Volume 27, Issue 8, Pages 1192-1199 (April 2017) Compartmentalization of DNA Damage Response between Heterochromatin and Euchromatin Is Mediated by Distinct H2A Histone Variants  Zdravko J. Lorković, Chulmin Park, Malgorzata Goiser, Danhua Jiang, Marie-Therese Kurzbauer, Peter Schlögelhofer, Frédéric Berger  Current Biology  Volume 27, Issue 8, Pages 1192-1199 (April 2017) DOI: 10.1016/j.cub.2017.03.002 Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 1 H2A.W.7 and H2A.X Occupy Distinct Nuclear Regions and Are Required for DNA Damage Response (A) Sequence alignment of N- and C-terminal tails of Arabidopsis H2A.W and H2A.X variants. Conserved KSPKK and SQEF motifs of H2A.W and H2A.X variants are highlighted. The peptide sequence of H2A.W.7, identified in a phosphoproteomic screen [19], is indicated in the red box with the phosphorylated SQ residues in red. The H2A.W.6 and H2A.W.7 peptides used to obtain variant-specific antibodies are boxed in blue. (B) Localization of H2A.W.7 and H2A.X in nuclei isolated from an h2a.w.7 mutant expressing H2A.W.7-HA. Note that there is co-localization of both variants in chromocenters, but not in the nucleoplasm. The graph on the right represents quantified levels (mean fluorescence intensities) of H2A.W.7 and H2A.X over chromocenters. Data are represented as means ± SD with n = 48 nuclei. The p value was determined by a two-tailed Student’s t test. See also Figure S1D for details. (C) Immunoprecipitation of H2A.W.7 and H2A.X from WT leaf nuclei digested with MNase. On the right is a profile of DNA isolated from an input extract demonstrating that most of the chromatin used for immunoprecipitation was digested to mononucleosomes. (D) Sensitivity of WT and h2a.w.7, h2a.x, and indicated double and triple mutant lines to zeocin determined by true leaf development assay. Seeds were germinated on MS plates containing 50 μg/mL of zeocin, and true leaves were scored 10 days after germination. Data are represented as means ± SD of two independent experiments with n = 150 seedlings. For confocal images, the scale bar represents 4 μm. See also Figure S1. Current Biology 2017 27, 1192-1199DOI: (10.1016/j.cub.2017.03.002) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 2 In Vivo Phosphorylation of H2A.W.7 Is Induced by DNA Damage (A) Time course of H2A.W.7 and H2A.X phosphorylation in the presence of bleomycin. WT seedlings were treated with 20 μg/mL of bleomycin for the indicated times, and nuclear extracts were analyzed by western blotting with antibodies directed against phosphorylated H2A.W.7 and H2A.X (γH2A.W.7 and γH2A.X). (B) Dose-dependent phosphorylation of H2A.W.7 and H2A.X. WT seedlings were treated for 2 hr with the indicated concentrations of bleomycin, and nuclear extracts were analyzed by western blotting with antibodies directed against γH2A.W.7 and γH2A.X. (C) Sequence of H2A.W.7 C-terminal tail with serine to alanine (S135A) and serine to aspartate (S135D) mutations (boxed). (D) The SQ motif of H2A.W.7 is required for response to DNA damage. Sensitivity of h2a.w.7 mutants expressing WT H2A.W.7 or S135A and S135D mutant proteins to genotoxic stress were determined by true leaf development assay in the presence of 50 μg/mL of zeocin and scored 10 days after germination. For each construct, two independent T3 transgenic lines were used. Data are represented as means ± SD of two independent experiments with n = 120 seedlings. (E) H2A.W.7 and H2A.X phosphorylation depends on ATM, but not ATR. Phosphorylation of H2A.W.7 and H2A.X was analyzed in seedlings from WT, atm, and atr mutant lines treated with or without bleomycin (20 μg/mL) for 2 hr. (F) Phosphorylation of H2A.W.7 in response to DNA damage is independent of H2A.X. Seedlings from WT and h2a.w.7, h2a.x.3, h2a.x.5 single, double, and triple mutant lines were treated for 2 hr with bleomycin, and nuclei were extracted and analyzed by western blotting using antibodies against γH2A.W.7 and γH2A.X. In (A), (B), (E), and (F), western