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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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Overexpression of p300 or CBP restores E1A 12S-repressed CR3 transactivation. (A) U2OS or HeLa cell extracts from cells transfected with empty vector, E1A 12S expression plasmid or E1A 13S expression plasmid were assayed for levels of endogenous p300 and E1A. (B) U2OS cells were co-transfected with plasmids expressing GAL4-CR3 (600 ng) and 600 ng of vectors expressing the indicated acetyltransferases, together with a GAL4-luciferase reporter plasmid (600 ng) and 60 ng of E1A 12S as indicated. Luciferase activity was assayed 24 h after transfection and the data was plotted versus CR3 alone (shown in grey), which was set to 1. Overexpressed p300, CBP, CBP AT- and pCAF were detected using anti-HA antibody for p300 and anti-FLAG M2 for CBP, CBP AT- and pCAF.
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Figure 5: Overexpression of p300 or CBP restores E1A 12S-repressed CR3 transactivation. (A) U2OS or HeLa cell extracts from cells transfected with empty vector, E1A 12S expression plasmid or E1A 13S expression plasmid were assayed for levels of endogenous p300 and E1A. (B) U2OS cells were co-transfected with plasmids expressing GAL4-CR3 (600 ng) and 600 ng of vectors expressing the indicated acetyltransferases, together with a GAL4-luciferase reporter plasmid (600 ng) and 60 ng of E1A 12S as indicated. Luciferase activity was assayed 24 h after transfection and the data was plotted versus CR3 alone (shown in grey), which was set to 1. Overexpressed p300, CBP, CBP AT- and pCAF were detected using anti-HA antibody for p300 and anti-FLAG M2 for CBP, CBP AT- and pCAF.

Mentions: The extensive mutational analysis presented above indicates that reduction of transcriptional activity by E1A 12S relies on binding to p300; although, other factors cannot be fully excluded from playing a role. We initially determined if E1A 12S had an effect on the steady state level of p300 by Western blot analysis in both U2OS and HeLa cells (Figure 5A). E1A 12S expressing cells showed similar levels of endogenous p300 expression as compared to vector transfected cells or cells expressing E1A 13S. This indicates that the mechanism by which E1A 12S blocks transcriptional activation by E1A 13S is not mediated by degradation of p300.Figure 5.


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)

Overexpression of p300 or CBP restores E1A 12S-repressed CR3 transactivation. (A) U2OS or HeLa cell extracts from cells transfected with empty vector, E1A 12S expression plasmid or E1A 13S expression plasmid were assayed for levels of endogenous p300 and E1A. (B) U2OS cells were co-transfected with plasmids expressing GAL4-CR3 (600 ng) and 600 ng of vectors expressing the indicated acetyltransferases, together with a GAL4-luciferase reporter plasmid (600 ng) and 60 ng of E1A 12S as indicated. Luciferase activity was assayed 24 h after transfection and the data was plotted versus CR3 alone (shown in grey), which was set to 1. Overexpressed p300, CBP, CBP AT- and pCAF were detected using anti-HA antibody for p300 and anti-FLAG M2 for CBP, CBP AT- and pCAF.
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Figure 5: Overexpression of p300 or CBP restores E1A 12S-repressed CR3 transactivation. (A) U2OS or HeLa cell extracts from cells transfected with empty vector, E1A 12S expression plasmid or E1A 13S expression plasmid were assayed for levels of endogenous p300 and E1A. (B) U2OS cells were co-transfected with plasmids expressing GAL4-CR3 (600 ng) and 600 ng of vectors expressing the indicated acetyltransferases, together with a GAL4-luciferase reporter plasmid (600 ng) and 60 ng of E1A 12S as indicated. Luciferase activity was assayed 24 h after transfection and the data was plotted versus CR3 alone (shown in grey), which was set to 1. Overexpressed p300, CBP, CBP AT- and pCAF were detected using anti-HA antibody for p300 and anti-FLAG M2 for CBP, CBP AT- and pCAF.
Mentions: The extensive mutational analysis presented above indicates that reduction of transcriptional activity by E1A 12S relies on binding to p300; although, other factors cannot be fully excluded from playing a role. We initially determined if E1A 12S had an effect on the steady state level of p300 by Western blot analysis in both U2OS and HeLa cells (Figure 5A). E1A 12S expressing cells showed similar levels of endogenous p300 expression as compared to vector transfected cells or cells expressing E1A 13S. This indicates that the mechanism by which E1A 12S blocks transcriptional activation by E1A 13S is not mediated by degradation of p300.Figure 5.

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