Figure 5.
The Histone Chaperone ANP32E Promotes H2A.Z Removal from UV Damage Sites
(A and B) Distribution of the indicated H2A variants (total levels, TMR pulse or H2A.Z Nt/Ct antibody) analyzed 1 hr after local UVC irradiation in U2OS cells expressing SNAP-tagged histones (A) and treated with the indicated siRNAs (B) (siLUC, control; siFACT, siSPT16 + siSSRP1). Arrowheads indicate UV irradiation sites. Fluorescence intensities are measured along the dotted lines. Scatterplots show mean ± SD from at least 40 cells (1 representative experiment out of 3). The significance of H2A.Z loss or enrichment at UV sites is indicated.
(C) Enrichment of new H2A.X and loss of total H2A.Z at UV sites (marked by CPD) analyzed at the indicated time points post 500 J/m2 local UVC irradiation in U2OS H2A.X-SNAP cells. Scatterplots show mean ± SD from at least 39 UV spots (1 representative experiment out of 2). Significance is given compared to a theoretical mean of 1.
(D and E) Accumulation of FACT subunit SSRP1 (D) and new H2A.X (E) at repair sites analyzed as in Figures 4B–4D. Percentages of cells recruiting FACT to repair sites are shown (from at least 250 cells scored in 2 independent experiments).
Scatterplots show mean ± SD from at least 70 cells (1 representative experiment out of 2). Knockdown efficiencies were verified by western blot. Scale bars, 10 μm. ns, non-significant; ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. See also Figures S5 and S6.