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Received 13 Jan 2016 | Accepted 14 Jun 2016 | Published 20 Jul 2016

Lei Li1,*, Lan Shi1,*, Shangda Yang2, Ruorong Yan1, Di Zhang1, Jianguo Yang1, Lin He1, Wanjin Li1, Xia Yi1, Luyang Sun1, Jing Liang1, Zhongyi Cheng3, Lei Shi2, Yongfeng Shang1,2 & Wenhua Yu1

Although SIRT7 is a member of sirtuin family proteins that are described as NAD -dependent class III histone deacetylases, the intrinsic enzymatic activity of this sirtuin protein remains to be investigated and the cellular function of SIRT7 remains to be explored.

Here we report that SIRT7 is an NAD -dependent histone desuccinylase. We show that SIRT7 is recruited to DNA double-strand breaks (DSBs) in a PARP1-dependent manner and catalyses desuccinylation of H3K122 therein, thereby promoting chromatin condensation and DSB repair. We demonstrate that depletion of SIRT7 impairs chromatin compaction during DNA-damage response and sensitizes cells to genotoxic stresses. Our study indicates SIRT7 is a histone desuccinylase, providing a molecular basis for the understanding of epigenetic regulation by this sirtuin protein. Our experiments reveal that SIRT7-catalysed H3K122 desuccinylation is critically implemented in DNA-damage response and cell survival, providing a mechanistic insight into the cellular function of SIRT7.

DOI: 10.1038/ncomms12235 OPEN

SIRT7 is a histone desuccinylase that functionally links to chromatin compaction and genome stability

1 Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education), Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Peking University Health Science Center, Beijing 100191, China. 2 2011 Collaborative Innovation Center of Tianjin for Medical Epigenetics, Tianjin Key Laboratory of Medical Epigenetics, Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Tianjin Medical University, Tianjin 300070, China. 3 Jingjie PTM BioLab Co. Ltd., Hangzhou Economic and Technological Development Area, Hangzhou 310018, China.* These authors contributed equally to this work. Correspondence and requests for materials should be addressed to Y.S. (email: mailto:[email protected]

Web End [email protected] ) or to W.Y. (email: mailto:[email protected]

Web End [email protected] ).

NATURE COMMUNICATIONS | 7:12235 | DOI: 10.1038/ncomms12235 | http://www.nature.com/naturecommunications

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ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms12235

Silent information regulator 2 (Sir2) proteins, or sirtuins, were originally discovered for their role in transcriptional repression of several genomic loci in Saccharomyces

cerevisiae1. Mammalian genomes encode seven members of the sirtuin family, SIRT17, all possessing a highly conserved catalytic domain and a nicotinamide adenine dinucleotide (NAD )-binding site while exhibiting different subcellular localization, enzymatic activity, molecular target(s) and tissue specicity2. Intriguingly, although SIRT proteins have been described as class III histone deacetylases (HDACs)3, recent studies suggest that these proteins might possess additional enzymatic activities. For example, it is reported that SIRT3 acts as a decrotonylase to regulate histone lysine crotonylation and gene transcription4, and that SIRT6 is able to remove fatty acyl modication on lysine (K)19 and K20 of tumour necrosis factor a (ref. 5), while SIRT5, a well-characterized mitochondrial sirtuin protein6, is shown to negatively regulate several acylations, including succinylation7, malonylation8 and glutarylation9 of both intra- and extra-mitochondrial proteins.

SIRT7 has been identied in the nucleolus and reported to regulate RNA polymerase I transcription10. Subsequent studies found that SIRT7 acts as an NAD -dependent H3K18 deacetylase11. In addition, SIRT7 has also been reported to target several non-histone proteins, including p53 (ref. 12), PAF53 (ref. 10), NPM1 (ref. 13), GABP-b1 (ref. 14) and U3-55k (ref. 15) for deacetylation, and has been implicated in hepatic lipid metabolism16, mitochondrial homeostasis17 and adipogenesis18. However, the enzymatic activity and cellular function of SIRT7 needs further elucidation.

Nucleosome is the fundamental repeating units for packing the large eukaryotic genome into the nucleus while still ensuring appropriate access to it. It consists of 147 bp of DNA wrapped around a protein octamer made up of two copies of each of the four histones H2A, H2B, H3 and H4 (ref. 19). There are two structurally and functionally distinct domains in a histone octamer: the globular domain forming the nucleosomal core around which DNA is wrapped and the unstructured tails to which various post-translational modications are added20. The accessibility of the DNA that is coiled around the histone octamer is a critical parameter for processes such as transcription, replication, recombination and DNA repair. Among various factors that control DNA accessibility, histone modication represents a prominent mechanism by which the nucleosome plasticity is regulated and chromatin conguration is shaped21,22.

A plethora of histone modications have been described and, in last decade, various histone modications, including phosphorylation, acetylation, methylation, ubiquitination, sumoylation and ADP-ribosylation have been the subjects of extensive study in the eld of epigenetics23. It is believed that factors involved in the deposition (writer), binding (reader) and removal (eraser) of these histone modications (marks) are at the epicentre of the regulatory circuits controlling the chromatin dynamics2224. It is proposed that various histone modications in combination constitute distinct histone languages to encode for different chromatin-related events25. In effect, chromatin modiers (writers or erasers) act in an interdependent manner and coordinated fashion to load or remove histone marks to control the chromatin conguration and to determining the biological consequence2224. Accordingly, identication and functional characterization of these chromatin modiers have been the major theme in the understanding of epigenetic regulation. Strikingly, recent studies identied a series of new types of histone modications, including biotinylation, citrullination, crotonylation, glutathionylation, propionylation, malonylation and succinylation9,2629, adding to the complexity of the already sophisticated epigenetic regulatory network.

Ultimately, the understanding of the biological signicance of these new modications is still dependent on identication of the writers, readers and erasers of these histone marks.

In this study, we report that SIRT7 is an NAD-dependent histone desuccinylase. We show that SIRT7 is recruited to double-strand break (DSB) sites in a PARP1-dependent manner and catalyses desuccinylation of H3K122 at DSB sites, thereby promoting chromatin condensation and efcient DSB repair. We demonstrate that depletion of SIRT7 impaired chromatin compaction and DNA repair, and sensitized cells to genotoxic stresses.

ResultsSIRT7 is a histone desuccinylase. As stated above, sirtuin family proteins were initially characterized as class III histone deacetylases3 but recently reported to possess additional deacylation activities7,8,30. To better understand the enzymatic activity and cellular function of SIRT7, we started by proling the expression of SIRT7 in various cell lines. The results showed that SIRT7 was widely expressed in different cell lines (Fig. 1a). SIRT7 was then stably knocked down in SIRT7 highly expressed MCF-7 cells, and the alteration in the levels of histone lysine crotonylation, succinylation and malonylation was assessed by an integrated approach of stable isotope labelling by amino acids in cell culture (SILAC) and mass spectrometry-based quantitative proteomics (Fig. 1b). Triplicate experiments showed that knockdown (KD) of SIRT7 did not result in evident changes in the level of histone lysine malonylation, and that the level of histone crotonylation rather decreased on SIRT7 depletion (Fig. 1c), suggesting that these histone marks might not be targeted directly by SIRT7. However, highly reproducible increases in the levels of succinylation of H2BK46, H2BK108, H4K31 and H4K77, especially H3K122, were detected on SIRT7 depletion (Fig. 1c), suggesting that SIRT7 is functionally associated with the regulation of histone succinylation.

To strengthen the functional connection between SIRT7 and histone succinylation, we generated three SIRT7 mutants: SIRT7H187Y, substitution of the highly conserved histidine residue (His187) in the predicted catalytic domain with tyrosine11; SIRT7S111A, substitution of the highly conserved serine residue (Ser111) in the NAD -binding pocket with alanine31,32; and a double mutant SIRT7H187Y/S111A (SIRT7DM). Wild-type SIRT7 and its mutants were then expressed in HEK293T cells. Histone succinylation was analysed by western blot with antibodies against pan-lysine succinylation. The specicity of these antibodies were veried by dot blot (Supplementary Fig. 1). Overexpression of either wild-type SIRT7 or SIRT7S111A was associated with a decrease in histone H3 lysine pan-succinylation, whereas overexpression of SIRT7H187Y, SIRT7DM or SIRT6 did not result in evident changes in histone H3 pan-lysine succinylation (Fig. 1d and Supplementary Fig. 2a,b). Similarly, overexpression of either wild-type SIRT7 or SIRT7S111A was associated with a decrease in H3K18ac, whereas overexpression of SIRT7H187Y, SIRT7DM or SIRT6 did not result in detectable changes in this modication (Fig. 1d and Supplementary Fig. 2a,b). In addition, KD of SIRT7 led to a more than twofold increase in the average uorescent intensity of lysine succinylation (Fig. 1e) while overexpression of green uorescent protein (GFP)-SIRT7 resulted in a decrease in pan-lysine succinylation (Supplementary Fig. 2c). Together, these experiments indicate that SIRT7 regulates histone succinylation in a catalytic activity-dependent manner, supporting a notion that SIRT7 functions as a potential histone desuccinylase.

