ASF1a Promotes Non-homologous End Joining Repair by Facilitating Phosphorylation of MDC1 by ATM at Double-Strand Breaks  Kyung Yong Lee, Jun-Sub Im, Etsuko Shibata, Anindya Dutta  Molecular Cell  Volume 68, Issue 1, Pages 61-75.e5 (October 2017) DOI: 10.1016/j.molcel.2017.08.021 Copyright © 2017 Elsevier Inc. Terms and Conditions

Molecular Cell 2017 68, 61-75.e5DOI: (10.1016/j.molcel.2017.08.021) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 1 ASF1a Is Required for NHEJ and Resistance to DSBs (A) Immunoblots of the NHEJ/DsRed293B lysates transfected with two different ASF1a-targeting siRNAs 48 hr after transfection of HA-I-SceI plasmids. HA-I-SceI was detected by an anti-HA antibody. (B) ASF1a knockdown reduces NHEJ efficiency. NHEJ efficiency is measured as described in the STAR Methods and represented as mean ± SD of triplicates. ∗∗∗p < 0.005, ∗p < 0.05. (C) ASF1a overexpression increases NHEJ efficiency. 293B cells having stable overexpression (o/e) of ASF1a were compared with WT 293B cells for ASF1a expression level in the immunoblot (top) and for NHEJ efficiency (bottom). Data are mean ± SD from triplicate measurements. (D) Rescue of NHEJ in siASF1a-transfected 293B cells by expression of siRNA-resistant ASF1a. Empty vector (+EV) or an ASF1a-expressing vector resistant to siASF1a (+ASF1a) was co-transfected with HA-I-SceI. Shown are immunoblots (top) and quantitation of NHEJ efficiency (bottom). Data are mean ± SD of triplicates. (E) Depletion of ASF1a renders cells sensitive to ionizing radiation (IR). Cell viability was quantified and presented as mean ± SD from triplicate measurements (bottom). Representative images (top) are shown. (F) Dose-dependent sensitivity to bleomycin of ASF1a-depleted cells. The indicated dose of bleomycin was administered for 24 hr, 48 hr after the first siRNA transfection. Data are mean ± SD from triplicates. Molecular Cell 2017 68, 61-75.e5DOI: (10.1016/j.molcel.2017.08.021) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 2 Knockout of ASF1A Reduces NHEJ and Promotes HR (A) A schematic of the targeting strategy for ASF1A knockout in 293B or HeLa DR13-9 cells using the CRSPR/Cas9 system. The sgRNAs targeting the ASF1A gene (top) and the region interrogated to identify the deletion (bottom) are shown. (B) A representative image of the PCR product from the 293B clones: WT and BA123 (with a homozygous deletion of the ASF1A gene). (C) A western blot showing the ASF1a protein level in 293B WT and ASF1A-null clones. (D) Knocking out ASF1A suppresses NHEJ efficiency. The percentage of DsRed-positive cells in each cell line was normalized to that of WT cells transfected with HA-I-SceI. Data are mean ± SD from triplicates. (E and F) ASF1a expression in ASF1A knockout cells rescues NHEJ efficiency. ASF1a was stably expressed in ASF1A knockout cell lines using retroviral infection. Shown are immunoblots of those lysates (E) and the NHEJ assay (F). Data are mean ± SD from triplicates. (G) Decrease of γH2AX after DSB is slowed in ASF1A knockout cells. The ratio of γH2AX to RPA70 signal was quantitated at each time point and normalized to the ratio at the 0-hr point. See also Figure S1A. (H) Representative image of PCR on genomic DNA from HeLa DR13-9 clones: WT and 35D9. (I) A western blot showing the ASF1a protein level in WT and ASF1A-null clones of HeLa DR13-9 cells. (J) Knocking out ASF1A promotes HR repair. HR was measured as described in the STAR Methods. Data are mean ± SD from triplicates. See also Figures S1B–S1D. Molecular Cell 2017 68, 61-75.e5DOI: (10.1016/j.molcel.2017.08.021) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 3 ASF1a Is Required for the Recruitment of 53BP1 and RAP80 at DSBs (A–C) Decrease of 53BP1 foci upon depletion of ASF1a in U2OS cells or in ASF1A−/− cells