Figure 7.

E6 downregulates SET7-mediated p53 methylation and stability. (a) Knocking down SET7 relieves the E6-mediated repression of p21 expression. U2OS cells transfected with 16E6 in the absence (lanes 1 and 2) or presence (lanes 3 and 4) of si-SET7 were treated with Adr for 6 h, followed by RT–PCR for p21 mRNA level and western blotting. (b) E6 inhibits p53 methylation at K372 in vivo. 18E6-transfected U2OS cells (lanes 1 and 2) or si-18E6-transfected HeLa cells (lanes 3 and 4) were treated by UV irradiation/MG132 or Adr/MG132, respectively, followed by western blotting. (c) 18E6 depletion from HeLa cells increases Adr-dependent p53 accumulation. The siRNA-transfected U2OS cells (lanes 1–3) and HeLa cells (lanes 4–7) were treated with or without Adr, followed by western blotting. The protein level of p53 was quantified and is shown in the bar chart below the western blots. (d) E6 preferentially degrades K372-unmethylated p53. Equal amounts of p53 from 40 μl of mock-treated cells or 5 μl of Adr-treated cells were subjected to IP using Ab against p53 mono-methylated at K372 (p53K372me1; see Materials and methods for Ab information) (lanes 1 and 2) or Ab against total p53 (FL-393; Santa Cruz), followed by E6-mediated in vitro degradation assay (lanes 3 and 4). The protein level of total p53 of each sample was then analyzed by western blotting using anti-p53 (DO-1) (sc-126; Santa Cruz). (e) SET7 and SAM together partially prevent E6-mediated p53 degradation. (f) E6 does not degrade p53 pre-methylated by SET7. (g) SET7 does not protect the p53K372R mutant from E6-mediated degradation. (e–g) Both p53 and 16E6 were in vitro transcribed and translated. p53 was incubated with or without 16E6, purified SET7 or SAM in the order shown in the scheme, followed by western blotting to show the p53 protein level at the indicated stage.