Figure 6: ISGylation promotes p53 transactivity.
From: Positive feedback regulation of p53 transactivity by DNA damage-induced ISG15 modification
(a) Wild-type p53 (Wt), its 2KR mutant, and an empty vector (Mock) were expressed in H1299 cells with PG13-Luc, p21-Luc and BAX-Luc. After exposure to ultraviolet (UV), they were incubated for various periods, and subjected to assay for the luciferase activity. Transfection efficiency was normalized by using β-galactosidase constructs. The luciferase activity seen without any treatment (that is, ‘0’ time in Mock) was expressed as 1.0 and the others were as its relative values. Error bar, ±s.d. (n=3). (b) shNS, shISG15 or shEFP was expressed in p53+/+ HCT116 cells with and without shRNA-insensitive Flag-ISG15 or Myc-EFP. After exposure to ultraviolet, the cells were incubated for 24 h and subjected to the assay for the luciferase activity as in a. Error bar, ±s.d. (n=3). (c) HisMax-p53 and ISG15-conjugating system (Es/Flag-ISG15) were expressed in p53−/− HCT116 cells with and without Flag-UBP43. The cell lysates were subjected to ChIP assay using anti-p53 antibody or anti-mouse IgG. Bound DNAs were subjected to PCR using the probes for p53REs of CDKN1, MDM2, BAX and ISG15. (d) HisMax-p53 and HisMax-2KR were expressed in p53−/− HCT116 cells with or without ISG15-conjugating system. The cell lysates were subjected to ChIP as in c. (e) shNS or shISG15 were expressed in p53+/+ HCT116 cells with and without shRNA-insensitive Flag-ISG15. After exposure to ultraviolet, the cells were incubated for 24 h, and then subjected to ChIP as in c. Note that shISG15 was directed to a 5′-UTR region. a, b and e, similar results were obtained when the cells were treated with doxorubicin or camptothecin in place of ultraviolet.