Since succinylation is a newly identied histone modication and there are no commercially available antibodies against this

2 NATURE COMMUNICATIONS | 7:12235 | DOI: 10.1038/ncomms12235 | http://www.nature.com/naturecommunications

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NATURE COMMUNICATIONS | DOI: 10.1038/ncomms12235 ARTICLE

Ksucc Kcro Kmal

HEK293T

Rep 1

Rep 2

Rep 3

Rep 1

Rep 2

Rep 3

Rep 1

Rep 2

Rep 3

SY5Y

HepG2

HeLa

MCF-7

MDA-MB-231

Log2(control/ knockdown)

Not identified or quantifiable

A375

K36 K95 K101 K115

K5 K20 K34 K46 K85 K108 K116 K120

K18 K14 K23 K56 K64 K79 K122

K12 K31 K77 K79 K91

kDa

U2OS

SIRT7

Tubulin

H2A

H2B

MCF-7 shControl

13C-Lysine ( )

Histone extraction Trypsin digestion

MCF-7 shSIRT7-1

12C-Lysine ( )

Mix peptide 1:1

Immunoprecipitation

Ksucc

1.2

Fold of change

(control/knockdown)

0.8

0.6

Identify and quantify by LCMS/MS

Intensity

0.4

H2BK116

H3K79

H2BK120

m/z

Database search

H2AK95

H4K91

H3K56

H2BK46

H2BK108

H3K122

H4K31

H4K77

Lysine succinylation

Lysine crotonylation

Lysine malonylation

Other modifications

Pan anti-succinyllysine antibody conjugated agarose beads

Pan anti-malonyllysine antibody conjugated agarose beads

Pan anti-crotonyllysine antibody conjugated agarose beads

One Two Three

shControl

shSIRT7-1

Tubulin

kDa

SIRT7

Vector

SIRT7wt

SIRT7S111A

SIRT7H187Y

Vector

SIRT7DM

SIRT6

kDa

Pan-succ DAPI Merge

6,000

1.0

0.5

0.3

0.6

0.4

1.2

0.9

1.3

0.7

1.0

0.9

1.0

Pan-succ(H3)

H3K18ac

FLAG

Tubulin

shControl shSIRT7-1

5,000

Intensity of pan-succ

4,000

3,000

2,000

1,000

shControl

shSIRT7-1

Figure 1 | SIRT7 regulates histone succinylation. (a) Western blotting analysis of SIRT7 expression in different cell lines. (b) The workow of the integrated SILAC labelling, afnity enrichment and mass spectrometry-based quantitative proteomics to quantify dynamic changes of histone lysine crotonylation (Kcro), succinylation (Ksucc) and malonylation (Kmal) in control or SIRT7-depleted MCF-7 cells. (c) Heatmap of the changes in the levels of Kcro, Ksucc and Kmal of histones detected by SILAC labelling and mass spectrometry-based quantitative proteomics. All experiments were carried out in triplicate and the results are presented as base 2 logarithmic value of the ratio of control/KD. Results of detectable succinylation in one, two or three replicates are also presented as histogram. The efciency of SIRT7 KD in MCF-7 cells was monitored by western blotting. (d) HEK293T cells were transfected with wild-type SIRT7, SIRT7 mutants or SIRT6. Histones were extracted and pan-succinylation of H3 and H3K18ac were analysed by western blotting. The bands were quantied with ImageJ software. The numbers indicate the relative levels of the indicated modications. Whole-cell lysate was prepared for monitoring the efciency of overexpression of SIRT7 and SIRT6 by western blotting. (e) High-throughput microscopic analysis of the mean relative uorescence intensity of pan-succinylation in control or SIRT7-depleted MCF-7 cells. Scale bar, 10 mm. Each bar represents the means.d. for triplicate experiments. **Po0.01 (two-tailed unpaired Students t-test).

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ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms12235

modication yet, we thus generated polyclonal antibodies against two succinylation modications, H3K122 succinylation (H3K122succ) and H2BK120 succinylation (H2BK120succ), based on our observation that SIRT7 KD resulted in the most evident changes in the levels of H3K122succ and H2BK120succ in the SILAC/liquid chromatography coupled tandem mass spectrometry (LCMS/MS) experiments. The specicity of these antibodies were veried by peptide competition assays using synthesized peptides (Fig. 2a and Supplementary Fig. 3ac). In addition, FLAG-tagged wild-type H3 (FLAG-H3wt) or lysine 122-mutated and succinylation-resistant33 H3 mutant

(FLAG-H3K122R) were also used to validate the specicity of the antibodies against H3K122succ (Fig. 2a). Overexpression of SIRT7 led to a decrease in H3K122succ level in HEK293T cells and KD/knockout of SIRT7 resulted in an increase in H3K122succ level in MCF-7, HCT116, HeLa or U2OS cells, whereas the levels of H2BK120succ and H3K122ac showed no obvious changes in these cells, regardless of overexpression or KD or knockout of SIRT7 (Fig. 2b and Supplementary Fig. 4ac). Meanwhile, overexpression of SIRT6 in HEK293T cells or KD of SIRT6 in MCF-7 cells did not result in evident changes in H3K122succ and H2BK120succ (Fig. 2b and Supplementary

HEK293T

MCF-7

Vector

FLAG

SIRT7

shControl

shSIRT7-1

kDa

H3K122succ

H3K122succ peptides

+H3K122succ

1.0

0.2

1.2

1.0

1.8

1.1

0.9

H3K122succ

FLAG

H3K122succ

SIRT7

H3 H2A/H2B

Histones from U2OS cells

kDa

15 H3K122ac

H3K122ac

CBB

H2BK120succ

Tubulin

H2BK120succ

Tubulin

H3wt

H3K122R

H3wt

H3K122R

H3wt

kDa

H3K122succ

HEK293T

MCF-7

H3K122succ

peptides

+H3K122succ

H3K122 control

peptides

+H3K122succ

Vector

FLAG

SIRT6

shControl

shSIRT6

1.0

1.1

0.9

kDa

H3K122succ

FLAG

1.0

0.9

H3K122succ

SIRT6

H2BK120succ

Tubulin

H2BK120succ

H2BK120succ peptides +H2BK120succ

H3 H2A/H2B

Histones from U2OS cells

kDa

15 CBB

Tubulin

+ + +

3.4

TSA

Sodium butyrate

NAM

kDa

1.0

1.8

7.5

1.4

1.8

3.4

1.0

H3K122succ

H3K18ac

Pan-ac (H3)

1.0

2.0

1.4

Figure 2 | SIRT7 regulates histone H3K122 succinylation in vivo. (a) Conrmation of the specicity of H3K122succ (upper) or H2BK120succ (lower) antibodies by peptide competition experiments. Different amounts of soluble histones extracted from U2OS cells were resolved on SDSpolyacrylamide gel electrophoresis (SDSPAGE) gels, probed with anti-H3K122succ or anti-H2BK120succ with or without excessive H3K122succ or H2BK120succ peptides. The extracted histones were also resolved on SDSPAGE and stained with Coomassie brilliant blue (CBB). FLAG-H3wt or FLAG-H3K122R was puried from U2OS cells, resolved on SDSPAGE gels and probed with the anti-H3K122succ antibody alone or anti-H3K122succ antibody pre-adsorbed with H3K122succ peptides or H3K122 control peptides, with H3 analysis as an internal control (middle). (b) SIRT7 or SIRT6 was overexpressed in HEK293T cells or knocked down in MCF-7 cells. Histones were extracted and the succinylation and acetylation were analysed by western blotting with the indicated antibodies. The efciency of overexpression or KD of SIRT7 and SIRT6 was monitored by western blotting of whole-cell lysate, with corresponding antibodies. (c) MCF-7 cells were treated with 10 mM trichostatin A (TSA), 4 mM of sodium butyrate or 10 mM NAM. Twenty four hours later, cells were collected and soluble histones were prepared and subjected to western blotting with antibodies as indicated.

