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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 CR3 transactivation by E1A 12S maps to the N-terminal/CR1 region. (A) Schematic representation of E1A 12S and the locations of fragments used as GFP fusions in the squelching assay. (B) U2OS cells were co-transfected with 0.1 μg of the indicated GFP-E1A fragment fusions, GAL4-CR3 (1 μg) and a GAL4-luciferase reporter (1 μg). Luciferase activity was assayed 24 h after transfection and the results were plotted versus GAL4-CR3 (shown in grey), which was set to 1.
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Figure 3: Repression of CR3 transactivation by E1A 12S maps to the N-terminal/CR1 region. (A) Schematic representation of E1A 12S and the locations of fragments used as GFP fusions in the squelching assay. (B) U2OS cells were co-transfected with 0.1 μg of the indicated GFP-E1A fragment fusions, GAL4-CR3 (1 μg) and a GAL4-luciferase reporter (1 μg). Luciferase activity was assayed 24 h after transfection and the results were plotted versus GAL4-CR3 (shown in grey), which was set to 1.

Mentions: In order to determine which region of E1A 12S mediates the silencing effect, a set of GFP fusion constructs that collectively encompass most of the E1A 12S protein (Figure 3A) were co-expressed with the GAL4-CR3 fusion protein in the presence of a GAL4-luciferase reporter plasmid (Figure 3B). Neither the CR2 region (residues 93–139), nor the C-terminal region (residues 187–289) of E1A were capable of silencing CR3 transactivation. Indeed, the C-terminal region, which tightly binds CtBP, had the opposite effect, inducing the reporter 4-fold. This result is consistent with our previous observations that the interaction of CtBP with CR3 limits CR3-dependent activation (41). The only region of E1A capable of silencing CR3-mediated transactivation mapped to the N-terminal 82 residues of E1A. Interestingly, this region is the only portion of 12S E1A that functions as a transcriptional activation domain when fused to a heterologous DBD (29). This suggests that the N-terminus of E1A and CR3 may be binding a common factor(s). Sequestration or degradation of this factor(s) by E1A 12S presumably causes the observed reduction in transcription by E1A 13S.Figure 3.


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 CR3 transactivation by E1A 12S maps to the N-terminal/CR1 region. (A) Schematic representation of E1A 12S and the locations of fragments used as GFP fusions in the squelching assay. (B) U2OS cells were co-transfected with 0.1 μg of the indicated GFP-E1A fragment fusions, GAL4-CR3 (1 μg) and a GAL4-luciferase reporter (1 μg). Luciferase activity was assayed 24 h after transfection and the results were plotted versus GAL4-CR3 (shown in grey), which was set to 1.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 3: Repression of CR3 transactivation by E1A 12S maps to the N-terminal/CR1 region. (A) Schematic representation of E1A 12S and the locations of fragments used as GFP fusions in the squelching assay. (B) U2OS cells were co-transfected with 0.1 μg of the indicated GFP-E1A fragment fusions, GAL4-CR3 (1 μg) and a GAL4-luciferase reporter (1 μg). Luciferase activity was assayed 24 h after transfection and the results were plotted versus GAL4-CR3 (shown in grey), which was set to 1.
Mentions: In order to determine which region of E1A 12S mediates the silencing effect, a set of GFP fusion constructs that collectively encompass most of the E1A 12S protein (Figure 3A) were co-expressed with the GAL4-CR3 fusion protein in the presence of a GAL4-luciferase reporter plasmid (Figure 3B). Neither the CR2 region (residues 93–139), nor the C-terminal region (residues 187–289) of E1A were capable of silencing CR3 transactivation. Indeed, the C-terminal region, which tightly binds CtBP, had the opposite effect, inducing the reporter 4-fold. This result is consistent with our previous observations that the interaction of CtBP with CR3 limits CR3-dependent activation (41). The only region of E1A capable of silencing CR3-mediated transactivation mapped to the N-terminal 82 residues of E1A. Interestingly, this region is the only portion of 12S E1A that functions as a transcriptional activation domain when fused to a heterologous DBD (29). This suggests that the N-terminus of E1A and CR3 may be binding a common factor(s). Sequestration or degradation of this factor(s) by E1A 12S presumably causes the observed reduction in transcription by E1A 13S.Figure 3.

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