Activation of CREB/ATF sites by polyomavirus large T antigen

Abstract

Polyomavirus large T antigen (LT) has a direct role in viral replication and a profound effect on cell phenotype. It promotes cell cycle progression, immortalizes primary cells, blocks differentiation, and causes apoptosis. While much of large T function is related to its effects on tumor suppressors of the retinoblastoma susceptibility (Rb) gene family, we have previously shown that activation of the cyclin A promoter can occur through a non-Rb-dependent mechanism. Here we show that activation occurs via an ATF/CREB site. Investigation of the mechanism indicates that large T can synergize with CREB family members to activate transcription. Experiments with Gal4-CREB constructs show that synergy is independent of CREB phosphorylation by protein kinase A. Examination of synergy with Gal4-CREB deletion constructs indicates that large T acts on the constitutive activation domain of CREB. Large T can bind to CREB in vivo. Genetic analysis shows that the DNA-binding domain (residues 264 to 420) is sufficient to activate transcription when it is localized to the nucleus. Further analysis of the DNA-binding domain shows that while site-specific DNA binding is not required, non-site-specific DNA binding is important for the activation. Thus, CREB binding and DNA binding are both important for large T activation of CREB/ATF sites. In contrast to previous models where large T transactivation occurred indirectly, these results also suggest that large T can act directly at promoters to activate transcription.

FIG. 1.

Cyclin A promoter regulation by large T. (A) Large T activation of the −37/−33 mE2F cyclin A promoter. 3T3 cells were transfected with 1.5 μg of the indicated reporters and 1.0 μg of either WT or Rb- pCMV LT. Cells were placed in 0.2% CS at 24 h after transfection. The luciferase activity was measured 48 h posttransfection. Values represent the activation (n-fold) (Fold Act.) above control (CON) levels set to 1. Error bars indicate the standard errors of the means. (B) Schematic of the cyclin A reporter construct. The −89 to +11 portion of the cyclin A promoter was fused to the luciferase gene (65). The various transcription factor-binding sites and their positions are noted. (C) Mapping large T activation to the ATF site. Cells were transfected as described for panel A except with the indicated mutant cyclin A reporters and WT pCMV LT only. Cells were kept in 10% CS throughout the transfection and harvested 48 h later.

FIG. 2.

Large T activates CREB/ATF sites. (A) Large T activation of a 4× CRE-containing reporter. Cells maintained under growing conditions (10% CS) were cotransfected with pCMV LT (1.0 μg) and pCRE-Luc (1.5 μg) and assayed for luciferase activity 48 h posttransfection. (B) Large T activation of ATF-containing promoters with different basal and upstream elements. Cells were maintained as described for panel A and transfected with pCMV LT (1.0 μg) and the indicated reporter constructs (3.0 μg). At 48 h posttransfection, a chloramphenicol acetyltransferase assay was performed and the percentage of acetylated [¹⁴C]chloramphenicol was measured with thin-layer chromatography and a PhosphorImager. Values represent activation (n-fold) (Fold Act.) above control (CON) levels.

FIG. 3.

Large T effects on CREB. (A) Large T synergizes with CREB to stimulate transcription. Cells were cotransfected with 1.0 μg of Gal4TK-Luc reporter and 1.0 μg of Gal4 DBD (DBD), Gal4-CREB (CREB), or Gal4-CREB S119A (CREB S119A). pSV-PKA (0.5 μg) and pCMV LT (1.0 μg) were also transfected as indicated. Cells were harvested 48 h posttransfection and assayed for luciferase activity. Activation (n-fold) (Fold Act.) above control levels is represented. (B) Large T and CREB interact in vivo. 100-mm-diameter dishes of 3T3 cells were transfected with 3 μg of pCMV LT and harvested 48 h later. Extracts were immunoprecipitated with anti-CREB antibody or normal rabbit serum (NRS) and immunoblotted for LT with PN116 antibody. “Input” represents 2.5% the amount of extract used for immunoprecipitation (IP). (C) Mapping large T/CREB synergy to specific CREB sequences. Cells were cotransfected with 1.0 μg of pCMV LT, 1.5 μg of Gal4TK-Luc reporter, and 1.0 μg of the depicted CRG constructs (57). The luciferase assay was done and quantitated as described above.