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NATURE COMMUNICATIONS | DOI: 10.1038/ncomms12235 ARTICLE

Fig. 4a). As stated above, the sirtuin proteins utilize NAD as a cofactor for their catalytic activity. To further substantiate the role of SIRT7 in histone desuccinylation, MCF-7 cells were treated with the general sirtuin inhibitor nicotinamide (NAM), or the HDAC inhibitors trichostatin A or sodium butyrate. Although H3K122succ levels increased slightly following trichostatin A and sodium butyrate treatment, NAM treatment was associated with a marked increase in H3K122succ level (Fig. 2c and Supplementary Fig. 4d). Collectively, these results suggested that SIRT7 is a NAD-dependent H3K122succ desuccinylase.

To further support this proposition, FLAG-SIRT7wt, FLAG-SIRT7H187Y, FLAG-SIRT5 or FLAG-SIRT6 were expressed in and puried from HEK293T cells (Fig. 3a) for in vitro desuccinylation assays. Incubation of H3K122succ peptides with these proteins revealed that the level of H3K122succ signicantly decreased when FLAG-SIRT7wt and NAD was included (Fig. 3b). The level of H3K122succ did not change when FLAG-SIRT7H187Y was used (Fig. 3b). Notably,

SIRT5 and SIRT6 also showed more or less H3K122succ desuccinylase activity (Fig. 3b). In vitro desuccinylation assays were also performed using calf thymus histones as substrates. Incubation of calf thymus histones with FLAG-SIRT7wt resulted in an overt decrease in the level of H3K122succ in a dose-dependent manner, an effect could be abolished by NAM, whereas no obvious changes were observed in the levels of H2BK120succ and H3K122ac (Fig. 3c and Supplementary Fig. 5a). Incubation of calf thymus histones with FLAG-SIRT7H187Y, FLAG-SIRT5 or FLAG-SIRT6 did not result in evident changes in the levels of H3K122succ, H3K122ac and H2BK120succ (Fig. 3c and Supplementary Fig. 5a). The discrepancy concerning H3K122succ desuccinylation by SIRT5 and SIRT6 on H3K122succ peptides versus calf thymus histones is unknown but could be possibly due to the overall structural difference between the synthesized peptides and the natural calf thymus histones. Mass spectrometric analysis also showed that SIRT7 exhibited robust H3K122succ desuccinylase activity while had no effect on H2BK120succ, H3K122ac and H3K18ac (Fig. 3df). In vitro desuccinylation assays were also performed with mononucleosomes isolated from HeLa cells, consistent with the results obtained with calf thymus histones, incubation of mononucleosomes with FLAG-SIRT7wt resulted in a marked and NAD-dependent decrease in the level of H3K122succ, an effect that could be abolished by NAM, whereas FLAG-SIRT7H187Y had no evident effect on this modication (Fig. 3g and Supplementary Fig. 5b). In addition, our data indicate that SIRT7 showed no substrate preference between nucleosomes and calf thymus histones (Supplementary Fig. 5c). Together, these results further demonstrated that SIRT7 is a NAD -dependent

H3K122succ desuccinylase.

SIRT7 is transiently recruited to DNA-damage sites. To explore the biological signicance of SIRT7-mediated histone desuccinylation, we next used immunopurication and mass spectrometry to identify proteins that are potentially associated with SIRT7 in vivo. The results showed that SIRT7 could be co-puried with a number of proteins, including DNA-PKcs, RAD50, PARP1, Ku80 and Ku70, that are known to be involved in DNA repair34, as well as proteins implicated in other cellular processes such as chromatin remodelling and ribosomal biogenesis (Fig. 4a).

The co-purication of DNA repair proteins with SIRT7 suggests that SIRT7 may play a role in DNA-damage response. In this regard, it is interesting to note that it has been reported that Sirt7-decient primary cardiomyocytes exhibit an increase in basal apoptosis and a signicantly diminished resistance to

oxidative and genotoxic stress12, and that SIRT7 was shown to promote cellular survival following genomic stress13. In addition, it was noted that K122Q and K122A mutants of H3K122 were much more sensitive to DNA-damaging reagents than wild type35. To test the hypothesis that SIRT7 is involved in DNA repair and in the maintenance of genome integrity, ultraviolet laser microirradiation system was utilized to generate localized DNA damage36 in GFP-SIRT7-expressing human U2OS cells. Real-time imaging of living cells showed accumulation of GFP-SIRT7 at the sites of DNA damage immediately after microirradiation. The uorescence intensied quickly, reaching to maximum at about 45 min, and attenuated thereafter, receding to pre-damage level at about 15 min (Fig. 4b), suggesting that the recruitment of SIRT7 to DNA-damage sites is a transient process. Notably, the accumulation of SIRT7 at DSBs represented only a fraction of the nuclear pool of this protein, the bulk of which remained concentrated in the nucleolus (Fig. 4b). Time-lapse analysis revealed that the accumulation of GFP-SIRT7 at the DSB sites reached half-maximum within 1 min after microirradiation (Fig. 4b). Collectively, these results indicate that SIRT7 is recruited to DNA-damage sites rapidly and transiently, suggesting that SIRT7 might function at an early and priming stage of DNA-damage response.

To gain a mechanistic insight into the rapid and transient recruitment of SIRT7 to DNA damage, we next investigated the functional relationship between SIRT7 and the two important factors involved in the early steps of DNA-damage response: PARP1 and ATM37, especially PARP1 was identied to be co-puried with SIRT7 (Fig. 4a). To this end, U2OS cells were pre-treated with PARP1 inhibitor PJ-34 (ref. 38), or ATM inhibitor KU-55933 (ref. 39), followed by laser microirradiation and immunostaining. Immunouorescent microscopy showed that, consistent with GFP-SIRT7, endogenous SIRT7 in U2OS cells without pre-treatment was efciently recruited to the sites of laser-induced DNA breaks 5 min after laser microirradiation, as marked by gH2AX staining, whereas chemical inhibition of PARP1 enzyme, but not ATM, resulted in a complete abrogation of SIRT7 accumulation at laser-induced damage sites (Fig. 4c). Consistently, KD of PARP1 also resulted in a complete abrogation of SIRT7 accumulation at laser-induced damage sites (Supplementary Fig. 6a,b). Co-immunoprecipitation experiments showed that PARP1 was efciently co-immuno-precipitated with SIRT7, and, remarkably, the interaction was intensied when cells were exposed to ionizing radiation (IR) (Fig. 4d). These results suggest that the recruitment of SIRT7 to DSB sites is dependent on PARP1.

SIRT7-catalyzed H3K122 desuccinylation in DSB repair. In order to explore the functional signicance of the recruitment of SIRT7 to DNA-damage sites, we examined the effect of SIRT7 on the repair efciency of two major DSB repair pathways, non-homologous end joining (NHEJ) and homologous recombination (HR), using EJ5-GFP-HEK293 and DR-GFPU2OS systems, respectively, as we described previously38. For NHEJ repair, depletion of SIRT7 expression was associated with a dramatic reduction of the relative percentage of GFP-positive cells by about 90%, which was comparable to the effect of depletion of Ku80, an essential component of NHEJ repair34 (Fig. 5a and Supplementary Fig. 7a). The KD efciency of SIRT7 and Ku80 were monitored by western blotting (Fig. 5a). Meanwhile, the effect of SIRT7 on HR repair was evaluated in U2OS cells using the DR-GFP system. The results showed that depletion of SIRT7 resulted in a marked decrease in the percentage of GFP-positive cells, an effect comparable to that of KD of BRCA1, a key regulator of HR repair pathway34 (Fig. 5b and Supplementary

NATURE COMMUNICATIONS | 7:12235 | DOI: 10.1038/ncomms12235 | http://www.nature.com/naturecommunications

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ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms12235

