Activation of DNA damage response signaling by condensed chromatin

Abstract

The DNA damage response (DDR) occurs in the context of chromatin, and architectural features of chromatin have been implicated in DNA damage signaling and repair. Whereas a role of chromatin decondensation in the DDR is well established, we show here that chromatin condensation is integral to DDR signaling. We find that, in response to DNA damage chromatin regions transiently expand before undergoing extensive compaction. Using a protein-chromatin-tethering system to create defined chromatin domains, we show that interference with chromatin condensation results in failure to fully activate DDR. Conversely, forced induction of local chromatin condensation promotes ataxia telangiectasia mutated (ATM)- and ATR-dependent activation of upstream DDR signaling in a break-independent manner. Whereas persistent chromatin compaction enhanced upstream DDR signaling from irradiation-induced breaks, it reduced recovery and survival after damage. Our results demonstrate that chromatin condensation is sufficient for activation of DDR signaling and is an integral part of physiological DDR signaling.

Figure 1. Chromatin undergoes rapid expansion and compaction after DNA damage, and interference with these chromatin changes attenuates DDR signaling

(A) Snapshots of a damaged chromatin region and recruitment of MRE11 at indicated timepoints. PAGFP-H2A, green; mCherry-tagged MRE11, red. Scale bar 3 μm. (B) Average area of damaged chromatin regions in vector control (green line) versus ASH2-overexpressing cells (black line) over time, an undamaged chromatin region is shown in red (no Hoechst sensitization).. p-values of ASH2 vs. control are shown in heat map below. Red, p< 0.01; orange, p<0.05; green, not significant. N >15 regions for each point. (C) Schematic of chromatin protein tethering system: 256 copies of the lac operator (lacO) and 96 copies of tet (tetO) flank an I-SceI cut site (I-SceIcs). Lac repressor fusions to either mCherry alone (LacR) or to chromatin proteins bind to the lac operator arrays after transient expression. (D) Maximum intensity projections of LacR or ASH2-tethered arrays (red) stained for γ-H2AX (green) after DSB induction by CFP-GR-I-SceI. Scale bar, 5 μm. Values show median integrated intensity of γ-H2AX at arrays ± median absolute deviation. (E) Percentage of LacR or ASH2-tethered arrays with γ-H2AX or 53BP1 enrichment 20 min post-DSB induction. Columns depict mean and error bars, SD. N ≥ 100 for each condition. *p < 0.05. (F) Ligation-mediated qPCR detecting the quantity of DSBs in I-SceI induced cells. Shown is the average ± SD of two independent experiments, each performed in triplicate. **p < 0.001. See also Supplemental Figure S1.

Figure 2. Compacted chromatin triggers DDR signaling

(A) Maximum intensity projections of cells transfected with indicated mCherry-LacR constructs (red) stained with anti-γ-H2AX (green), and DAPI (blue). Green arrows indicate arrays enriched in γ-H2AX that are magnified 2X in the inset image. Gray arrows indicate arrays without significant γ-H2AX. Scale bar, 5 μm. Percentages ± SD of arrays staining positive for γ-H2AX are shown in the top center of each panel. (B) Images of cells as in (A), but stained with anti-53BP1 (green) (C) Quantification of γ-H2AX and 53BP1 colocalization frequency at the arrays. Values represent fold change in averages ± SD from 3 experiments (N ≥ 300 for each condition). * p < 0.05 compared to LacR alone. (D) Images of cells as in (A), but expressing indicated chromatin expansion factors or facultative heterochromatin proteins fused to the LacR protein. Scale bar, 5 μm. See also Supplemental Figure S2.

