DNA double-strand break movement in heterochromatin depends on the histone acetyltransferase dGcn5

Abstract

Cells employ diverse strategies to repair double-strand breaks (DSBs), a dangerous form of DNA damage that threatens genome integrity. Eukaryotic nuclei consist of different chromatin environments, each displaying distinct molecular and biophysical properties that can significantly influence the DSB-repair process. DSBs arising in the compact and silenced heterochromatin domains have been found to move to the heterochromatin periphery in mouse and Drosophila to prevent aberrant recombination events. However, it is poorly understood how chromatin components, such as histone post-translational modifications, contribute to these DSB movements within heterochromatin. Using irradiation as well as locus-specific DSB induction in Drosophila tissues and cultured cells, we find enrichment of histone H3 lysine 9 acetylation (H3K9ac) at DSBs in heterochromatin but not euchromatin. We find this increase is mediated by the histone acetyltransferase dGcn5, which rapidly localizes to heterochromatic DSBs. Moreover, we demonstrate that in the absence of dGcn5, heterochromatic DSBs display impaired recruitment of the SUMO E3 ligase Nse2/Qjt and fail to relocate to the heterochromatin periphery to complete repair. In summary, our results reveal a previously unidentified role for dGcn5 and H3K9ac in heterochromatic DSB repair and underscore the importance of differential chromatin responses at heterochromatic and euchromatic DSBs to promote safe repair.

Graphical Abstract

Figure 1.

H3K9ac is increased at heterochromatic DSBs in a dGcn5-dependent manner. (A) Schematic representation of the DR-white system in Drosophila. The DR-white construct is integrated in a single euchromatic or heterochromatic locus in the fly genome to generate different lines (nomenclature as in (30)). The DR-white construct contains an 18-bp DNA sequence targeted by the I-SceI endonuclease, resulting in the formation of a single DSB (30). (B) Scheme detailing the DSB induction protocol in DR-white fly lines. Chromatin was extracted 6 h after DSB induction. DSBs were induced by a 1 h heat-shock (37°C) of third instar DR-white larvae containing a heat-shock promoter-driven I-SceI transgene (hsp.I-SceI). To control for heat-shock effects, all conditions (without/with hsp.I-SceI) are heat-shocked. (C) ChIP-qPCR analysis of H3K9ac levels at indicated DR-white loci. The qPCR primers reside 1.4 kb downstream of the I-SceI cut site (‘3xp3’ in Figure 1A). Fold change is calculated by dividing the H3K9ac enrichment levels in the damaged (+DSB, +I-SceI, Supplementary Figure S1A) samples by those in the controls (−DSB, no (−) I-SceI Supplementary Figure S1A). The grey dotted line represents a fold change of 1, in which H3K9ac levels are unchanged upon DSB induction. All qPCR results are relative to an internal control region (yellow), which is consistently enriched for H3K9ac. Error bars represent mean ± SD from ≥4 independent experiments. (D) Representative time-lapse images of non-irradiated (no IR) and irradiated (5 min after 5 Gy gamma-radiation) Kc cells with fluorescently tagged Mu2 (green, DSB marker), dGcn5 (blue) and HP1a (magenta, heterochromatin marker). Dashed lines outline the nuclei. Insets are zoom-in views of Mu2, dGcn5 and HP1a colocalization. Scale bar = 2 μm. (E, F) Quantification of H3K9ac intensity levels in heterochromatin (E, H3K9me3-enriched, immunofluorescence stainings performed as in Supplementary Figure S2H) and euchromatin (F, low H3K9me3, see Supplementary Figure S2H) at DSBs in non-irradiated versus irradiated control (yellow dsRNA), dGcn5-depleted and dKDM4A-depleted Kc cells. Irradiated cells were fixed 5-10 min after damage induction. Error bars represent mean + SD from ≥3 independent experiments. (*) P-value ≤ 0.05, (**) P-value ≤ 0.01, (***) P-value ≤ 0.001, paired t-test. If not shown, P-value not significant (>0.05). Figure 1A was created with Biorender.com.

