Fig. 1: USP7 deubiquitinates UHRF1-mediated ubiquitination of H3 and H2B in S phase of the cell cycle.

a Schematic diagram showing USP7 structural organization and mutants used in this study. b WB analysis showing that ectopically expressed USP7 and UHRF1 oppositely controlled ubiquitination on histones H2B and H3 in HEK293T cells. Note that USP7 catalyzed deubiquitination of H2B and H3 depending on its deubiquitinase activity. All cells were synchronized to S phase by aphidicolin treatment followed by release in fresh medium for 4 h. Core histones were prepared by acid extraction. Also shown are short exposure images of H3 (H3-SE) and H2B (H2B-SE). c WB analysis showing that knockdown of USP7 or UHRF1 by siRNA oppositely affected H3 and H2B ubiquitination in HEK293T cells in S phase. d WB analysis showing that ectopically expressed histones were deubiquitinated by USP7 and ubiquitinated by UHRF1. Note that deubiquitination by USP7 is dependent on its deubiquitinase activity. e UHRF1 ubiquitinated H2B at site(s) other than K120. HA-UHRF1 and FLAG-H2B or H2B-K120R mutant were co-expressed in HEK293T cells. The core histones were prepared by acid extraction and subjected to WB analysis using either anti-FLAG or anti-H2B-K120Ub antibody as indicated. f WB analysis showing that purified USP7 but not USP7m mutant deubiquitnated H2B and H3 in vitro. g In vitro pull-down assay showing DNMT1 preferentially bound both ubiquitinated H3 and H2B. The core histones were prepared from HEK293T cells transfected with FLAG-H3 or FLAG-H2B together with UHRF1 and synchronized to the S phase of cell cycle as above. WB was performed with anti-FLAG antibody.