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Transcriptional control by adenovirus E1A conserved region 3 via p300/CBP.

Pelka P, Ablack JN, Torchia J, Turnell AS, Grand RJ, Mymryk JS - Nucleic Acids Res. (2009)

Bottom Line: This is supported by the observation that the repressive effect of E1A 12S is reversed by expression of exogenous p300 or CBP, but not by a CBP mutant lacking actyltransferase activity.Furthermore, we show that transcriptional activation by 13S E1A is greatly reduced by siRNA knockdown of p300 and that CR3 binds p300 independently of the well-characterized N-terminal/CR1-binding site.These results identify a new functionally significant interaction between E1A CR3 and the p300/CBP acetyltransferases, expanding our understanding of the mechanism by which this potent transcriptional activator functions.

View Article: PubMed Central - PubMed

Affiliation: Department of Oncology, The University of Western Ontario, London Regional Cancer Centre, London, Ontario, Canada. peter.pelka@gmail.com

ABSTRACT
The human adenovirus type 5 (HAdV-5) E1A 13S oncoprotein is a potent regulator of gene expression and is used extensively as a model for transcriptional activation. It possesses two independent transcriptional activation domains located in the N-terminus/conserved region (CR) 1 and CR3. The protein acetyltransferase p300 was previously identified by its association with the N-terminus/CR1 portion of E1A and this association is required for oncogenic transformation by E1A. We report here that transcriptional activation by 13S E1A is inhibited by co-expression of sub-stoichiometric amounts of the smaller 12S E1A isoform, which lacks CR3. Transcriptional inhibition by E1A 12S maps to the N-terminus and correlates with the ability to bind p300/CBP, suggesting that E1A 12S is sequestering this limiting factor from 13S E1A. This is supported by the observation that the repressive effect of E1A 12S is reversed by expression of exogenous p300 or CBP, but not by a CBP mutant lacking actyltransferase activity. Furthermore, we show that transcriptional activation by 13S E1A is greatly reduced by siRNA knockdown of p300 and that CR3 binds p300 independently of the well-characterized N-terminal/CR1-binding site. Importantly, CR3 is also required to recruit p300 to the adenovirus E4 promoter during infection. These results identify a new functionally significant interaction between E1A CR3 and the p300/CBP acetyltransferases, expanding our understanding of the mechanism by which this potent transcriptional activator functions.

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Repression of E1A 13S- and CR3-mediated transactivation by E1A 12S is dose-dependent and independent of the method by which E1A is targeted to the promoter. (A) HeLa cells were co-transfected with plasmids expressing E1A 13S (1.5 μg) and E1A 12S (as indicated) together with an adenovirus E3-luciferase reporter plasmid (0.5 μg). Luciferase activity was assayed 48 hours after transfection. TF, transcription factor. (B) HeLa cells were co-transfected with plasmids expressing E1A 13S (1.5 μg) and E1A 12S (as indicated) together with an adenovirus E4-luciferase reporter plasmid (0.5 μg). Luciferase activity was assayed 48 h after transfection. (C) U2OS cells were co-transfected with plasmids expressing a GAL4 DNA-binding domain-CR3 (1 μg) fusion and E1A 12S (as indicated) together with a GAL4 responsive luciferase reporter (1 μg). Luciferase activity was assayed 24 h after transfection.
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Figure 2: Repression of E1A 13S- and CR3-mediated transactivation by E1A 12S is dose-dependent and independent of the method by which E1A is targeted to the promoter. (A) HeLa cells were co-transfected with plasmids expressing E1A 13S (1.5 μg) and E1A 12S (as indicated) together with an adenovirus E3-luciferase reporter plasmid (0.5 μg). Luciferase activity was assayed 48 hours after transfection. TF, transcription factor. (B) HeLa cells were co-transfected with plasmids expressing E1A 13S (1.5 μg) and E1A 12S (as indicated) together with an adenovirus E4-luciferase reporter plasmid (0.5 μg). Luciferase activity was assayed 48 h after transfection. (C) U2OS cells were co-transfected with plasmids expressing a GAL4 DNA-binding domain-CR3 (1 μg) fusion and E1A 12S (as indicated) together with a GAL4 responsive luciferase reporter (1 μg). Luciferase activity was assayed 24 h after transfection.

Mentions: E1A 13S can strongly activate transcription from a luciferase reporter gene under the control of the adenovirus E3 regulatory region (Figure 2A). Intriguingly, co-transfection of limiting amounts of E1A 12S repressed activation of luciferase expression by E1A 13S (Figure 2A). Indeed, activation was reduced by ∼50% when one-tenth the amount of 12S expression vector was present. The effect of E1A 12S on 13S transactivation was not due to a reduction of 13S expression levels as determined by Western blot analysis. We also tested an adenoviral E4 promoter construct (Figure 2B). This reporter showed similar response to the E3 reporter, with robust activation by E1A 13S and a pronounced decrease in activation when sub-stoichiometric amounts of E1A 12S were co-expressed with E1A 13S.Figure 2.


