11:3347–3354, 1992). Here we show that CR3-mediated transactivation of all adenovirus early promoters and the HSP70 promoter requires the AR1 element. We further show that AR2 can substitute for AR1 only when artificially juxtaposed to CR3. AR1 consists of six tandem glutamic acid-proline (EP) repeats and is positioned immediately downstream of CR3. Genetic dissection of AR1 showed that the number of EP repeats in AR1 is critical for CR3 function. Thus, reducing or increasing the number of EP repeats reduces the CR3 transactivation capacity. Furthermore, the introduction of amino acid substitutions into AR1 suggested that the net negative charge in AR1 is of critical importance for its function as an enhancer of CR3-mediated transcriptional activation. Using an in vitro binding approach, we showed that the AR1 element is not part of the CR3 promoter localization signal mediating contact with the Sp1, ATF-2, or c-Jun upstream-binding transcription factors. Previous studies have suggested that the 49-amino-acid sequence constituting CR3 represents the minimal domain required for E1A-induced activation of viral early promoters. Since AR1 was required for efficient CR3-mediated transcriptional activation of all tested promoters, we suggest that the carboxy-terminal boundary for the CR3 transactivation domain should be extended to include the AR1 element.

scholarly journals Acetylation-Mediated Transcriptional Activation of the ETS Protein ER81 by p300, P/CAF, and HER2/Neu

2003 ◽  
Vol 23 (17) ◽  
pp. 6243-6254 ◽  
Author(s):  
Apollina Goel ◽  
Ralf Janknecht
Keyword(s):  
Protein Kinase ◽  
Transcriptional Activation ◽  
Binding Activity ◽  
Tumor Formation ◽  
Mitogen Activated Protein Kinase ◽  
Transactivation Domain ◽  
Dna Binding Activity ◽  
Mitogen Activated Protein

ABSTRACT The regulated expression of the ETS transcription factor ER81 is a prerequisite for normal development, and its dysregulation contributes to neoplasia. Here, we demonstrate that ER81 is acetylated by two coactivators/acetyltransferases, p300 and p300- and CBP-associated factor (P/CAF) in vitro and in vivo. Whereas p300 acetylates two lysine residues (K33 and K116) within the ER81 N-terminal transactivation domain, P/CAF targets only K116. Acetylation of ER81 not only enhances its ability to transactivate but also increases its DNA binding activity and in vivo half-life. Furthermore, oncogenic HER2/Neu, which induces phosphorylation and thereby activation of ER81, was less able to activate acetylation-deficient ER81 mutants, indicating that both acetyltransferase and protein kinase-specific regulatory mechanisms control ER81 activity. Importantly, HER2/Neu overexpression stimulates the ability of p300 to acetylate ER81, likely by inducing phosphorylation of p300 through the Ras→Raf→mitogen-activated protein kinase pathway. This represents a novel mechanism by which oncogenic HER2/Neu, Ras, or Raf may promote tumor formation by enhancing acetylation not only of ER81 but also of other downstream effector transcription factors as well as histones.

scholarly journals Region of Yeast TAF 130 Required for TFIID To Associate with Promoters

2001 ◽  
Vol 21 (4) ◽  
pp. 1145-1154 ◽  
Author(s):  
Mario Mencı́a ◽  
Kevin Struhl
Keyword(s):  
Rna Polymerase Ii ◽  
Transcriptional Activation ◽  
Thermal Inactivation ◽  
Yeast Cells ◽  
Activation Domains ◽  
Core Promoters ◽  
Transcriptional Activation Domains ◽  
Tfiid Complex

