Fig 2: HIRA homooligomerizes in cells and forms a homotrimer in vitro. a YFP constructs of human HIRA and mutants. The WD40 repeat (aa 1–369), B (aa 439–475) and C (aa 763–963) domains involved in UBN, ASF1a, and CABIN1 interactions, respectively, are shown. Star indicates single amino acid substitution with alanine. The co-immunoprecipitation efficiency of endogenous HIRA or HIRA-HA (HIRA coIP) is indicated for each construct, “+” indicates that the efficiency of coIP is similar to the one obtained with the wt HIRA construct and “−” indicates that the efficiency of the coIP is decreased. b Western blot analysis of anti-GFP-immunoprecipitates from U2OS nuclear extracts expressing YFP-tagged proteins. c Western blot analysis of anti-GFP-immunoprecipitates from U2OS nuclear extracts expressing both YFP-tagged and HIRA-HA proteins. d Western blot analysis of anti-GFP-immunoprecipitates from U2OS nuclear extracts expressing YFP-tagged proteins. In b, c and d, input corresponds to 10% of nuclear extract used for each experiment. e Equilibrium sedimentation of recombinant proteins HIRA(661–1017), CABIN1 full length (FL) and HIRA(661–1017) + CABIN1 FL. Theoretical (open symbols) and experimental (closed symbol) curves are shown

Fig 3: H3K56 acetylation is required for the dephosphorylation of γ-H2AX. (A) Depletion of ASF1A results in the retention of γ-H2AX for a longer time at sites of UV damage. OSU-2 cells were transfected with ASF1A shRNA and after 24 h irradiated with 10 J m−2 UV through micro-pore filters placed on the cell monolayers. Cells were recovered at indicated times and fixed with paraformaldehyde. Immunofluorescence was performed using anti-γ-H2AX antibody. Images shown are from a representative of multiple experiments. (B) Quantitation of γ-H2AX foci in ASF1A-depleted cells. Triplicate experiments were performed as described above in (A) and the number of cells containing γ-H2AX foci in shRNA-treated and -untreated cells were quantitated as follows. An average of 150 merged nuclear foci from at least five different microscopic fields were used for the quantitation of γ-H2AX foci for each experiment. The graph depicts the average number of cells containing γ-H2AX foci from the three independent experiments. The error bars show the mean standard deviation. (C) H3K56R mutation results in the retention of γ-H2AX foci at sites of UV damage. H1299-H3.1 and H1299-H3K56R cells were exposed to 10 J m−2 of UV radiation through micro-pore filters. Cells were fixed at the indicated intervals and immunofluorescence was performed to detect γ-H2AX foci. Images shown are from a representative experiment. (D) Quantitation of γ-H2AX foci in H1299 cells. Quantitation of γ-H2AX foci in H1299-H3.1 and H1299-H3K56R cells was essentially performed as described in (B) from two independent experiments. (E) H3K56 acetylation-deficient cells are defective in γ-H2AX dephosphorylation. H1299-H3.1 and H1299-H3K56R cells were irradiated at 10 J m−2 UV and whole cell lysates prepared at 4, 8, 24 and 48 h. Equal quantities of cell lysates were resolved on SDS–PAGE and the levels of γ-H2AX determined by western blotting. Total H2AX and β-ACTIN were used as loading controls.

Fig 4: ASF1a is overexpressed in a variety of primary human tumors.a Relative ASF1a mRNA levels were determined using qPCR in hepatocellular carcinoma (HCC) (T: tumor, n = 51) samples and adjacent non-tumorous liver tissues (N: non-tumorous, n = 29) (data are presented as the mean ± SD; P < 0.0001). b ASF1a mRNA expression (transcripts per million in log scale) in liver hepatocellular carcinoma (LIHC) (T, n = 369; N, n = 160), prostate adenocarcinoma (PRAD) (T, n = 408; N, n = 152), stomach adenocarcinoma (STAD) (T, n = 408; N, n = 221), and breast invasive carcinoma (BRCA) (T, n = 1085; N, n = 291) (data are presented as the mean ± SD; P < 0.05). qPCR, quantitative real-time PCR

Fig 5: Restoration of H3K56 acetylation is necessary for recovery from UV-induced cell-cycle checkpoint arrest. (A) ASF1A-deficient cells exhibit cell-cycle checkpoint recovery defect. OSU-2 cells were arrested in G1 phase of the cell-cycle by serum starvation for 48 h. The arrested cells were transfected with ASF1A shRNA and after 24 h irradiated with 5 J m−2 UV and released into the cell-cycle by addition of media containing serum. Transfection with ASF1A shRNA was performed again to maintain the knockdown up to 50 h. Cells were harvested at the indicated times, fixed with 70% ethanol and the DNA content determined by FACS. (B) H3K56R mutant cells are defective in recovery from UV-induced cell-cycle checkpoint arrest. H1299-H3.1 and H1299-H3K56R cells were synchronized by serum starvation for 24 h. Cells were then irradiated with 10 J m−2 UV and released into the cell-cycle by addition of media containing serum. Cells were harvested at the indicated time points, fixed and DNA content determined by FACS.