Figure 2.

dGcn5 promotes repair of heterochromatic DSBs. (A) Representative images of Kc cells depleted for yellow (control) and dGcn5 fixed at indicated time points following 5 Gy irradiation. Cells were stained for γH2Av (green, DSB marker) and DAPI (magenta). White arrows indicate γH2Av foci in DAPI bright domain (heterochromatin). Dashed lines enclose the nuclei. Cells at time point 0 were fixed without prior irradiation. Scale bar = 2 μm. (B,C) Quantification of images as in (A). Average number of γH2Av foci in DAPI bright (B) and DAPI weak (C) is shown at indicated time points. Shade bars represent mean ± SD from three independent experiments. (D,E) Quantification of γH2Av foci inside DAPI bright (D) and DAPI weak (E) in non-irradiated versus 120 min after irradiation control (yellow dsRNA), dGcn5-depleted, dKDM4A-depleted or dGcn5 and dKDM4A co-depleted Kc cells. HET = heterochromatin, EU = euchromatin. Error bars represent mean + SD from three independent experiments. (*) P-value ≤ 0.05, (**) P-value ≤ 0.01, paired t-test. If not shown, P-value not significant (>0.05).

Figure 3.

dGcn5 is required for DSB movement outside the HP1a domain. (A) Representative time-lapse images of ATRIP foci (green, HR protein) within the HP1a domain (magenta) in irradiated (5 Gy) control (yellow dsRNA) or dGcn5 depleted Kc cells. Insets are zoom-in views of heterochromatic ATRIP foci. Dashed lines enclose the nuclei. Scale bars = 2 μm. (B) Quantification of time-lapse movies as in (A). ATRIP foci movement and kinetics plotted from their appearance in the HP1a domain (0′, magenta) to the timepoint they moved outside of the HP1a domain (‘out’, grey) or resolved (‘disappear’, black). Each row indicates quantification of one ATRIP focus.(C) Quantification of the mean residence time of ATRIP foci within the HP1a domain. (*) P-value ≤ 0.05, paired t-test. Error bars represent mean + SD from three independent experiments.

Figure 4.

dGcn5 promotes Qjt recruitment at heterochromatic DSBs. (A) Representative time-lapse images of Kc cells expressing HP1a (magenta, heterochromatin) and Qjt (Nse2, green) with indicated conditions. Arrowhead indicates Qjt focus residing inside the HP1a domain. Dashed lines enclose the nuclei. Scale bars = 2 μm. (B,C) Quantification of Qjt foci inside the HP1a domain (B) and in euchromatin (C, outside HP1a domain) in non-irradiated versus 10 min after irradiation control (yellow dsRNA) and dGcn5-depleted Kc cells. HET = heterochromatin, EU = euchromatin. Error bars represent mean + SD from three independent experiments. (*) P-value ≤ 0.05, (***) P-value ≤ 0.001, one way ANOVA test followed by Tukey's multiple comparison. If not shown, P-value not significant (>0.05).

Figure 5.

dGcn5 mutant is synthetically lethal with ATR mutant. (A) Schematic representation of the dGcn5 [E333st] mutation. dGcn5 consists of three exons (black rectangles) and two introns (lines in between the black rectangles). The premature stop codon (E333st, orange arrow in first exon) is introduced in the three nucleotides encoding for the 333^rd amino acid (58). (B) Representative images of control and dGcn5 [E333st]/+ wing disc cells, fixed without prior irradiation (0 min) or 120 min after 5 Gy irradiation. Cells were stained for γH2Av (green, DSB marker) and H3K9me3 (magenta, heterochromatin marker). Dashed white lines indicate nuclei. Arrowheads indicate γH2Av foci inside the heterochromatic domain. Scale bar = 2 μm. (C) Quantification of images as in (B). Average number of γH2Av foci in heterochromatin is shown at indicated time points. Error bars represent mean + SD from three independent experiments. (*) P-value ≤ 0.05, one way ANOVA test followed by Tukey's multiple comparison. If not shown, P-value not significant (>0.05). (D) Synthetic lethality assay of dGcn5 mutant with ATR mutant. N = 310 flies for the control line (dGcn5 mutant, wild-type ATR allele (mei41+; dGcn5 [E333st]/+)) and n = 344 flies for the cross between dGcn5- and ATR- mutant flies (mei41 [29D]; dGcn5 [E333st]/+). Unpaired t-test. (E) Model for the role of dGcn5-mediated H3K9ac in heterochromatic DSB repair. The local transfer of an acetyl group onto H3K9 by dGcn5 at heterochromatic break sites leads to recruitment of SMC5/6-Nse2 (Qjt) which in turn promotes movement and repair of heterochromatic DSBs. The deposition of new H3K9ac marks by dGcn5 is independent of dKDM4A-mediated demethylation of H3K9me3. Figure 5E was created with Biorender.com.