ABSTRACT TFIID, a multiprotein complex comprising the TATA-binding protein (TBP) and TBP-associated factors (TAFs), associates specifically with core promoters and nucleates the assembly the RNA polymerase II transcription machinery. In yeast cells, TFIID is not generally required for transcription, although it plays an important role at many promoters. Understanding of the specific functions and physiological roles of individual TAFs within TFIID has been hampered by the fact that depletion or thermal inactivation of individual TAFs generally results in dissociation of the TFIID complex. We describe here C-terminally deleted derivatives of yeast TAF130 that assemble into normal TFIID complexes but are transcriptionally inactive in vivo. In vivo, these mutant TFIID complexes are dramatically reduced in their ability to associate with all promoters tested. In vitro, a TFIID complex containing a deleted form of TAF130 associates poorly with DNA, but it is unaffected for interacting with transcriptional activation domains. These results suggest that the C-terminal region of TAF130 is required for TFIID to associate with promoters.

scholarly journals AMF-1/Gps2 Binds p300 and Enhances Its Interaction with Papillomavirus E2 Proteins

2000 ◽  
Vol 74 (13) ◽  
pp. 5872-5879 ◽  
Author(s):  
Yu-Cai Peng ◽  
David E. Breiding ◽  
Francis Sverdrup ◽  
James Richard ◽  
Elliot J. Androphy
Keyword(s):  
Gene Expression ◽  
Transcriptional Activation ◽  
Functional Significance ◽  
Cellular Protein ◽  
Transcriptional Coactivator ◽  
Adenovirus E1a ◽  
Coactivator P300 ◽  
P300 Recruitment

ABSTRACT The cellular protein AMF-1 (Gps2) positively modulates gene expression by the papillomavirus E2 protein (D. E. Breiding et al., Mol. Cell. Biol. 17:7208–7219, 1997). We show here that AMF-1 also binds the transcriptional coactivator p300 in vitro and in vivo. E2 interacted weakly with p300. These observations led to a model in which AMF-1 recruits p300 into a complex with E2. Cotransfection of AMF-1 or p300 stimulated levels of E2-dependent transcription, while cotransfection of both AMF-1 and p300 showed an additive effect. The functional significance of p300 recruitment for E2 transactivation was evidenced by repression of E2-activated transcription by adenovirus E1A, which inhibits both coactivator and acetylase activities of p300. Antibodies to AMF-1 or E2 immunoprecipitated histone acetylase activity from cell lysates. Western blotting using antibody against acetyl-lysine failed to detect acetylation of AMF-1 or E2 in complex with p300. These results suggest that AMF-1 facilitates the recruitment of p300 and its histone acetylase activity into complexes with E2 and represents a novel mechanism of transcriptional activation.

scholarly journals Functional interaction of a novel cellular protein with the papillomavirus E2 transactivation domain.

1997 ◽  
Vol 17 (12) ◽  
pp. 7208-7219 ◽  
Author(s):  
D E Breiding ◽  
F Sverdrup ◽  
M J Grossel ◽  
N Moscufo ◽  
W Boonchai ◽  
...  
Keyword(s):  
Amino Acid ◽  
Dna Replication ◽  
Transcriptional Activation ◽  
Mammalian Cells ◽  
Point Mutations ◽  
Cellular Protein ◽  
Bovine Papillomavirus ◽  
Transactivation Domain

The transactivation domain (AD) of bovine papillomavirus type 1 E2 stimulates gene expression and DNA replication. To identify cellular proteins that interact with this 215-amino-acid domain, we used a transactivation-defective mutant as bait in the yeast two-hybrid screen. In vitro and in vivo results demonstrate that the cDNA of one plasmid isolated in this screen encodes a 37-kDa nuclear protein that specifically binds to an 82-amino-acid segment within the E2 AD. Mutants with point mutations within this E2 domain were isolated based on their inability to interact with AMF-1 and were found to be unable to stimulate transcription. These mutants also exhibited defects in viral DNA replication yet retained binding to the viral E1 replication initiator protein. Overexpression of AMF-1 stimulated transactivation by both wild-type E2 and a LexA fusion to the E2 AD, indicating that AMF-1 is a positive effector of the AD of E2. We conclude that interaction with AMF-1 is necessary for the transcriptional activation function of the E2 AD in mammalian cells.

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