Transcriptional control by adenovirus E1A conserved region 3 via p300/CBP.

Pelka P, Ablack JN, Torchia J, Turnell AS, Grand RJ, Mymryk JS - Nucleic Acids Res. (2009)

Repression of E1A 13S- and CR3-mediated transactivation by E1A 12S is dose-dependent and independent of the method by which E1A is targeted to the promoter. (A) HeLa cells were co-transfected with plasmids expressing E1A 13S (1.5 μg) and E1A 12S (as indicated) together with an adenovirus E3-luciferase reporter plasmid (0.5 μg). Luciferase activity was assayed 48 hours after transfection. TF, transcription factor. (B) HeLa cells were co-transfected with plasmids expressing E1A 13S (1.5 μg) and E1A 12S (as indicated) together with an adenovirus E4-luciferase reporter plasmid (0.5 μg). Luciferase activity was assayed 48 h after transfection. (C) U2OS cells were co-transfected with plasmids expressing a GAL4 DNA-binding domain-CR3 (1 μg) fusion and E1A 12S (as indicated) together with a GAL4 responsive luciferase reporter (1 μg). Luciferase activity was assayed 24 h after transfection.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2651774&req=5

Figure 2: Repression of E1A 13S- and CR3-mediated transactivation by E1A 12S is dose-dependent and independent of the method by which E1A is targeted to the promoter. (A) HeLa cells were co-transfected with plasmids expressing E1A 13S (1.5 μg) and E1A 12S (as indicated) together with an adenovirus E3-luciferase reporter plasmid (0.5 μg). Luciferase activity was assayed 48 hours after transfection. TF, transcription factor. (B) HeLa cells were co-transfected with plasmids expressing E1A 13S (1.5 μg) and E1A 12S (as indicated) together with an adenovirus E4-luciferase reporter plasmid (0.5 μg). Luciferase activity was assayed 48 h after transfection. (C) U2OS cells were co-transfected with plasmids expressing a GAL4 DNA-binding domain-CR3 (1 μg) fusion and E1A 12S (as indicated) together with a GAL4 responsive luciferase reporter (1 μg). Luciferase activity was assayed 24 h after transfection.
Mentions: E1A 13S can strongly activate transcription from a luciferase reporter gene under the control of the adenovirus E3 regulatory region (Figure 2A). Intriguingly, co-transfection of limiting amounts of E1A 12S repressed activation of luciferase expression by E1A 13S (Figure 2A). Indeed, activation was reduced by ∼50% when one-tenth the amount of 12S expression vector was present. The effect of E1A 12S on 13S transactivation was not due to a reduction of 13S expression levels as determined by Western blot analysis. We also tested an adenoviral E4 promoter construct (Figure 2B). This reporter showed similar response to the E3 reporter, with robust activation by E1A 13S and a pronounced decrease in activation when sub-stoichiometric amounts of E1A 12S were co-expressed with E1A 13S.Figure 2.

Bottom Line: This is supported by the observation that the repressive effect of E1A 12S is reversed by expression of exogenous p300 or CBP, but not by a CBP mutant lacking actyltransferase activity.Furthermore, we show that transcriptional activation by 13S E1A is greatly reduced by siRNA knockdown of p300 and that CR3 binds p300 independently of the well-characterized N-terminal/CR1-binding site.These results identify a new functionally significant interaction between E1A CR3 and the p300/CBP acetyltransferases, expanding our understanding of the mechanism by which this potent transcriptional activator functions.

View Article: PubMed Central - PubMed

Affiliation: Department of Oncology, The University of Western Ontario, London Regional Cancer Centre, London, Ontario, Canada. peter.pelka@gmail.com

ABSTRACT
The human adenovirus type 5 (HAdV-5) E1A 13S oncoprotein is a potent regulator of gene expression and is used extensively as a model for transcriptional activation. It possesses two independent transcriptional activation domains located in the N-terminus/conserved region (CR) 1 and CR3. The protein acetyltransferase p300 was previously identified by its association with the N-terminus/CR1 portion of E1A and this association is required for oncogenic transformation by E1A. We report here that transcriptional activation by 13S E1A is inhibited by co-expression of sub-stoichiometric amounts of the smaller 12S E1A isoform, which lacks CR3. Transcriptional inhibition by E1A 12S maps to the N-terminus and correlates with the ability to bind p300/CBP, suggesting that E1A 12S is sequestering this limiting factor from 13S E1A. This is supported by the observation that the repressive effect of E1A 12S is reversed by expression of exogenous p300 or CBP, but not by a CBP mutant lacking actyltransferase activity. Furthermore, we show that transcriptional activation by 13S E1A is greatly reduced by siRNA knockdown of p300 and that CR3 binds p300 independently of the well-characterized N-terminal/CR1-binding site. Importantly, CR3 is also required to recruit p300 to the adenovirus E4 promoter during infection. These results identify a new functionally significant interaction between E1A CR3 and the p300/CBP acetyltransferases, expanding our understanding of the mechanism by which this potent transcriptional activator functions.

Show MeSH
Related in: MedlinePlus