FLAG-SIRT5

FLAG-SIRT6

FLAG-SIRT7wt

FLAG-

SIRT7H187Y

SIRT7wt

SIRT7H187Y

SIRT5

SIRT6

H3K122 succinylation

NAD+

140

100

2.0E5

1.5E5

1.0E5

0.5E5

4.0E4

2.0E4

25 ng

16 ng

8 ng

25 ng

16 ng

8 ng

H3K122succ

H3K122 succinyl peptide

Control

SIRT7

Abundance Abundance Relative abundance

1 0.41 1 0.89 1 0.57 1 0.73 1 0.44 1 0.99 1 0.70 1 0.74 1 0.37 1 1.07 1 0.59 1 0.50

21 22

Calf thymus histones NAD+

FLAG-SIRT7wt (g)

NAM

+ + + + + + + + + +0.25 0.25 1 2.5 2.5

Calf thymus histones NAD+

FLAG-SIRT7H187Y (g)

NAM

+ + + + + + + + + +0.25 0.25 1 2.5 2.5

0.5

1.0

0.7

0.3

1.2

0.9

0.2

1.1

0.8

0.7

1.1

0.9

0.8

0.9

1.0

0.9

1.2

1.0

0.9

1.0

1.1

kDa

H3K122succ

SIRT7

Retention time (min)

15 H3K122ac

H3K122succ

SIRT7

H2BK120succ

1.0

V T I M P D IQ L A R

K(succ)

H3K122ac

1.0

100

200

60 b3-H2O y8++

Calf thymus histones NAD+

FLAG-SIRT5 (g)

NAM

+ + + + + + + + + +0.25 0.25 1 2.5 2.5

Calf thymus histones NAD+

FLAG-SIRT6 (g)

NAM

+ + + + + + + + + +0.25 0.25 1 2.5 2.5

228.10779

y10

y5 y6 y7

1.0

1.2

0.9

1.2

1.1

1.3

1.2

kDa

400

600

800

1,000

1,200

1,400

H3K122succ

FLAG

1.3

H3K122succ

FLAG

1.0

0.9

1.2

0.9

0.8

1.0

0.8

kDa

m/z

H2BK120succ

Modification

Peak area ratio (SIRT7/control)

H3K122succ

0.09

H2BK120succ

0.88

Nucleosomes NAD+

FLAG-SIRT7wt (g)

NAM

+ + + + + + + +0.5 0.5 5 5

Nucleosomes

NAD+

FLAG-SIRT7H187Y (g)

NAM

+ + + +

+ + +

0.5 0.5 5 5

H3K56succ

0.01

H3K79succ

1.0

0.01

1.0

0.8

1.1

0.2

0.8

0.9

0.7

0.8

1.1

kDa

1.1

1.3

1.2

1.5

1.2

kDa

H3K122succ

SIRT7

H3K122succ

SIRT7

H3K122ac

0.92

H3K18ac

0.99

H2BK120succ

H3K122ac

H2BK120succ

H3K122ac

Figure 3 | SIRT7 catalyses histone H3K122 desuccinylation in vitro. (a) FLAG-SIRT5, FLAG-SIRT6, FLAG-SIRT7wt or FLAG-SIRT7H187Y was expressed in and puried with anti-FLAG M2 afnity gel from HEK293T cells and stained with Coomassie brilliant blue. (b) In vitro desuccinylation assays with synthesized H3K122succ peptides. Two micrograms of puried FLAG-SIRT7wt, FLAG-SIRT7H187Y, FLAG-SIRT5 or FLAG-SIRT6 were incubated with 500 ng H3K122succ peptides in the presence or absence of 1.0 mM NAD. The reaction mixtures containing 8, 16 or 25 ng peptides were boiled and subjected to dot blot analysis with anti-H3K122succ or anti-H3. The dots were quantied by densitometry with ImageJ software. The numbers indicate the relative levels of the indicated modications. (c) In vitro desuccinylation assays with calf thymus histones. Different amounts of puried FLAG-SIRT7wt, FLAG-SIRT7H187Y, FLAG-SIRT5wt or FLAG-SIRT6wt were incubated with 1 mg calf thymus histones in the presence of 1.0 mM NAD and/or 10 mM NAM.

The reaction mixtures were boiled and analysed by western blotting with the indicated antibodies. (d) The base peaks of H3K122succ in control and SIRT7-treated calf thymus histones. The peak areas were used for the quantication of H3K122succ in the two samples. (e) The MS/MS spectra for the identication of H3K122succ. b and y ions indicate peptide backbone fragment ions containing the N and C terminal, respectively. indicates doubly

charged ions. (f) The quantication ratios of several succinylation and acetylation sites in histones by comparing the peak areas in SIRT7-treated and control samples. (g) In vitro desuccinylation assays with mononucleosomes. Different amounts of puried FLAG-SIRT7wt or FLAG-SIRT7H187Y were incubated with 1 mg HeLa cell-derived mononucleosomes in the presence or absence of 1.0 mM NAD and/or 10 mM NAM. The reaction mixture was analysed by western blotting with the indicated antibodies.

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NATURE COMMUNICATIONS | DOI: 10.1038/ncomms12235 ARTICLE

Marker

Vector

FLAG

SIRT7

SIRT7 Merge

H2AX

KDa

DNA-PKcs

Nucleolar pre-ribosomal-associated protein 1

60S ribosomal protein L5; 40S ribosomal protein S6

CHD7CHD4 URB2

DMSO

Myosin-10

RBBP7 RBBP4

225

115

35 30 25

15 10

RAD50 PARP1 Ku80, GNL2

Ku70

SIRT7

PJ-34 KU-55933

d IgG IR

IP: SIRT7

ATP-dependent RNA helicase DDX3X; GTPBP4, MTA2

hnRNPK

DMSO

PJ-34

KU-55933

kDa

140

GFP-SIRT7

PARP1

SIRT7

Tubulin

0 min 1 min 5 min 10 min

Time after microirradiation

kDa

100

140

Fluorescence instensity

IPInput

PARP1

SIRT7

PARP1

Tubulin

140

T1/2=~1 min

H2AX

0 2 4 6 8 10 12

Time (min)

GFP-SIRT7

Figure 4 | SIRT7 is transiently recruited to DNA-damage sites in a PARP1-dependent manner. (a) Whole-cell extracts from HEK293T cells expressing vector or FLAG-SIRT7wt were subjected to afnity purication with anti-FLAG M2 afnity gel. The puried protein complex was resolved on SDSpolyacrylamide gel electrophoresis and silver stained. The bands were retrieved and analysed by mass spectrometry. (b) U2OS cells stably expressing GFP-SIRT7 were subjected to laser microirradiation using micropoint system and analysed for the accumulation of GFP-SIRT7 in DSBs by uorescent microscopy (upper). Scale bar, 10 mm. The real-time recruitment of GFP-SIRT7 was also analysed in 30 independent cells (lower). Error bars indicate means.e.m. (c) U2OS cells were pre-treated with PARP1 inhibitor PJ-34 or ATM inhibitor KU-55933 for 1 h and subjected to laser microirradiation and immunouorescent analysis of SIRT7 and gH2AX at 5 min after microirradiation (upper). Scale bar, 10 mm. The expression of SIRT7 was analysed by western blotting (lower). (d) U2OS cells were exposed to 6 Gy of IR. Whole-cell lysate was immunoprecipitated with antibodies against SIRT7 followed by immunoblotting with the indicated antibodies.

Fig. 7b). Together, these results indicate that SIRT7 is required for efcient repair of DSBs.

To further investigate whether the catalytic activity of SIRT7 is required for efcient DSB repair, rescue experiments were performed by using SIRT7 siRNA-1 resistant SIRT7 (rSIRT7). The results revealed that rSIRT7wt was able to rescue the decreased NHEJ repair efciency induced by depletion of endogenous SIRT7, whereas rSIRT7H187Y was not (Fig. 5c). Similarly, rSIRT7wt expression was associated with a restoration of HR repair in SIRT7-depleted cells, whereas rSIRT7H187Y was not (Fig. 5c). Furthermore, quantitative chromatin immunoprecipitation (qChIP) assays in DR-GFP-U2OS cells showed that both rSIRT7wt and rSIRT7H187Y were effectively recruited to break sites, with rSIRT7H187Y even exhibiting a stronger binding capacity than rSIRT7wt (Fig. 5d), excluding the possibility that failing to rescue the decreased NHEJ and HR repair efciency by rSIRT7H187Y resulted from decient recruitment of rSIRT7H187Y to DSB sites. The efciency of KD of SIRT7 and overexpression of I-SceI, rSIRT7wt, or rSIRT7H187Y was monitored by western blotting (Fig. 5e). The specicity of SIRT7 KD by siRNA was also validated (Fig. 5f). Collectively, these results suggest that the catalytic activity of SIRT7 is required for its function in DNA repair.

