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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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p300 is recruited to the adenovirus E4 promoter only in the presence of E1A 13S. Top panel. A schematic representation of the right end of the adenoviral genome with the locations of primer hybridization indicated, numbering refers to the adenovirus type 2 genome. Bottom panel. HeLa cells were infected with the indicated adenoviruses and chromatin immunoprecipitation was carried out 16 h after infection with M73 anti-E1A antibody, RW128 anti-p300 antibody and mouse anti-rabbit antibody as a negative control. Immunoprecipitated DNA was then subjected to PCR analysis using E4-specific primers and the product was resolved on a 2% agarose gel.
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Figure 8: p300 is recruited to the adenovirus E4 promoter only in the presence of E1A 13S. Top panel. A schematic representation of the right end of the adenoviral genome with the locations of primer hybridization indicated, numbering refers to the adenovirus type 2 genome. Bottom panel. HeLa cells were infected with the indicated adenoviruses and chromatin immunoprecipitation was carried out 16 h after infection with M73 anti-E1A antibody, RW128 anti-p300 antibody and mouse anti-rabbit antibody as a negative control. Immunoprecipitated DNA was then subjected to PCR analysis using E4-specific primers and the product was resolved on a 2% agarose gel.

Mentions: During viral infection, E1A facilitates transcriptional initiation at the viral early promoters (42). To determine whether p300 is recruited to the adenovirus E4 promoter during viral infection, we performed chromatin immunoprecipitations with E1A and p300 antibodies (Figure 8) on infected HeLa cells. In the context of a viral genome, E1A and p300 were found occupying the viral E4 promoter only in the presence of the E1A 13S isoform (pm975 and dl309 viruses), but not when the cells were infected with an E1A 12S only virus (dl520).Figure 8.


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)

p300 is recruited to the adenovirus E4 promoter only in the presence of E1A 13S. Top panel. A schematic representation of the right end of the adenoviral genome with the locations of primer hybridization indicated, numbering refers to the adenovirus type 2 genome. Bottom panel. HeLa cells were infected with the indicated adenoviruses and chromatin immunoprecipitation was carried out 16 h after infection with M73 anti-E1A antibody, RW128 anti-p300 antibody and mouse anti-rabbit antibody as a negative control. Immunoprecipitated DNA was then subjected to PCR analysis using E4-specific primers and the product was resolved on a 2% agarose gel.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 8: p300 is recruited to the adenovirus E4 promoter only in the presence of E1A 13S. Top panel. A schematic representation of the right end of the adenoviral genome with the locations of primer hybridization indicated, numbering refers to the adenovirus type 2 genome. Bottom panel. HeLa cells were infected with the indicated adenoviruses and chromatin immunoprecipitation was carried out 16 h after infection with M73 anti-E1A antibody, RW128 anti-p300 antibody and mouse anti-rabbit antibody as a negative control. Immunoprecipitated DNA was then subjected to PCR analysis using E4-specific primers and the product was resolved on a 2% agarose gel.
Mentions: During viral infection, E1A facilitates transcriptional initiation at the viral early promoters (42). To determine whether p300 is recruited to the adenovirus E4 promoter during viral infection, we performed chromatin immunoprecipitations with E1A and p300 antibodies (Figure 8) on infected HeLa cells. In the context of a viral genome, E1A and p300 were found occupying the viral E4 promoter only in the presence of the E1A 13S isoform (pm975 and dl309 viruses), but not when the cells were infected with an E1A 12S only virus (dl520).Figure 8.

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