after treatment with bleomycin for 1 hr before fixation. Representative images (A) and quantitation (B and C) are shown. Cells with more than 20 foci of 53BP1 were counted. Data are mean ± SD of triplicates. ∗∗∗p < 0.005. Scale bar, 10 μm. See also Figure S2. (D) Rescue of 53BP1 foci in ASF1a-depleted U2OS cells by siRNA-resistant ASF1a. Representative images (top) are shown, and data are mean ± SD of triplicates. ∗∗∗p < 0.005, ∗p < 0.05 (bottom). (E and F) No change in BRCA1 foci upon depletion of ASF1a. Representative images (E) and quantitation (F) are shown. (G) RAP80 foci are decreased in ASF1a-depleted U2OS cells. (H) CtIP foci are increased in ASF1a-depleted U2OS cells. The quantitations in (F) and (G) are the same as in (B), and cells having more than five foci of CtIP were counted in (H). Data in (F)–(H) are mean ± SD of triplicates. Molecular Cell 2017 68, 61-75.e5DOI: (10.1016/j.molcel.2017.08.021) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 4 ASF1a Promotes the Localization of ATM and Phosphorylation of MDC1 at DSBs by Facilitating the ATM-MDC1 Interaction (A) Immunoblots with the indicated antibodies show no decrease in DNA damage-induced autophosphorylation of ATM or in ATM phosphorylation of NBS1, Chk2, and H2AX after depletion of ASF1a. (B and C) Decreased pS1981-ATM foci after depletion of ASF1a. Shown are representative images of immunostaining (B) and quantification of focus-positive cells (C). Scale bar, 10 μm. Data are mean ± SD of triplicates. ∗∗∗p < 0.005. (D) siRNA-resistant ASF1a rescues pS1981-ATM focus formation. Shown are representative images (top) and quantification of cells with more than 10 pS1981-ATM foci (bottom). Data are mean ± SD of triplicates. (E) A rapid disappearance of pS1981-ATM focus formation upon ASF1A knockout. Cells having more than five pS1981-ATM foci in WT or ASF1A−/− HeLa DR13-9 were counted as positive cells at the indicated time points after pulse treatment with 40 μg mL−1 bleomycin for 20 min. Data are mean ± SD of triplicates. ∗p < 0.05, ∗∗∗p < 0.005. (F) Defective interaction of MDC1 with phospho-ATM after ASF1a knockdown. (G and H) ASF1a is required for ATM phosphorylation on MDC1. HEK293T cells transfected with the indicated siRNAs (G) and 293B WT and ASF1A−/− cells (H) were transfected with the HA-MDC1 plasmid. DMSO, bleomycin, or cisplatin was added for 14 hr before harvest. Molecular Cell 2017 68, 61-75.e5DOI: (10.1016/j.molcel.2017.08.021) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 5 ASF1a Interacts with MDC1, and the FHA Domain of MDC1 Is Required for ASF1a Localization to DSBs (A) HA-ASF1a precipitates contain endogenous MDC1. Shown are immunoblots of immunoprecipitates or input lysate. (B) Histone binding by ASF1a is not required for the interaction with MDC1. The WT or Val94Arg mutant of HA-ASF1a was immunoprecipitated with anti-HA antibody. (C) MDC1 is required for ASF1a localization at the I-SceI cut site. The ChIP assay was performed as described in the STAR Methods. Data are mean ± SD of triplicates. (D) MDC1-dependent association of ASF1a with chromatin after DNA damage. U2OS cells treated with bleomycin for 1 hr, followed by pre-extraction to remove soluble proteins, were fixed and immunostained. Scale bar, 10 μm. (E) Co-localization of MDC1 and ASF1a to the I-SceI cut site. The eluate from the anti-MDC1 ChIP was applied to an anti-ASF1a ChIP. Data are mean ± SD of triplicates. (F) Co-localization of ASF1a and MDC1 foci at DSBs. (G) Untagged ASF1a co-immunoprecipitates MDC1 but not phospho-ATM. (H) ASF1a-MDC1 interaction needs the FHA domain of MDC1. The HA-MDC1 WT or FHA deletion mutant (Δ55-124 amino acids [aa]) was overexpressed in HEK293T cells, followed by bleomycin treatment for 14 hr. (I and J) MDC1 interaction with