FIG. 4.

Activation of cyclin A by large T domains. (A) Schematic of the large T molecule. The N-terminal and C-terminal domains are depicted as NT and CT, respectively. Numbers represent amino acid positions in the molecule. The subdomains within NT and CT are shown. (B) Effect of extending NT to include the DBD. Cells were cotransfected with 1.5 μg of −37/−33 mE2F cyclin A reporter and 1.0 μg of pCMV LT, NT, or 1-420. Luciferase activity was measured 48 h after transfection. Values represent activation (n-fold) (Fold Act.) above control (CON) levels. Extracts were blotted with PN116 antibody to detect LT, NT, and 1-420 protein levels. (C) The DBD alone activated the cyclin A promoter. Cells were cotransfected with 1.5 μg of −37/−33 mE2F cyclin A reporter and 1.0 μg of pOZ HA-LT, 750 ng (low dose) or 3.0 μg (high dose) of pCMV HA-CT, 3.0 μg of pCMV HA-CT 264-420, or 3.0 μg of pCMV NLS HA-CT 264-420. Luciferase assays were done as described above. Extracts were blotted with HA11 antibody to detect LT, CT, and 264-420. (D) Synergy of large T domains with CREB. Cells were cotransfected with 1.0 μg of Gal4TK-Luc reporter, 1.0 μg of Gal4-DBD or Gal4-CREB (CREB), and 333 ng of pCMV LT, NT, or 1-420 or 3 μg of pCMV HA-CT or pCMV NLS HA-CT 264-420. Luciferase activity was measured 48 h posttransfection.

FIG. 5.

Analysis of large T mutants defective in DNA binding. (A) The LT mutant, S306P, fails to bind to the polyomavirus origin. Cells transfected with 3.0 μg of pCMV LT or pCMV S306P were incubated with ³²P end-labeled restriction fragments of an EcoRI-DdeI double digest of pUCori, a plasmid containing the polyomavirus origin. The mixtures were then immunoprecipitated with anti-T serum, and the DNA associated with the large T immune complexes was isolated. Binding to the origin is marked by the presence of the 454-bp EcoRI fragment, denoted by an arrow. Expression of large T and S306P in the cell extracts is shown as a blot probed with PN116 antibody. (B) The S306P/V358A double mutant has a lowered affinity for DNA. Total DNA from E. coli cells expressing either WT or S306P/V358A GST fusion proteins was purified and run out on an agarose gel and subjected to ethidium bromide staining. Expression of the GST fusion proteins is shown by Coomassie staining. (C) Non-sequence-specific DNA binding is important for large T transactivation of the cyclin A promoter. Cells were assayed as described for Fig. 4B. Values shown represent activation (n-fold) (Fold Act.) above control (CON) levels. Extracts were blotted with PN116 to detect large T protein.

FIG. 6.

The role(s) of the DBD. (A) Large T, when tethered to a promoter, acts as a coactivator. Cells were cotransfected with 1.5 μg of TK-Gal4-Luc reporter and 1.0 μg of Gal4-NT, Gal4-CT, or Gal4-CT S306P/V358A. Luciferase activity was measured 48 h posttransfection, and the values were quantitated as activation (n-fold) (Fold Act.) above control (CON) levels. (B) The role of DBD in E2F site activation is different from CREB/ATF site activation. Cells were cotransfected with either 1.5 μg of −37/−33 mE2F cyclin A reporter or 1.5 μg of E2F reporter and 1.0 μg of pCMV LT, pCMV NT, or pCMV NT/DnaB. Luciferase activity was measured as described for panel A. Values shown represent activation (n-fold) (Fold Act.) above control levels.

FIG. 7.

The DBD contributes to CREB binding. (A) Cells were transfected with 3 μg of pCMV NT, LT, 1-420, or S306P/V358A and immunoprecipitated (IP) with anti-CREB antibody. Samples were analyzed with PN116 as described for Fig. 3B to detect large T species. (B) Cells were transfected with 3 μg of pCMV HA-CT or pCMV HA-CT 264-420 and assayed as described above except that HA-11 antibody was used to detect the T antigens.