Figure 3. DDR signaling from arrays is not due to array breakage or replication defects

(A) Quantification of arrays showing positive TUNEL signals. I-SceI transfected cells and MIS18α-arrays provide positive controls for DNA end detection. Values represent averages ± SD from at least 3 experiments. * p < 0.05 compared to lac alone, N > 200 for each. (B) Southern blot of genomic DNA isolated from indicated tethering conditions, using a probe to the lac array. I-SceI transfected cells provide positive control for breaks, unrelated lanes from the blot between I-SceI and HP1α omitted for simplicity. (C) Ligation-mediated PCR assay detecting damage within the lac array. I-SceI used as a positive control for breaks, this same DNA was used in the negative control reaction with no adaptor. Normalized signal intensity of PCR reactions are depicted by averages ± SD from 2 independent trials. (D) Cells pre-arrested in G1 by double thymidine block or (E) serum-starved prior to transfection with lac or HP1γ tethering constructs. Values represent averages ± SD of γ-H2AX or 53BP1 recruitment measured in cyclin A- or Ki67-negative cells, respectively, from 2-5 independent experiments. N > 150 per condition. *p < 0.05 compared to cycling cells. See also Supplemental Figure S3.

Figure 4. Activation of upstream DDR signaling by condensed chromatin

(A) Recruitment of MDC1 and NBS1 to condensed arrays, quantified as in Fig. 2C. Values represent average fold change ± SD from 3 independent experiments (N ≥ 200 for each condition). * p < 0.05 compared to LacR alone. (B) Maximum intensity projections of phospho-ATM (S1981) immunostaining at LacR, HP1- or SUV3-9 arrays. Green arrows, arrays enriched in phospho-ATM; gray arrows, no enrichment. Scale bar, 5 μm. (C) γ-H2AX formation in condensed chromatin following siRNA depletion of ATM and/or ATR, depicted as average fold change to control siRNA (siControl) transfected with mCherry-LacR ± SD from 2 experiments (N = 40-150), *p < 0.05. Knockdown is shown in Supplemental Figure S4. (D) DNA-PK, ATM and ATR were inhibited with KU55933 (ATMi), VE-821 (ATRi), or NU7441 (DNA-PKi), after tethering. Average ± SD of γ-H2AX recruitment compared to DMSO-treated cells transfected with LacR from 3 independent trials. N > 175 per condition, *p < 0.05, **p < 0.01. (E) Immunoblot analysis of activated DDR factors detected by phospho-specific antibodies. Loading control, Lamin A/C. (F) Cyclin A staining and (G) FACS cell cycle profiling of cell populations with tethered chromatin factors. Shown is average percentage of cells positive (S/G2, M phases), or negative (G1) for cyclin A staining, or as determined by DNA content.

Figure 5. Mitotic chromosome condensation activates chromatin-induced DDR

(A) Individual γ-H2AX foci quantified by integrated density measurements. Box: quartiles 1 - 3, whiskers: value range, red bar: median values, as follows: 487 (interphase), 1317 (mitosis). ***p < 0.001, N > 300 foci for each. (B) Mitotic cells were harvested by shake-off, attached to slides and stained for γ-H2AX (red), and either MDC1 or 53BP1 (green). Shown are maximum intensity projections with DAPI overlay (blue). (C) Increased γ-H2AX foci in cells treated with 50 nM CalA for 60 min, fixed and immunostained for γ-H2AX (red), and DAPI (blue). Scale bar, 10 μm. (D) Total nuclear intensity of γ-H2AX from cells in indicated treatments. Box and whiskers as in A. One single outlier (>1.5 times outside the interquartile range) is marked with a gray dot, and the red bars indicate median values. *p < 0.05, ** p < 0.01. See also Supplemental Figure S5.

Figure 6. Persistent condensation of chromatin enhances upstream signaling from IR-induced breaks, but reduces cell survival after damage

(A) Immunoblot of phospho-NBS1 in control or SUV3-9 overexpressing cells ±5 Gy γ-irradiation. β-actin shown as a loading control. (B) Quantification of phospho-NBS1 levels, normalized to β-actin. Values represent averages ± SD from 3 experiments. Unirradiated NBS1 levels are normalized to 1. **p < 0.01 compared to irradiated control. (C) Blot of phospho-CHK2 in conditions as in panel A and depicted identically. (D) Quantification of phospho-CHK2 as in B. *p < 0.05 compared to irradiated control. (E) Clonogenic survival assays of cells expressing SUV3-9. Surviving colonies were normalized to unirradiated controls. Values represent median ± median average deviation from 3 experiments. *p < 0.05 compared to irradiated control. See also Supplemental Figure S6.