To investigate whether SIRT7-catalyzed H3K122succ desuccinylation was involved in DNA-damage response, MCF-7 cells were treated with DNA damaging reagents etoposide (VP16) or camptothecin (CPT), histone succinylation and acetylation levels were measured by western blotting. The results showed that treatment with either VP16 or CPT resulted in evident decrease in the levels of H3K122succ and H3 pan-succinylation, whereas the levels of H3K122ac, H2BK120succ, H3K18ac and H3 pan-acetylation showed no obvious changes (Fig. 6a). However, when SIRT7 was knocked down in MCF-7 cells, treatment with CPT was no longer associated with decreases in H3K122succ level (Fig. 6b and Supplementary Fig. 8a). Similar result was also observed in U2OS cells (Fig. 6b and Supplementary Fig. 8b). Furthermore, U2OS cells were treated with IR and histones were extracted at different time points after IR for western blotting analysis of H2K122succ. We found that, compared with that at 0 h, the level of H3K122succ was signicantly decreased at 1 h after IR, and gradually recovered at 4 h after IR, meanwhile no obvious changes were detected in the levels of H3K122ac and H2BK120succ (Fig. 6c and Supplementary Fig. 8c). However, when SIRT7 was depleted, treatment with IR no longer resulted in decrease in H3K122succ level, the levels of H3K122ac and H2BK120succ also had no obvious changes (Fig. 6c and Supplementary Fig. 8c). These results suggest that

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NHEJ

siSIRT7-2 siControl

siControl

GFP+ percentage

12 Control

I-SceI

** **

Control I-SceI

** **

siControl

siSIRT7-1

siSIRT7-2

siKu80-1

siBRCA1-1

siKu80-2

kDa

GFP+ percentage

I-SceI I-SceI

Ku80

SIRT7

Tubulin

siControl

siSIRT7-1

siSIRT7-2

siControl

siKu80-1

0 siKu80-2

Control I-SceI

** **

GFP+ percentage

Control I-SceI

** **

siBRCA1-2

kDa

260

BRCA1

SIRT7

Tubulin

siControl

siSIRT7-1

siControl

siBRCA1-1

siBRCA1-2

NHEJ

** **

GFP+ percentage

** **

GFP+ percentage

0 + + + + + + + + + + + +

Control siRNA SIRT7 siRNA-1 rSIRT7wt rSIRT7H187Y pcDNA3.1

+ + + + + + + + + + + +

+ + + + + + + + + + + + + + + + + +

Control siRNA SIRT7 siRNA-1 rSIRT7wt rSIRT7H187Y pcDNA3.1

Relative enrichment

** **

Relative enrichment

IgG SIRT7

Control siRNA SIRT7 siRNA-1 rSIRT7wt rSIRT7H187Y pcDNA3.1

I-SceI

+ + + + + + + + + + + +

20 30 40 50 60

Time after transfection (h)

IgG SIRT7

kDa

70 FLAG

Tubulin

Control siRNA SIRT7siRNA-1 FLAG-SIRT7 FLAG-rSIRT7wt FLAG-rSIRT7H187Y HA-I-SceI

kDa

HA-I-SceI

+ + + + + + + + + + + +

SIRT7

Tubulin

Control siRNA SIRT7siRNA -1 rSIRT7wt rSIRT7H187Y pcDNA3.1

I-SceI

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structure of U2OS cells stably transfected with these constructs was analysed by micrococcal nuclease (MNase) sensitivity assays41.We found that compared with stable expression of H3wt, stable expression of H3K122E resulted in an increase, albeit moderately, in MNase accessibility in chromatin, whereas stable expression of H3K122R led to a decrease in MNase sensitivity (Fig. 7a and Supplementary Fig. 9a,b), suggesting that H3K122 succinylation is associated with an increased chromatin accessibility, whereas desuccinylation of H3K122succ is linked to a condensed chromatin state.

HistoneDNA interactions are very stable; histones are only released from chromatin by NaCl concentrations in excess of1.5 M (refs 42,43). To substantiate the relationship between H3K122succ and chromatin compaction, nucleosome stability assay44 was performed to investigate the difference of histoneDNA interactions in H3wt, H3K122E and H3K122R stably expressing U2OS cells. Compared with H3wt, the NaCl solubility of histone H3 in H3K122E stably expressing cells increased markedly, while the NaCl solubility of histone H3 in H3K122R cells decreased signicantly (Fig. 7b). It suggests that succinylation of H3K122 was associated with a reduction of histoneDNA interactions while desuccinylation of H3K122succ stabilizes the nucleosomes, consistent with our hypothesis that H3K122 succinylation is associated with an increased chromatin accessibility, whereas desuccinylation of H3K122succ is linked to a condensed chromatin state. In addition, cells overexpressing GFP-SIRT7 had smaller nuclear size compared with cells that expressed GFP only (Supplementary Fig. 9c). In agreement with this observation, detailed quantication of proliferation patterns in shControl and shSIRT7 cells demonstrated that although SIRT7 KD group had a similar number of cells in S phase to that in control group, the number of cells in late S phase on SIRT7 KD decreased (Supplementary Fig. 9d). These observations altogether point to a role of SIRT7-meidated H3K122succ desuccinylation in chromatin compaction.

To strengthen this argument, we performed MNase sensitivity assays in control or SIRT7-depleted U2OS cells under treatment of IR. Knockdown of SIRT7 resulted in an, albeit moderate, increase in MNase sensitivity of chromatin, and the effect was signicantly augmented following IR treatment (Fig. 7c and Supplementary Fig. 9e,f). These data suggest that loss of function of SIRT7 is associated with chromatin decompaction thus compromises subsequent chromatin relaxation, consistent with a previous report that decompaction of chromatin by overexpression or tethering H3K4 methyltransferase ASH2 dampens DNA-damage response and alters local chromatin dynamics45. We also performed nucleosome stability assays in control or SIRT7-depleted U2OS cells under IR treatment.The results showed that DNA damage increased the NaCl

Figure 5 | The catalytic activity of SIRT7 is required for efcient DSB repair. (a) NHEJ efciency was determined by FACS in SIRT7- or Ku80-decient EJ5-HEK293 cells. Each bar represents the means.d. for triplicate experiments. KD efciency of SIRT7 and Ku80 was examined by western blotting. (b) HR efciency was determined by FACS in SIRT7- or BRCA1-decient DR-GFP-U2OS cells. Each bar represents the means.d. for triplicate experiments. KD efciency of SIRT7 and BRCA1 was examined by western blotting. (c) Rescue experiments for NHEJ or HR deciency induced by SIRT7 depletion. EJ5-GFP-HEK293 cells (left) or DR-GFP-U2OS cells (right) stably expressing SIRT7 siRNA-1-resistant SIRT7wt (rSIRT7wt) or siRNA-1-resistant SIRT7H187Y (rSIRT7H187Y) were transfected with control siRNA or siSIRT7-1 as indicated. Twenty four hours later, the cells were transfected with pcDNA3.1 vector or I-SceI for 48 h, and collected and analysed by FACS. Each bar represents the means.d. for triplicate experiments. (d) SIRT7 occupancy at chromatin anking DSB generated by endonuclease I-SceI. DR-GFP-U2OS cells transfected with I-SceI were collected at different time points and subjected to ChIP assay, with antibodies against SIRT7. The nal DNA extractions were amplied by quantitative real-time PCR using primer that covers the DNA sequences anking the I-SceI site. Each bar represents the means.d. for triplicate experiments (left). Control or DR-GFP-U2OS cells stably expressing rSIRT7wt or rSIRT7H187Y were transfected with control siRNA or siSIRT7-1 as indicated. Twenty four hours later, the cells were transfected with pcDNA3.1 vector or I-SceI for 40 h and subjected to qChIP analysis with antibodies against SIRT7. Each bar represents the means.d. for triplicate experiments (right). (e) The efciency of SIRT7 KD and overexpression of I-SceI, rSIRT7wt or rSIRT7H187Y in DR-GFP-U2OS cells. Tubulin was analysed as an internal control. (f) The KD specicity of SIRT7 siRNA-1 for FLAG-tagged wild-type SIRT7, siRNA-1-resistant rSIRT7wt or rSIRT7H187Y in DR-GFP-U2OS cells. The haemagglutinin (HA)-tagged I-SceI and tubulin were used as loading controls. **Po0.01 (two-tailed unpaired Students t-test).

