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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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Knockdown of p300 by siRNA impairs activation by E1A 13S and CR3. (A) HeLa cells were transfected with siRNA for p300 or a negative control siRNA and subsequently with 1 μg of plasmid expressing E1A 13S and 1 μg of E4-luciferase reporter. Luciferase activity was assayed 5 days after the initial siRNA transfection. (B) U2OS cells were transfected with siRNA for p300 or a negative control siRNA and subsequently with 1 μg of plasmid expressing the GAL4-CR3 fusion and 1 μg of GAL4-luciferase reporter. Luciferase activity was assayed 5 days after the initial siRNA transfection.
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Figure 6: Knockdown of p300 by siRNA impairs activation by E1A 13S and CR3. (A) HeLa cells were transfected with siRNA for p300 or a negative control siRNA and subsequently with 1 μg of plasmid expressing E1A 13S and 1 μg of E4-luciferase reporter. Luciferase activity was assayed 5 days after the initial siRNA transfection. (B) U2OS cells were transfected with siRNA for p300 or a negative control siRNA and subsequently with 1 μg of plasmid expressing the GAL4-CR3 fusion and 1 μg of GAL4-luciferase reporter. Luciferase activity was assayed 5 days after the initial siRNA transfection.

Mentions: To definitively determine if p300 played a role in CR3-mediated transactivation, p300 was knocked down in human HeLa cervical cancer cells (for the E4/E1A 13S assay) or human U2OS osteosarcoma cells (for the GAL4-CR3 assay) using siRNA previously shown to be specific to p300 mRNA (35). p300 levels were greatly reduced in cells transfected with siRNA specific for p300, but not in those that were transfected with a negative control siRNA (Figure 6). Knockdown of p300 had a dramatic effect on transactivation by both 13S and the GAL4-CR3 fusion, without affecting the expression levels of either activator (Figure 6). This effect was not due to a general reduction of transactivation or loss in viability in cells in which p300 was knocked down because a reporter not regulated by E1A or CR3 was largely unaffected by the knockdown (data not shown), in agreement with what was previously observed with this siRNA (35). This result conclusively demonstrates that p300 is required for transactivation by CR3.Figure 6.


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)

Knockdown of p300 by siRNA impairs activation by E1A 13S and CR3. (A) HeLa cells were transfected with siRNA for p300 or a negative control siRNA and subsequently with 1 μg of plasmid expressing E1A 13S and 1 μg of E4-luciferase reporter. Luciferase activity was assayed 5 days after the initial siRNA transfection. (B) U2OS cells were transfected with siRNA for p300 or a negative control siRNA and subsequently with 1 μg of plasmid expressing the GAL4-CR3 fusion and 1 μg of GAL4-luciferase reporter. Luciferase activity was assayed 5 days after the initial siRNA transfection.
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Related In: Results  -  Collection

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Figure 6: Knockdown of p300 by siRNA impairs activation by E1A 13S and CR3. (A) HeLa cells were transfected with siRNA for p300 or a negative control siRNA and subsequently with 1 μg of plasmid expressing E1A 13S and 1 μg of E4-luciferase reporter. Luciferase activity was assayed 5 days after the initial siRNA transfection. (B) U2OS cells were transfected with siRNA for p300 or a negative control siRNA and subsequently with 1 μg of plasmid expressing the GAL4-CR3 fusion and 1 μg of GAL4-luciferase reporter. Luciferase activity was assayed 5 days after the initial siRNA transfection.
Mentions: To definitively determine if p300 played a role in CR3-mediated transactivation, p300 was knocked down in human HeLa cervical cancer cells (for the E4/E1A 13S assay) or human U2OS osteosarcoma cells (for the GAL4-CR3 assay) using siRNA previously shown to be specific to p300 mRNA (35). p300 levels were greatly reduced in cells transfected with siRNA specific for p300, but not in those that were transfected with a negative control siRNA (Figure 6). Knockdown of p300 had a dramatic effect on transactivation by both 13S and the GAL4-CR3 fusion, without affecting the expression levels of either activator (Figure 6). This effect was not due to a general reduction of transactivation or loss in viability in cells in which p300 was knocked down because a reporter not regulated by E1A or CR3 was largely unaffected by the knockdown (data not shown), in agreement with what was previously observed with this siRNA (35). This result conclusively demonstrates that p300 is required for transactivation by CR3.Figure 6.

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