ASF1a decreases as its interaction with ATM increases after DNA damage. Shown are immunoblots with the indicated antibodies (I) and quantification of co-precipitated ATM or ASF1a with HA-MDC1 after pulse treatment with bleomycin (J). Molecular Cell 2017 68, 61-75.e5DOI: (10.1016/j.molcel.2017.08.021) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 6 ASF1a Is Required for RNF8, but not MDC1, Recruitment at DSBs (A–C) Decrease in bleomycin-induced RNF8 foci, but not MDC1 foci, in ASF1A knockout cells. HeLa DR13-9 WT or ASF1A−/− cells were incubated with 20 μg mL−1 bleomycin for 40 min, followed by fixation. Shown are representative images of immunostaining with the indicated antibodies (A and B) and quantification of cells with more than five foci of RNF8 or MDC1 (C). See co-localization of RNF8 and MDC1 foci in Figure S3. Scale bar, 10 μm. Data are mean ± SD of triplicates. ∗∗∗p < 0.005. (D) Decreased interaction of MDC1 with RNF8 after DNA damage in ASF1A knockout cells. A plasmid expressing FLAG-RNF8 was transfected in 293B WT and ASF1A−/− cells, followed by bleomycin treatment before harvest. The asterisk indicates immunoglobulin heavy chain. (E–I) ASF1a is required for RNF8/168 localization, not MDC1, at the I-SceI cut site. HeLa DR13-9 cells transfected with the indicated siRNAs (E–G) and WT and ASF1A−/− cells (H and I) were applied to a ChIP assay using anti-RNF8 (E and H), anti-MDC1 (F and I), and anti-RNF168 (G) antibodies. Data are mean ± SD of triplicates. ∗∗∗p < 0.005, ∗p < 0.05. Molecular Cell 2017 68, 61-75.e5DOI: (10.1016/j.molcel.2017.08.021) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 7 DSB-Dependent Ubiquitination on Histones and FK2 Focus Formation Require ASF1a (A) DSB-induced H2A or H2AX mono-ubiquitination is dependent on ASF1a. HEK293T cells were transfected with the indicated siRNAs and treated with bleomycin. (B) Expression of siRNA-resistant ASF1a restores γH2AX mono-ubiquitination in cells depleted of endogenous ASF1a. The indicated siRNAs were transfected into either WT U2OS or U2OS cells with stable overexpression of siRNA-resistant ASF1a. (C) Decrease in RNF8/168-dependent ubiquitination of H1.2-, H2A- and γH2AX-, but not PRC1-dependent, ubiquitination of H2A K119 in ASF1A knockout cells. HeLa DR13-9 WT or ASF1A−/− cells were incubated with 20 μg mL−1 bleomycin for 80 min. (D–F) Immunostaining with anti-FK2 antibody in ASF1a-depleted cells. U2OS cells were transfected with the indicated siRNA and immunostained following treatment with 5 μg mL−1 bleomycin for 1 hr (D and E) or 90 min post-IR (2 Gy) (F). Shown are representative images (D and F) and quantification of (D) in (E). Data are mean ± SD of triplicates. ∗∗∗p < 0.005. (G) Rescue of mono-ubiquitination of H2AX by WT or Val94Arg mutant ASF1a in ASF1a-depleted U2OS cells. (H) Rescue of FK2 foci by WT or Val94Arg mutant ASF1a in ASF1a-depleted U2OS cells. The indicated siRNAs were transfected into either WT U2OS or U2OS cells with stable overexpression of siRNA-resistant ASF1a WT or V94R mutant. Cells were immunostained after treatment with 10 μg mL−1 bleomycin for 1 hr. Data are mean ± SD of triplicates. See also Figure S5. (I) High frequency of homozygous deletion of the ASF1A gene in cancers. Cbioportal (http://cbioportal.org) was analyzed, and each blue or gray bar represents a patient with or without a homozygous deletion in the indicated gene, respectively. To save space, a large number of gray bars were omitted from the rows in SkCM, prostate AdCa, and PancAdCa. The number of patients whose tumors had homozygous deletion of at least one of these repair genes (excluding ASF1B) is shown below for each cancer. Molecular Cell 2017 68, 61-75.e5DOI: (10.1016/j.molcel.2017.08.021) Copyright © 2017 Elsevier Inc. Terms and Conditions