DNA-damage reagent-induced decrease of H3K122succ was functionally linked to SIRT7.

We then performed laser microirradiation and immunouorescent assays using anti-H3K122succ to address whether the decrease of H3K122succ is SIRT7- and PARP1-dependent and specic to the DSB sites. In control U2OS cells, H3K122succ level at DSB sites decreased 5 min after microirradiation, while in SIRT7-depleted U2OS cells, H3K122succ level at DSB sites did not change (Fig. 6d,f). We then analysed H3K122succ level at DSB sites in U2OS cells transfected with siControl or siPARP1. The results showed that in siControl cells H3K122succ level at DSB sites decreased 5 min after microirradiation, while in siPARP1 cells H3K122succ level at DSB sites had no detectable change (Fig. 6e,f). It is worthy of noting that ultraviolet laser microirradiation and ionizing radiation are different DNA insults. Thus, these stimuli-evoked DNA-damage responses might be different, at least in terms of time course and factor recruitment, which could potentially contribute to the difference of the dynamic changes of H3K122succ level observed in these microirridiation experiments versus that in IR experiments in Fig. 6c. These results suggest that SIRT7-mediated H3K122succ desuccinylation after microirradiation is PARP1-dependent and specic to DSB sites.qChIP assays were then performed and the level of H3K122succ at DNA-damage sites was measured using HR repair system in DR-GFP-U2OS cells. The results showed that transfection of the cells with I-SceI led to a marked decrease in the level of H3K122succ around the break sites (Fig. 6g). However, when SIRT7 was knocked down, I-SceI transfection no longer resulted in a decrease in the level of H3K122succ at the break sites (Fig. 6g), and ectopic expression of rSIRT7wt, but not rSIRT7H187Y, could fully restore the downregulated level of H3K122succ (Fig. 6g). These results further indicate that SIRT7 is recruited to DSB sites and catalysed H3K122succ desuccinylation therein.

H3K122 desuccinylation by SIRT7 in chromatin condensation. The lateral surface site H3K122 has been implicated in DNA-damage repair35, and H3K122ac has been reported to contribute to a relaxed chromatin state associated with active transcription40. In comparison, succinylation is a more acidic modication, which could change the charge on lysine from 1 to 1 under

physiological conditions30. Therefore, we speculated that succinylation of H3K122 is associated with chromatin relaxation while desuccinylation of H3K122succ linked to a more condensed chromatin state. To test this hypothesis, we generated FLAG-tagged constructs of wild-type H3 (H3wt) or H3 mutants H3K122E, which mimic the negative charge state of the succinyl group33, and non-succinylated H3K122R (ref. 33). The chromatin

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ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms12235

solubility of histones gH2AX, H2AX and H3 within damaged chromatin (Fig. 7d). Importantly, when SIRT7 was depleted, the DNA damage-dependent increase in NaCl solubility of histones was enhanced (Fig. 7d), suggesting that SIRT7-mediated H3K122succ desuccinylation promotes the stability of histone DNA interactions within nucleosomes during DNA-damage response, thereby creating a condensed chromatin domain.

To further investigate the importance of H3K122succ desuccinylation in chromatin condensation thus efcient DSB repair, analysis of NHEJ repair efciency in EJ5-GFP-HEK293 cells stable expressing FLAG-H3wt, FLAG-H3K122E or FLAG-H3K122R by uorescence-activated cell sorting (FACS) showed that expression of H3K122E and H3K122R reduce NHEJ repair efciency (Fig. 7e and Supplementary Fig. 10a,b). Signicant

a b

MCF-7

DMSO

VP16

MCF-7

U2OS

shSIRT7-2

H2BK120succ

H2AX

shControl shSIRT7-1

H2AX

Tubulin

shControl shSIRT7-1

CPT

DMSO

kDa

CPT

1.0 0.1 1.0 0.5

1.0 0.8 1.0 1.0

1.0 0.1 1.0 0.3

1.0 1.0 1.0 1.1

1.0 0.8 1.0 0.8

1.0 34.2 1.0 5.0

H3K122succ

H3K122ac

1.0

0.4

0.9

1.2

1.0

0.9

0.8

kDa

1.0

0.3

0.8

0.9

1.0

1.3

0.8

1.0

2.4

1.0

0.9

H3K122succ

SIRT7

H3K122succ

SIRT7

H2BK120succ

H3K18ac

H2BK120succ

Pan-succ(H3)

Pan-ac (H3)

H3K122ac

1.0

8.2 1.5 18.8

1.0

7.3 5.9 8.0 5.5 9.6

H2AX

Tubulin

H2AX

c d

U2OS

DAPI

shControl0 1 4 8 0 1 4 8 IR/hours

shSIRT7-1

kDa

shControl

H3K122succ Merge

H2AX

1.0

0.4

0.9

1.5

1.6

1.0

1.3

1.4

0.9

1.5

1.0

1.1

1.2

0.7

0.9

1.2

0.6

1.1

1.3

H3K122succ

SIRT7

H2BK120succ

H3K122ac

shSIRT7-1

1.0 64.9 13.4 9.8 9.5 37.9 6.1 1.5

H2AX

Tubulin

H2AX

siControl

H3K122succ Merge

H2AX

siPARP1-1+2

2 Bulk Laser path

1.5

Fold of change

Relative enrichment

IgG H3K122succ

** ** **

0.5

shControl

shSIRT7-1

siControl

0 siPARP1

0 + + + + + + + + + + + +

I-SceIControl siRNA SIRT7 siRNA-1 rSIRT7wt rSIRT7H187Y pcDNA3.1

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NATURE COMMUNICATIONS | DOI: 10.1038/ncomms12235 ARTICLE

pre-rRNA synthesis by wild-type SIRT7 (ref. 47), whereas our study is the rst report on the mutagenesis study of SIRT7 desuccinylase activity for histone lysine succinylation; (2) the crystal structures of SIRT2 from Archaeoglobus fulgidus (SIR2-Af1)32 and Caenorhabditis elegans (SIRT2)31 indicate the corresponding residue of SIRT7 S111 lies in the NAD-binding pocket, which is not in direct contact with the observed NAD molecule in the structure31,32. In fact, it is buried deep in the active site of the enzyme and does not appear to be structurally important. It is possible that binding of histone succinylated lysine may lead to conformation change of the NAD-binding domain in SIRT7, and hence the position or the conformation of S111 is changed so that it is not directly involved in the desuccinylation reaction.

H3K122 is located on the lateral surface of the histone octamer closing to the dyad symmetry axis40, the regions with the strongest DNAhistone interaction within the nucleosome48. Modications on nucleosome lateral surface have the potential to facilitate nucleosomal mobilization or histone eviction, thereby modulating chromatin accessibility49,50. Moreover, succinyl group differs from acetyl moiety in that succinyl group is two carbons longer and with an additional terminal carboxyl group, potentially generating a stronger stereospecic blockage than acetylation. In addition, compared with acetylation, succinylation is a more acidic modication that disfavours ionic interactions between positively charged lysine side chains and a negatively charged chemical moiety in other molecules, such as DNAs or proteins30. It is reasonable to infer that succinylation of the globular histone residues would result in stronger disturbance for the regular nucleotide structure of chromatin than acetylation of the residues on the same site or sites outside the globular domain do, and vice versa. Therefore, succinylation of H3K122 would be potentially associated with a loose chromatin state while desuccinylation of H3K122succ could contribute to the condensation of chromatin structure. Indeed, our results showed that SIRT7-catalysed H3K122succ desuccinylation is linked to chromatin condensation and to efcient DSB repair.

