Fig. 6

Vpr induces chromatin remodelling through histone H2B ubiquitination. a Increased mobility of H2B under Vpr expression. The FRAP assay was performed using Mit-23 cells with H2B-GFP. The collected values for the recovery rate of each GFP signal were subjected to statistical analysis. Error bars indicate ± SEM. b Vpr-induced mobilization of H2B depends on ubiquitination. Mean percentages of H2B-GFP recovery after 180 s of photo-bleaching were compared. **P < 0.05. c H2B was ubiquitinated by Vpr-Wt expression. For this experiment, we newly established cell lines (HT1080vRxt-Vpr), in which expression of Vpr-Wt (lanes 3 and 4), Vpr-Q65R (lanes 5 and 6), Vpr-R77Q (lanes 7 and 8), Vpr-R80A (lanes 9 and 10) and Vpr-Ct4RA (lanes 11 and 12) can be controlled by the tetracycline promoter, and ubiquitination of H2B on K120 was examined after Dox treatment (5 μg/ml, 2 days). As a negative control, a clone expressing Luciferase (Luc) was also included (lanes 1 and 2). Total H2B and Dox-induced expression of Vpr were detected. d Mobility of H2B was not increased by the Q65R mutant of Vpr. Graph shows mean percentages of H2B-GFP recovery after 180 s of photo-bleaching. e Defect of the Q65R mutant for RPA70 loading was rescued by TSA. After treatment with TSA (50 nM, 16 h), RPA70 loading onto the LacO repeats was quantitated by ChIP assay. *P < 0.01