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Figure 1 E6.16 interacts with p300/CBP in vivo. (A) Cos-1 cells were transfected with an expression vector alone or one that contains E6.16 with a flag tag (Flag-E6). Whole-cell extracts were prepared 48 h after transfection and they were immunoprecipitated with either IgG, RW144 (recognizes both CBP and p300) or anti-flag antibodies. The immunoprecipitated proteins were separated on a 4–20% gradient gel and blotted with either a combination of RW128 and MN11 (upper panels) or anti-flag (lower panels). (B) Bacterially derived 6-His-tagged E6 (6His–E6) was incubated with the indicated p300/CBP–GST fusion protein. The bound complexes were removed from solution with glutathione beads, separated on a 15% SDS–polyacrylamide gel and blotted with an anti-histidine antibody. An anti-GST Western blot of the GST–p300 fusion proteins used in the upper panel is shown below. (C) Whole-cell extracts from U2OS were incubated with either E6–GST fusion protein or GST alone. The bound complexes were removed with glutathione beads, separated on a 6% SDS–polyacrylamide gel and blotted with a mixture of RW128 and MN11 monoclonal antibodies against p300/CBP.
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 | Figure 2 Interaction of E6.16 and E6.6 with different domains of CBP. (A) A diagram of the domains of CBP and p300. The CH1, KIX and CH3 domains are indicated by the hatched boxes. The HAT domain indicates the region that contains the histone acetylase activity. The percentage homology between the two proteins is indicated (Giles et al., 1998). The reported interaction regions of the various transcription factors and transcription accessory proteins with p300/CBP are listed below. (B) Radiolabeled in vitro-translated E6.16 or E6.6 proteins were added to the GST fusion proteins of the regions of CBP indicated: GST–KIX (amino acids 461–662), GST–C/H3 (amino acids 1621–1877) and GST–CT (C-terminal domain, amino acids 1990–2441) (left and center panels). The N-terminal region of p300, amino acids 1–595, was also used (right panel). The bound complexes were removed from solution with glutathione beads, separated on a 15% SDS–polyacrylamide gel and were quantified using a PhosphorImager and Imagequant software (Molecular Dynamics). The radiolabeled imput of each TNT protein is indicated. Note that E6.6 migrates slightly more quickly than E6.16.
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Figure 3 Mapping the interaction of E6.16 and E6.6 with the N-terminus of p300, which contains the CH1 domain. (A) Radiolabeled in vitro translated E6.16 and E6.6 were added to the indicated GST–p300 fusion proteins. The bound complexes were separated on a 15% SDS–polyacrylamide gel and were analyzed with phosphoimagery. Summary of the binding of E6.16 and p53 to the p300 C/H1 domain. +/- indicates that binding is above background but greatly reduced over wild-type constructs containing the C/H1 domain. *The results for the binding of p53 to C/H1 domain are taken from Grossman et al. (1998). (B) A Coomassie Blue stained gel of the GST–p300 fusion proteins used in (A).
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 | Figure 4 Interaction of different p300 domains with E6.16 mutants. Radiolabeled in vitro translated wild-type E6.16, E6.6 and the indicated E6.16 mutants proteins were incubated with either (A) GST–C/H3, (B) GST–C/H1 or (C) GST–CT3 and –CT4 fusion proteins. The bound complexes were removed with glutathione beads, separated on a 15% SDS–polyacrylamide gel and quantified using a PhosphorImager and Imagequant software (Molecular Dynamics). See Table I. The input is 10% of that added to the test reactions. The Coomassie Blue-stained gel of the GST–CT3 and –CT4 fusion proteins used in (C) is shown below the gel.
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Figure 5 E6.16 inhibits c-Fos activation in a mammalian two-hybrid system. (A) A schematic of the mammalian two-hybrid system used. (B) U2OS cells were co-transfected with a reporter (Gal4–Luc) that contains five Gal4 binding sites upstream of the luciferase gene and a construct that has the activation domain of c-Fos fused to the Gal4DBD (Gal4–c-Fos). The co-activation construct contains the C/H3 domain of p300 fused to the VP16 activation domain (C/H3–VP16). The results shown represent one experiment from four carried out. The fold activation is luciferase activity of any test transfection assay over reporter alone. (C) The same co-transfections were performed as in (B) but increasing amounts of the C/H3–VP16 construct were added to relieve the inhibitory effect by E6.16.
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 | Figure 6 Full-length p300 has intrinsic transcriptional activation properties which are inhibited by E6.16. U2OS cells were co-transfected with a reporter (Gal4–Luc) that contains five Gal4 binding sites upstream of the luciferase gene and a construct (Gal4–p300) that contains full-length p300 fused in-frame to the Gal4DBD. Wild-type E6.16 and the two E6.16 mutants, E6.1645Y47Y49H and E6.16 128–132, are in the pcDNA vector. The results shown represent one experiment from three carried out. The fold activation is luciferase activity of any test transfection assay over reporter alone.
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Figure 7 Co-activation activity of different p300 domains is inhibited by E6.16, E6.6 and E6.16 mutants. (A) U2OS cells were co-transfected with a reporter (Gal4–Luc) that contains five gal4 binding sites upstream of the luciferase gene and an activation construct (Gal4–p300.1–743) that contains amino acids 1–743 (contains the C/H1 and KIX domains) of p300 fused in-frame to the Gal4DBD. The wild-type E6.16, E6.6 and the E6.16 mutants are in the pcDNA vector. The results shown represent one experiment from three carried out. The fold activation is luciferase activity of any test transfection assay over reporter alone. (B) The same co-transfection assays were performed as in (A) except that the activation construct (Gal4–p300245–1337) used contains the C/H3 and the C-terminal regions of p300 fused in-frame to the Gal4DBD.
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 | Figure 8 Both p53- and NF- B-responsive promoter elements are inhibited by E6.16. (A) Saos-2 (does not contain functional p53 proteins) were co-transfected with a reporter construct (pG13) that contains 13 p53 DNA binding sites upstream of the luciferase gene or 13 mutated p53 DNA binding sites (pMG13) and a p53 expression construct (pCMV-p53). The co-activation construct contains full-length p300 (pCMV-p300). The wild-type E6 and the E6 mutants' expression constructs are indicated. The results shown represent one experiment from three carried out. The fold activation is luciferase activity of any test transfection assay over reporter alone. (B) U2OS cells were co-transfected with a reporter construct (NF-B–Luc) that contains three NF-B sites upstream of the luciferase gene. The co-activation construct contains full-length p300 (pCMV–p300) and different amounts of this expression construct were added as indicated. The results shown represent one experiment. The luciferase activity of the reporter alone is set to one and the results are expressed relative to this value. (C) Wild-type E6.16, but not the mutants E6.1645Y47Y49H and E6.16123–127, cause the degradation of p53. Radiolabeled rabbit reticulocyte in vitro-translated E6.16, E6.1645Y47Y49H and E6.16123–127 were mixed with radiolabeled rabbit reticulocyte in vitro-translated p53. The mixtures were incubated at 25°C for 2 h. The proteins were separated on a 12% SDS–polyacrylamide gel and then quantified using a PhosphorImager and Imagequant software (Molecular Dynamics).
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