It is documented that chromatin modiers, including polycomb group proteins36, HDACs51, HP1 (ref. 52), nucleosome remodelling and deacetylase complex36, H3K9me3 methyltransferase SUV39H1 (ref. 53), PRDM2 (ref. 54) and macroH2A1.1 (refs 54,55) that are generally associated with chromatin compaction are also recruited to the sites of damaged DNA, suggesting that chromatin condensation is actively involved in DNA repair process45,53,54,56. It is proposed that chromatin condensation is an integral but transient part of the DNA-damage response; condensed chromatin enhances upstream signalling thus promotes DNA repair45. Therefore, it is possible that SIRT7-mediated transient H3K122succ desuccinylation at DSB site in the early stage of DNA damage response (DDR) might promote DNA repair by enhancing upstream signalling. It is also suggested that

Figure 6 | SIRT7 desuccinylates H3K122succ at DSB sites. (a) MCF-7 cells were treated with 40 nM VP16 or 1 mM CPT for 8 h. Histones were extracted for western blotting analysis with the indicated antibodies. (b) Control or SIRT7-depleted MCF-7 cells (left) or U2OS cells (right) were treated with 1 mM CPT for 8 h followed by histone extraction and western blotting analysis with the indicated antibodies. The efciency of SIRT7 KD and DNA-damage effect induced by

CPT were monitored by western blotting of whole-cell lysate using antibodies against SIRT7 and gH2AX, respectively. (c) Control or SIRT7-depleted U2OS cells were exposed to 10 Gy of IR and collected at different time points for histone extraction and western blotting analysis with the indicated antibodies. The efciency of SIRT7 KD and IR treatment was monitored by western blotting of whole-cell lysate using antibodies against SIRT7 and gH2AX, respectively.

(d) Control or SIRT7-depleted U2OS cells were subjected to laser microirradiation and immunouorescent analysis of H3K122succ and gH2AX at 5 min after microirradiation. Scale bar, 10 mm. (e) U2OS cells transfected with control siRNA or siPARP1 were subjected to laser microirradiation and immunouorescent analysis of H3K122succ and gH2AX at 5 min after microirradiation. Scale bar, 10 mm. (f) H3K122succ levels in e,f were measured at and beside damage sites using ImageJ. At least 30 independent cells were scored. Data are represented as means.e.m. **Po0.01 (two-tailed Students t-test). (g) Control or

DR-GFP-U2OS cells stably expressing rSIRT7wt or rSIRT7H187Y were transfected with control siRNA or siSIRT7-1 as indicated. Twenty four hours later, the cells were transfected with pcDNA3.1 vector or I-SceI for 40 h and subjected to qChIP analysis with antibodies against H3K122succ. Each bar represents the means.d. for triplicate experiments. **Po0.01 (two-tailed unpaired Students t-test).

decreases in HR repair efciency were also detected in DR-GFPU2OS cells expressing H3K122E or H3K122R (Fig. 7f). These results suggest that both persistent succinylation and non-succinylation of H3K122 could impair HR and NHEJ repair efciency, suggesting that SIRT7-mediated transient H3K122succ desuccinylation is an integral component of DSB repair process.

SIRT7-catalysed H3K122 desuccinylation in cell survival. To explore the biological signicance of SIRT7-mediated H3K122succ desuccinylation in DNA repair, we investigated the effect of loss of function of SIRT7 on cell survival and apoptosis in response to DNA-damaging agents. The results showed that KD of SIRT7 in MCF-7 cells resulted in a signicant increase in cell apoptosis, and exposure cells to VP16 or CPT severely aggravated this situation (Fig. 8a). Control or SIRT7-depleted MCF-7, U2OS, HCT116, HepG2 cells or SIRT7-knockout U2OS cells were then treated with or without IR at different dosage and subjected to clonogenic survival assays. The results showed that KD or knockout of SIRT7 signicantly compromised cell survival in response to IR treatment (Fig. 8bf). Moreover, clonogenic survival assays demonstrated that U2OS cells stably expressing H3K122E or H3K122R were much more sensitive to IR than cells stably expressing H3wt (Fig. 8g). Collectively, these results suggest that SIRT7-mediated H3K122succ desuccinylation is critically implemented in DNA-damage response and cell survival.

DiscussionRecent identication of a series of new histone acylations, including succinylation9,2629 indicates the epigenetic regulatory circuit is more complicated than we thought. Identication of the writers, readers and erasers of these new histone acylations including succinylation is critically needed for the understanding of the biological signicance of these modications. Neither histone succinylase nor desuccinylase has been described as of today. Although SIRT5 has been reported to be able to regulate succinylation7, its mitochondrial subcellular localization excludes the possibility for this sirtuin protein to act on nuclear histones.

It is interesting to note that SIRT7S111A mutation shows no obvious effect on desuccinylation of H3K122succ (Fig. 1d and Supplementary Fig. 2a,b) as the corresponding mutations inactivate other members of sirtuin family32,46,47. The possible explanations for this discrepancy are discussed as follows: (1) the mutagenesis studies of others reports focused on different substrates and different enzymatic activities. For example, the mutant of S36 on yeast HST2 (homologues of the SIR2 silencing gene 2) showed signicantly reduced NAD nicotinamide exchange and deacetylase activity for [3H]-acetylated BSA32, and loss of ADP-ribosyltransferase activity in another equivalent residue mutant mSIRT6S56A (ref. 46) SIRT7S112A (equivalent to S111A in our study) would abolish the stimulation effect for

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ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms12235

U2OS

Marker

H3wt

H3K122E

H3K122R

H3K122E

0.7

% of total signal

H3wt

H3wt H3K122E H3K122R

mono

tri

kDa

1.0 2.1 0.2

0.6

5,000 3,000 2,000

1,000

750 500 250

15 70

Upper

0.5

NaCl extract

WCL

0.4

Ponceau S

Tri

0.3

Tubulin

FLAG-H3

100

Mono

0.2

8 1 0.1 kbp

c e

Time post IR (h)

8,000 5,000 3,000 2,000

1,000

750500250100 Mono

0 0.25 1 4 8

IR (10 Gy)

Marker

shControl

shSIRT7-1

25 Control I-SceI

20 ** **

GFP+ percentage

NHEJ

Upper

5 H3wt

H3K122E

H3K122R

Tri

H3wt

H3K122R

1 2 3 4 5 6 7 8 9 10

Upper

kDa

70 15

HA (HA-I-SceI)

FLAG (FLAG-H3)

-actin

HA (HA-I-SceI)

FLAG (FLAG-H3)

-Actin

200

Relative instensity

150

100

16 Control I-SceI

12 ** ** HR

Mono

shControl

shSIRT7-1

shControl

shSIRT7-1

shControl

Tri

GFP+ percentage

shSIRT7-1

shControl

shSIRT7-1

shControl

shSIRT7-1

0 h 0.25 h 1 h 4 h 8 h

4 H3wt

H3K122E

H3K122R

H3wt

H3K122E

H3K122R

kDa

shControl

U2OS

shSIRT7-1

kDa

0 0.25 1 4 8 0 0.25 1 4 8 Time post IR (h)

H2AX

1.0 112.7 105.0 77.4 31.4 6.1 145.0 160.2 109.1 39.3

1.0 3.5 3.3 1.7 0.9 1.2 3.8 3.9 2.7 1.5

1.0 2.3 1.8 1.6 1.0 0.9 2.6 2.4 2.3 1.1

H2AX

Ponceau S

NaCl extract

Figure 7 | SIRT7-catalysed H3K122succ desuccinylation is linked to chromatin condensation during DSB repair. (a) Nuclei from U2OS cells stably expressing FLAG-H3wt, FLAG-H3K122E or FLAG-H3K122R were incubated with 40 gel units of MNase for 5 min followed by DNA extraction and ethidium bromide staining (left). The band densities were quantied using ImageJ software and expressed as percentage of signal minus background of the entire line from top to the bottom. Calibrated kilobase pair (kbp) sizes are indicated (right). (b) U2OS cells stably expressing FLAG-H3wt, FLAG-H3K122E or FLAG-H3K122R were extracted in lysate buffer containing 1.5 M NaCl, salt soluble proteins were separated by SDSpolyacrylamide gel electrophoresis (SDSPAGE), and H3 was detected by western bloting. Ponceau S staining indicates loading. The efciency of overexpression of FLAG-H3, H3 mutants or total H3 was monitored by western blotting of whole-cell lysate, with corresponding antibodies. (c) Control or SIRT7-depleted U2OS cells were exposed to10 Gy of IR and collected at different time points. Nuclei were prepared and subjected to MNase assays. Mononucleosome, dinucleosome and trinucleosome are indicated (upper). The band densities were quantied using ImageJ software and the intensity values were background subtracted (lower). (d) Control or SIRT7-depleted U2OS cells were exposed to 10 Gy of IR and collected at different time points. Cells were extracted in lysate buffer containing 1.0 M NaCl, salt-soluble proteins were separated by SDSPAGE, and gH2AX, H2AX and H3 were detected by western bloting. Ponceau S staining indicated loading. (e,f) Overexpression of H3K122 mutants affected the repair efciency of NHEJ and HR. EJ5-GFP-HEK293 (e) or DR-GFP-U2OS (f) cells stably expressing FLAG-H3wt, FLAG-H3K122E or FLAG-H3K122R were transfected with I-SceI for 48 h and analysed by FACS. Each bar represents the means.d. for triplicate experiments. **Po0.01 (two-tailed unpaired Students t-test). The efciency of overexpression of FLAG-H3, H3 mutants or

HA-I-SceI was monitored by western blotting.

