Global chromatin compaction limits the strength of the DNA damage response

Abstract

In response to DNA damage, chromatin undergoes a global decondensation process that has been proposed to facilitate genome surveillance. However, the impact that chromatin compaction has on the DNA damage response (DDR) has not directly been tested and thus remains speculative. We apply two independent approaches (one based on murine embryonic stem cells with reduced amounts of the linker histone H1 and the second making use of histone deacetylase inhibitors) to show that the strength of the DDR is amplified in the context of "open" chromatin. H1-depleted cells are hyperresistant to DNA damage and present hypersensitive checkpoints, phenotypes that we show are explained by an increase in the amount of signaling generated at each DNA break. Furthermore, the decrease in H1 leads to a general increase in telomere length, an as of yet unrecognized role for H1 in the regulation of chromosome structure. We propose that slight differences in the epigenetic configuration might account for the cell-to-cell variation in the strength of the DDR observed when groups of cells are challenged with DNA breaks.

Figure 1.

Reduced H1 levels lead to enhanced resistance to DNA-damaging agents. Colony-survival assay of H1^+/+, H1⁵⁰, and H1^50/rec ES lines showing the relative surviving fraction of cells exposed to IR (A), MMS (1-h exposure; B), or HU (4-h exposure; C). Data points denote means of three independent experiments performed in triplicate. (D) Cell cycle profiles of H1^+/+ and H1⁵⁰ ES cells. Numbers indicate the percentage at each cell cycle stage (G1, S, and G2). (E) Percentage of BrdU-positive cells in both genotypes after the same period of BrdU exposure as the one used for HU treatments in C. Error bars represent ±SD.

Figure 2.

Hyperactive checkpoint responses in ES cells with reduced levels of H1. (A) Cell cycle distribution of H1^+/+ and H1⁵⁰ ES cells before (control) and 1 h after treatment with 0.25 Gy. Propidium iodide staining (x axis) and H3-S10 phosphorylation (y axis) is used to distinguish mitotic (red) from G2 cells. The percentage of mitotic cells is shown for each case. (B) Percentage of mitotic cells (normalized to untreated cells) after exposure to several doses of IR was measured in H1^+/+, H1⁵⁰, and H1^50/rec ES cells as described in A. (C and D) Intra–S phase checkpoint activation in H1^+/+, H1⁵⁰, and H1^50/rec ES cells exposed to increasing doses of IR (C) or MMS (D). Error bars show mean ± SD.

Figure 3.

Enhanced signaling per DSB in H1⁵⁰ cells. (A) ATM, Chk2, Chk1, and H2AX phosphorylation were analyzed by Western blotting in H1^+/+ and H1⁵⁰ ES cells exposed to increasing doses of IR. Quantification of Chk1 (B) and ATM (C) phosphorylation from the data observed in A. Data were normalized by total Chk1 and ATM levels, respectively. (D) IR-induced phosphorylation of H2AX and Chk1 in the different genotypes. (E) Chk1 and H2AX phosphorylation in H1^+/+ and H1⁵⁰ ES cells exposed to increasing doses of HU. MMS- (F) or HU (G)-induced γ-H2AX formation of H1^+/+, H1⁵⁰, and H1^50/rec ES cells. (H) γ-H2AX (red) and 53BP1 (green) foci analyzed in H1^+/+ and H1⁵⁰ cells 45 min after exposure to 3 Gy. Bars, 50 μm. (I) Distribution of the γ-H2AX intensity per nucleus in control and irradiated populations of the different genotypes obtained by high-throughput microscopy. Black bars mark median values. (J) Quantification of the amount (integrated intensity) of γ-H2AX present per individual focus.

Figure 4.

Increased T-SCE and enlarged telomeres in H1-depleted cells. (A) Quantification of T-SCE in H1^+/+ and H1⁵⁰ cells. A significant increase in T-SCE was observed in all cultures of H1-depleted cells (χ² test, P < 0.01). Error bars correspond to experiments performed in two independent ES lines (n = 2). (B) CO-FISH allows for the differential staining of the telomeres coming from the leading (green) or the lagging (red) chromatid. The image illustrates a typical T-SCE event (arrow) that leads to two telomeric signals per chromosome coming from the same sister chromatid, in contrast to the normal pattern found in the absence of recombination events (right). Bar, 1 μm. (C and D) Telomere length distribution in two independently derived pairs of H1^+/+ and H1⁵⁰ ES lines as determined by Q-FISH.

Figure 5.

TSA pretreatment stimulates the DDR. (A) γ-H2AX foci formation (red) in control or TSA-pretreated MCF7 cells 20 min after exposure to 3 Gy. A white mask shows the location of nuclei (derived from the corresponding DAPI image) in the control cells. TSA treatment was of 0.1 μM for 5 h for all of the experiments shown in this study. Bars, 50 μm. (B) Distribution of the γ-H2AX intensity per nucleus in control and irradiated (3 Gy) populations of untreated or TSA-pretreated MCF7 cells obtained by high-throughput microscopy. Black bars mark median values. Arrows highlight the difference in median intensity between each condition. (C) ATM, SMC1, Chk1, and H2AX phosphorylation was analyzed by Western blotting in control or TSA-pretreated cells 30 min after exposure to several doses of IR. (D) H2AX phosphorylation kinetics in control or TSA-pretreated cells in response to 10 Gy of IR. (E) Representative image showing the pattern of single DSBs induced by I-SceI (as measured by 53BP1 foci) in nuclease-transduced MCF7^ISceI cells. (F) Representative image showing 53BP1 and γ-H2AX foci induced by I-SceI (arrows) in nuclease-transduced MCF7^ISceI cells either untreated or treated with TSA. 0.1 μM TSA (5 h) was added 24 h after viral transduction. Bars, 20 μm. (G) Quantification of the amount (integrated intensity) of γ-H2AX present per I-SceI–induced focus in control or TSA-pretreated cells was made as described previously (Fig. 3 J and see Materials and methods). (H) Model illustrates the pathways regulating the global chromatin relaxation process induced by DNA damage and how this modulates the outcome of the DDR.