Screen for DNA-damage-responsive histone modifications identifies H3K9Ac and H3K56Ac in human cells

Abstract

Recognition and repair of damaged DNA occurs within the context of chromatin. The key protein components of chromatin are histones, whose post-translational modifications control diverse chromatin functions. Here, we report our findings from a large-scale screen for DNA-damage-responsive histone modifications in human cells. We have identified specific phosphorylations and acetylations on histone H3 that decrease in response to DNA damage. Significantly, we find that DNA-damage-induced changes in H3S10p, H3S28p and H3.3S31p are a consequence of cell-cycle re-positioning rather than DNA damage per se. In contrast, H3K9Ac and H3K56Ac, a mark previously uncharacterized in human cells, are rapidly and reversibly reduced in response to DNA damage. Finally, we show that the histone acetyl-transferase GCN5/KAT2A acetylates H3K56 in vitro and in vivo. Collectively, our data indicate that though most histone modifications do not change appreciably after genotoxic stress, H3K9Ac and H3K56Ac are reduced in response to DNA damage in human cells.

Figure 1

Screen for DNA-damage-responsive histone modifications. (A) U2OS cells were untreated (Untr) or treated with 2 mM HU for 24 h or with 60 μg/ml phleomycin (Phleo) for 2 h. Cells were split into two samples, one being used for the acid extraction of histones that were analysed in (B) and one sample for whole cell Laemmli extracts, which were analysed by western blotting with the indicated antibodies in (A). (B) Coomassie staining of recombinant histones (Rec) and acid-extracted histones from either untreated (Untr), HU-treated or phleo-treated U2OS cells. (C) Antibody-based screen for DNA-damage-responsive histone PTMs. Samples from (B) were used for western blot analysis with the antibodies described in Supplementary Table 1. The results were categorized as described in the text.

Figure 2

Analysis of DDR-responsive histone PTMs. (A) Analysis of histone PTMs in U2OS or Hela cells. Left panels, U2OS cells were treated with the indicated DNA-damaging agents (see Materials and methods for details) and analysed by western blotting of whole cell Laemmli extracts with the indicated antibodies. Right panels, Hela cells were untreated or treated with 60 μg/ml of phleomycin and analysed as described for U2OS cells. (B) Cell-cycle analysis of histone PTMs. U2OS cells were synchronized at the G1/S-transition by a double-thymidine procedure and subsequently released into the cell cycle. Samples were taken at indicated time points and cell-cycle distributions were determined by FACS analyses. A sample from asynchronous (Asyn) cells is shown as a control. These samples were additionally subjected to western blot analyses of whole cell Laemmli extracts and probed with the indicated antibodies.

Figure 3

Mitotic H3 phosphorylations are unaltered by DNA damage. (A) H3 phosphorylation levels, but not acetylation levels, are unaffected by DNA damage. U2OS cells were arrested in pro-metaphase with nocodazole, then were either kept in nocodazole (Noc; M-phase) or released from nocodazole for 6 h (Noc & Rel; G1/S-phase) and subsequently either mock or phleomycin treated. Samples were analysed as in Figure 2. (B) Immunofluorescence analysis of H3 phosphorylations in mitosis. Nocodazole-arrested U2OS cells were left untreated or treated with phleomycin and analysed by immunofluorescence with the indicated antibodies. Induction of DNA damage was visualized by γH2AX staining and the nucleus by DAPI staining of DNA. (C) Analysis of H3S10p and H3T11p in interphase cells. U2OS cells were untreated or treated for 2 h with phleomycin and analysed by immunofluorescence for H3S10p and H3T11p. The enlarged section depicts punctuate co-localized staining of H3S10p and H3T11p. Note the loss of brightly stained mitotic cells in phleomycin-treated cell populations. (D) H3T11p is unaffected in cells containing concentrated tracks of DNA damage. U2OS cells were microirradiated to induce tracks containing DNA damage. Cells were allowed to recover for 2 h before fixation and subsequent dual staining for 53BP1 and H3T11p. Inset shows a zoomed section of the damaged area.

Figure 4

H3K9Ac and H3K56Ac decrease rapidly and reversibly upon DNA damage. (A) Transcriptional inhibition does not affect H3K9Ac and H3K56Ac levels. Cells were treated for 2 h with DRB, a Pol II transcription inhibitor, and analysed for H3K9Ac and H3K56Ac as described for Figure 2. (B) Dynamics of changes in H3K9Ac and H3K56Ac levels are reciprocal to those of γH2AX. Recovery dynamics of H3K9Ac and H3K56Ac levels were measured by acute treatment of U2OS cells with phleomycin for 2 h and subsequently releasing them into phleomycin-free medium. Samples were taken at the indicated time points and analysed using whole cell Laemmli extracts with the indicated antibodies. H3K14Ac was included to control for a DDR-independent histone acetylation, γH2AX to detect DNA-damage induction and H3 for loading. (C) H3K9Ac and H3K56Ac levels decrease rapidly upon DNA damage. U2OS cells were chronically treated with phleomycin and samples were taken at the indicated times and analysed as in A. (D) PIKK inhibition reduces H3K9 and H3K56 acetylation. U2OS cells were pre-incubated for 1 h with or without KU-55933, or wortmannin, then analysed as in A. (E) H3K56Ac levels are reduced at promoters of cell-cycle regulatory genes upon DNA damage. Schematic representation of CYCLIN B1 and CDK1 genes showing positions analysed by ChIP followed by real-time qPCR. U2OS cells were untreated or treated with phleomycin or 2 h, before analyses by ChIP. Data represents percentage of IPed H3K56Ac signal normalized to IPed H3 signal at the indicated genomic loci. Data represent the average of three independent experiments and error bars show the standard deviation between these experiments.

Figure 5

hGCN5 acetylates H3K9 and H3K56 in vitro and in vivo. (A) The H3K9 and H3K56 motifs share sequence similarity. (B) Human GCN5 acetylates H3K9 and H3K56 in vitro. HAT activity towards H3K9 and H3K56 was assessed with purified S. cerevisiae and S. pombe Rtt109 (ScRTT109 and SpRTT109, respectively) or human GCN5 (hGCN5) in an in vitro HAT-assay containing purified human recombinant histone H3.1 (Rec. H3) with subsequent analysis by western blotting with the indicated antibodies. SpClr4 was used as a non-HAT enzyme and a ponceau staining of Rec. H3 is shown as a loading control. (C) H3K9Ac antibody is specific. Purified SpRtt109 was incubated with Rec.H3 or Rec. H3 containing a Lys9 to Ala mutation (H3K9A) and analysed as in A. (D) H3K56Ac antibody is specific. Experiments were done as in B except Rec. H3K56A was used instead of Rec. H3K9A. (E) hGCN5 is involved in acetylation of H3K9 and H3K56 in vivo. U2OS cells were transfected with siRNAs against luciferase (siLuc) or GCN5 (siGCN5) and samples were analysed after 48 h by western blotting of whole cell Laemmli extracts with the indicated antibodies. (F) GCN5 depletion leads to the loss of H3K56Ac at promoters of cell-cycle regulatory genes. U2OS cells were transfected as in E and analysed as in Figure 4E.