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. 2011 Mar 3;471(7336):74-79.
doi: 10.1038/nature09803.

HDACs link the DNA damage response, processing of double-strand breaks and autophagy

Thomas Robert #  1 , Fabio Vanoli #  1 , Irene Chiolo #  1   2 , Ghadeer Shubassi  1 , Kara A Bernstein  3 , Rodney Rothstein  3 , Oronza A Botrugno  4 , Dario Parazzoli  5 , Amanda Oldani  5 , Saverio Minucci  4   6 , Marco Foiani  1   6
Affiliations

HDACs link the DNA damage response, processing of double-strand breaks and autophagy

Thomas Robert et al. Nature. .

Abstract

Protein acetylation is mediated by histone acetyltransferases (HATs) and deacetylases (HDACs), which influence chromatin dynamics, protein turnover and the DNA damage response. ATM and ATR mediate DNA damage checkpoints by sensing double-strand breaks and single-strand-DNA-RFA nucleofilaments, respectively. However, it is unclear how acetylation modulates the DNA damage response. Here we show that HDAC inhibition/ablation specifically counteracts yeast Mec1 (orthologue of human ATR) activation, double-strand-break processing and single-strand-DNA-RFA nucleofilament formation. Moreover, the recombination protein Sae2 (human CtIP) is acetylated and degraded after HDAC inhibition. Two HDACs, Hda1 and Rpd3, and one HAT, Gcn5, have key roles in these processes. We also find that HDAC inhibition triggers Sae2 degradation by promoting autophagy that affects the DNA damage sensitivity of hda1 and rpd3 mutants. Rapamycin, which stimulates autophagy by inhibiting Tor, also causes Sae2 degradation. We propose that Rpd3, Hda1 and Gcn5 control chromosome stability by coordinating the ATR checkpoint and double-strand-break processing with autophagy.

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Figures

Figure 1
Figure 1. VPA treatment counteracts DNA double-strand-break processing
a–c, RFA1::FLAG DDC2::MYC cells were arrested in G2 and released in YP galactose to induce HO endonuclease. After 30 min, the culture was split in two: +VPA and −VPA. a, Samples were processed for western blot using anti-Rad53, Srs2 and Ddc2 antibodies. b, A schematic diagram showing probe locations with respect to the HO cut site. The resection rate was calculated as the rate of HO cut band disappearance. c, Fold enrichment of the 0.2-kb fragment was calculated after ChIP of Rfa1–Flag, Ddc2–Myc. d, VPA effect on ectopic recombination (rec.) in wild-type (WT) cells. Error bars represent standard deviation (s.d.) calculated from four independent experiments.
Figure 2
Figure 2. VPA affects Sae2 and Exo1 but not Mre11 protein levels
a, MRE11::MYC cells were treated as in Fig. 1a. Cell samples were processed for ChIP analysis and the fold enrichment of the 0.2-kb fragment after ChIP of Mre11–Myc without (−VPA) or with (+VPA) VPA was calculated. b, EXO1::FLAG SAE2::PK MRE11::MYC cells were grown as in a. Cell samples were taken and processed for western blot analysis using anti-Flag, PK and Myc antibodies.
Figure 3
Figure 3. GFP–Atg8 Cherry–Ape1 cellular distributions in VPA-treated cells
a, Cherry::APE1 GFP::ATG8 cells were grown and shifted to YPD, nitrogen starvation (SD-N) or YPD+VPA medium for 3 h. Samples were processed for microscopy. The table shows numbers corresponding to the experiment. Percentage of fluorescence signals is presented and error bars represent the s.d. obtained from three independent experiments. DIC, differential interference contrast. Scale bars, 3 μm. b, Pho8Δ60 and Pho8Δ60 atg1Δ cells were grown as in a. Pho8Δ60 activity was calculated by measuring alkaline phosphatase levels. Error bars represent s.d. calculated from five independent experiments. c, GFP::ATG8 and GFP::ATG8 atg1Δ cells were grown as in a. Cell samples were processed for western blot using anti-GFP antibody. Quantification is presented in Supplementary Fig. 3.
Figure 4
Figure 4. Sae2 in VPA-treated cells
a, 4HA–Sae2 was immunoprecipitated ± VPA with anti-HA and subsequently ± anti-acetyl-Lysine. Eluate was analysed using anti-HA. Lane 1: input Sae2; 2: input Sae2–HA –VPA; 3: as in 2 but + VPA; 4: 3 μl elution Sae2–HA – VPA after anti-HA immunoprecipitation (IP; input AcK-IP); 5: double amount of 4; 6: IP anti-AcK elution from anti-HA IP of Sae2–HA – VPA; 7: as in 6 but + VPA. b, SAE2::PK erg6Δ cells were treated as in Fig. 2a. After 30 min induction, VPA and PMSF were added or not. Samples were processed for western blot using anti-PK. c, Wild-type SAE2::PK, SAE2::PK atg1Δ and SAE2::PK atg19Δ cells were grown as in b. After 30 min induction, VPA was added or not and samples treated as in b. d, Wild-type SAE2::PK cells were grown as in b. After 120 min induction, rapamycin (200 ng ml−1) was added or not and samples were treated as in b.
Figure 5
Figure 5. Gcn5, rpd3 and Hda1 influence Sae2 levels and cell survival in atg1 mutants in response to DNA damage
a, Survival of wild-type, rpd3Δ, hda1Δ and rpd3Δ hda1Δ strains after 4NQO, MMS and HU treatment. Error bars represent s.d. calculated from seven independent experiments. b, HO was induced in G2 wild-type, hdalΔ, rpdΔ and hdalΔ rpdΔ cells and western and Southern blot analyses were performed. c, Wild-type SAE2::PK and hdalΔ rpdΔ SAE2::PK cells were grown as in b and western blot was performed as in Fig. 4b. d, e, Wild-type SAE2::PK and gcn5Δ SAE2::PK strains were grown as in b and after 30 min of HO induction either VPA (d) or rapamycin (e) was added or not. Western blot was performed. f, Percentage of viability of wild-type, atg1Δ, rpd3Δ hdalΔ and atg1Δ rpd3Δ hdalΔ strains. Error bars represent s.d. calculated from four independent experiments.
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