blots with antibodies against histone H3 and non-modified H2A.W.7 and H2A.X were used as loading controls. The latter two also confirm the corresponding mutant genotypes shown in (F). Note that the antibody against H2A.X recognizes the identical epitopes on both H2A.X.3 and H2A.X.5, and as a consequence we were able to report only global levels of H2A.X in single h2a.x mutants. See also Figure S2. Current Biology 2017 27, 1192-1199DOI: (10.1016/j.cub.2017.03.002) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 3 Genome-wide Distribution of H2A.W.7 (A) Co-localization of H2A.W.7 and H2A.W.6 in nuclei isolated form h2a.w.7 mutant seedlings expressing HA-tagged H2A.W.7. Enlargements of regions delimited by white squares are provided below each image to show small non-overlapping patches marked by H2A.W.6 or H2A.W.7. (B) Western blot analysis of immunoprecipitates obtained with H2A.W.6- and H2A.W.7-specific antibodies from MNase-digested WT nuclei. On the right is a profile of DNA isolated from an input extract demonstrating that most of chromatin used for immunoprecipitation was digested to mononucleosomes. (C) Distribution of H2A.W.6 and H2A.W.7 over chromosome one. Chromatin immunoprecipitation sequencing (ChIP-seq) reads per million mapping reads for all five chromosomes were calculated in 100-kb bins and expressed relative to input. Position of pericentromeric heterochromatin is indicated by a black bar. See also Figure S3. (D) Profiles of H2A.W.6 and H2A.W.7 over heterochromatic regions defined by enrichment of H3K9me2. (E) Association of H2A.W.6- and H2A.W.7-enriched regions with the indicated genomic features. Regions with a ChIP:input ratio ≥2.5 were used for analysis. TSS, transcription start site; TES, transcription exit site. (F) Representative genome-browser views of H2A.W.7, H2A.W.6, and H3K9me2 in indicated regions of chromosomes 3 and 4. Genes (blue) and transposable elements (TEs) (black) are shown below. (G) Distribution of H2A.W.6 and H2A.W.7 over defined H2A.W.7 regions in the chromocenters. (H) Distribution of H2A.W.6 and H2A.W.7 over defined H2A.W.7 regions in the chromosome arms. For confocal images, the scale bar represents 4 μm. Current Biology 2017 27, 1192-1199DOI: (10.1016/j.cub.2017.03.002) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 4 Nuclear Distribution and Dynamics of γH2A.W.7 and γH2A.X (A) Nuclei were isolated from mock- or bleomycin-treated (20 μg/mL for 2 hr) seedlings and immunostained with antibodies specific for γH2A.X and γH2A.W.7. (B) Quantification of γH2A.X and γH2A.W.7 foci associated with chromocenters after 2, 4, and 8 hr of bleomycin treatment. Data are represented as means ± SD. p values were determined by two-tailed Student’s t test. See also Figure S4B for details. (C) Total levels of γH2A.W.7 and γH2A.X in WT seedlings analyzed after 2, 4, and 8 hr of bleomycin treatment (20 μg/mL). Bars (at the bottom) are average data for time points 2 (blue) and 8 (red) hr of treatment from three independent experiments ±SD. (D) Total levels of γH2A.W.7 and γH2A.X in h2a.w.7 and h2a.x seedlings analyzed after 2, 4, and 8 hr of bleomycin treatment (20 μg/mL). Bars (at the bottom) are average data for time points 2 (blue) and 8 (red) hr of treatment from three independent experiments ±SD. (E) Quantification of γH2A.W.7 and γH2A.X levels during recovery of DNA damage. Seedlings were treated for 2 hr with 20 μg/mL of bleomycin, and, after extensive washing, samples were collected at indicated time points and used for nuclei isolation. Western blots were probed with γH2A.X- and γH2A.W.7-specific antibodies, and band intensities were quantified with ImageDoc software (Bio-Rad) and normalized to the total levels of H2A.X and H2A.W.7. Data are represented as means ± SD of two independent experiments. For confocal images, scale bars represent 4 μm. See also Figure S4. Current Biology 2017 27, 1192-1199DOI: (10.1016/j.cub.2017.03.002) Copyright © 2017 Elsevier Ltd Terms and Conditions