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. 2012 Aug 21;31(19):3918–3934. doi: 10.1038/emboj.2012.232

Figure 4.

Figure 4

POH1 influences JMJD2A chromatin occupancy. (A) POH1-depleted cells are partially resistant to JMJD2A overexpression. U20S transfected with Non-T or POH1 siRNA for 24 h, before transfection with WT Flag-JMJD2A or JMJD2A with the mutation D939→R (DR) for a further 48 h. Cells were then exposed to 2 Gy irradiation and fixed 1 h later before incubation with anti-53BP1 and anti-Flag antibodies and imaged by confocal microscopy (left—expression of WT-JMJD2A only is shown). Red is Flag-JMJD2A, green is 53BP1, blue is DNA stained by Hoechst. Flag-JMJD2A expressing cells are illustrated with a white arrow. The graph (right) shows quantification of the presence of 53BP1 foci (>5 foci/cell) in Flag-JMJD2A expressing cells. (Cells were scored for 100 cells/condition, 2 repeats.) (B) POH1 promotes the maintenance of JMJD2A on chromatin. U20S treated with Non-T control or POH1 siRNAs before being left untreated or exposed to 2 Gy irradiation and the 200 mM NaCl-resistant chromatin fraction prepared after 1 h recovery. Chromatin (nuclear insoluble) was resuspended in SDS–PAGE buffer and boiled before immunoblotting with antibodies for JMJD2A (top panel), Histone-3, lysine 9 tri-methylation, H3K9me3 (middle panel) or Histone-3, H3, (third panel) as a loading control. In the bottom panel, the nuclear soluble extract was blotted for JMJD2A. (C) POH1 DUB activity promotes the maintenance of JMJD2A on chromatin. U20S transfected with Flag-POH1 wild type or Flag-POH1-JAMMM for 24 h then treated with Non-T control or POH1 siRNAs for a further 72 h and the insoluble chromatin fraction prepared. Chromatin was resuspended in SDS–PAGE buffer and boiled before immunoblotting with antibodies as for (B). In the bottom panel, the material in the nuclear extract removed on 200 mM NaCl was blotted for JMJD2A. (D) Lysine 63 but not lysine 48 of Ub is required to promote 53BP1 recruitment in cells treated with proteasome inhibitor. Quantification of 53BP1 foci in MG132-treated U20S cells transfected with Myc-LacZ (myc) or myc-tagged forms of Ub; wild-type (WT), lysine 63- to -arginine mutant (K63R), or lysine 48- to -arginine mutant (K48R). All cells were treated with 5 μM MG132 for 2 h before irradiation (5 Gy) and fixed after 1 h recovery then immunostained with antibodies to 53BP1 and to myc. 53BP1 foci were counted in myc-positive cells (n=100 cells/condition).