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Vehicle VP16 CPT

8.43%

5.75%

6.00%

7.32%

1.99%

50 shControl shSIRT7-1

20 ** **

14.44%

4.18%

32.10%

8.08%

% of apoptotic cells

shControl

FL2-HFL2-H

FL2-H

2.94%

13.80%

4.24%

shSIRT7-1

FL2-H

Vehicle

VP16

CPT

FL1-H

100

MCF-7

shControl shSIRT7-1

100

U2OS

shControl shSIRT7-1 shSIRT7-2

Survival fraction (log)

***

Survival fraction (log)

0.1

0 2 4 6

1 0 2 4 6

IR (Gy)

100

HCT116

shControl shSIRT7-1 shSIRT7-2

100

HepG2

shControl shSIRT7-1 shSIRT7-2

Survival fraction (log)

0.1

0 2 4 6

IR (Gy)

HCT116

HepG2

shControl

shSIRT7-1

shSIRT7-2

shSIRT7-1

shSIRT7-2

kDa

shControl

SIRT7

Tubulin

SIRT7

Tubulin

f g

U2OS

100

Survival fraction (log)

***

WT KO

H3wt H3K122E H3K122R

1 0 2 4 6

IR (Gy)

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for lentiviral production was subcloned with a C-terminal FLAG tag from pBOS-HA-H3.1. PITA-FLAG-H3wt was used as a template to obtain the PITA-FLAG-H3K122E and PITA-FLAG-H3K122R mutants by standard site-directed mutagenesis. All clones were conrmed by DNA sequencing.

Antibodies and reagents. The polyclonal antibodies against H3K122succ and H2BK120succ were generated by immunizing rabbits with a synthetic succinyl peptide corresponding to residues surrounding K122 of human histone H3 or K120 of human histone H2B. Antibodies were puried by protein A-conjugated agarose followed by afnity chromatography with K122 succinylated histone H3 or K120 succinylated histone H2B peptides. The sources of the other antibodies against the following proteins were as follows: FLAG (F3165), a-tubulin (clone B-5-1-2,

T6074) and b-actin (A1978) from Sigma; SIRT7 (sc-365344), Ku80 (sc-5280) and PARP1/2 (sc-7150) from Santa Cruz Biotechnology; gH2AX from Millipore (05-636) and Cell Signaling Technology (9718P); H3K122ac (ab33309), H3 (ab1791), H2AX (ab11175) and BrdU (ab8039) from Abcam; H3K18ac (PTM-114), pan-succinylation (PTM-401) and pan-acetylation (PTM-105) from PTM BioLabs; BRCA1 (22362-1-AP) from Proteintech; haemagglutinin (HA) (M180-3) from MBL; SIRT6 from Abgent (AP-6245a); and agarose beads conjugated with pan anti-succinyllysine (PTM-402), crotonyllysine (PTM-503) and malonyllysine (PTM-904) antibodies were purchased from PTM BioLabs.

VP16 (E1383), camptothecin (C9911), anti-FLAG M2 afnity gel (A2220),1 FLAG peptide (F3290), PJ-34 (P4365) and BrdU (B5002) were from Sigma.

KU-55933 (118500) and NAD (20-221) were from Millipore. NAM (N814605) was from Macklin. Protein A/G Sepharose CL-4B beads were from

Amersham Biosciences, and protease inhibitor mixture cocktail was from Roche Applied Science.

Cell culture and transfection. MCF-7, U2OS, HeLa, HepG2, HCT116 and HEK293T cells were from the American Type Culture Collection. DR-GFP-U2OS and EJ5-GFP-HEK293 cell lines were kindly provided by Dr Xingzhi Xu (Capital Normal University, Beijing). The cells were maintained in Dulbeccos modied Eagles medium supplemented with 10% fetal bovine serum (FBS). Transfections were carried out using Lipofectamine 2000 (Invitrogen) according to the manufacturers instructions. The sequences of siRNAs are given in Supplementary Table 1. SIRT7 siRNAs were synthesized by Sigma. Ku80 and BRCA1 siRNAs were synthesized by Suzhou GenePharma. siRNA oligonucleotides were transfected into cells using RNAiMAX (Invitrogen) according to the manufacturers instructions.

Lentiviral production and infection. RNAi lentivirus system was constructed using pLKO.1 according to protocols described online (http://www.addgene.org/tools/protocols/plko/#E

Web End =http://www.addgene.org/ http://www.addgene.org/tools/protocols/plko/#E

Web End =tools/protocols/plko/#E ). The sequences of short hairpin RNAs are given in Supplementary Table 2. In brief, short hairpin RNA sequences targeting human SIRT7 (TRCN0000359594, shSIRT7-1; TRCN0000359663, shSIRT7-2) or SIRT6 (TRCN0000232532) were cloned into pLKO.1. The recombinant constructs, as well as assistant vectors psPAX2 and pMD2.G, were co-transfected into HEK293T cells.Viral supernatants were collected 48 h later, claried by ltration through 0.45-mm lters and concentrated by ultracentrifugation. The concentrated viruses were used to infect 5 105 cells (2030% conuent) in a 60-mm dish with 8 mg ml 1

polybrene. Infected cells were selected with 1.5 mg ml 1 puromycin (Amresco).

The lentivirus carrying rSIRT7wt, rSIRT7H187Y, FLAG-H3.1wt, FLAG-

H3.1K122E and FLAG-H3.1K122R were packaged and collected similarly.

SILAC labelling and quantitative proteomics analysis. Control or SIRT7 KD MCF-7 cells were grown in Dulbeccos modied Eagles medium supplemented with 10% FBS and either the heavy form of [U-13C6]-L-lysine or light [U-12C6]-L-lysine for more than six generations before being collected to achieve more than 97% labelling efciency. After that, the cells were further expanded in SILAC media to desired cell number (B5 108) in 15 150-mm2 asks. The cells were then

collected and the core histones were isolated and digested. Lysine crotonylation (Kcro), succinylation (Ksucc) and malonylation (Kmal) peptides were then enriched by pre-washed antibody beads (PTM Biolabs, Hangzhou). The eluted peptides were cleaned with C18 ZipTips (Millipore) according to the manufacturers instructions, followed by analysis with LCMS/MS. The resulting MS/MS data were processed by using MaxQuant with integrated Andromeda search engine (version 1.4.1.2).

False discovery rate thresholds for protein, peptide and modication site were specied at 1%.

Figure 8 | SIRT7-mediated H3K122succ desuccinylation is implemented in cellular response to DNA damage. (a) Control or SIRT7-depleted MCF-7 cells were treated with 40 nM VP-16 or 1 mM CPT, and collected for annexin V and propidium iodide double staining. Cell apoptosis was determined by ow cytometry. Data were represented as means.d. (be) Control or SIRT7-depleted MCF-7 (b), U2OS (c), HCT116 (d) or HepG2 (e) cells were treated with or without IR at the indicated doses and then subjected to clonogenic survival assays. The efciency of SIRT7 KD in HCT116 (d) or HepG2 (e) cells was monitored by western blotting of whole-cell lysate, with corresponding antibodies. (f) Control or SIRT7-knockout U2OS cells were treated with or without IR at the indicated doses and then subjected to clonogenic survival assays. (g) U2OS cells stably expressing FLAG-H3wt, FLAG-H3K122E or FLAG-H3K122R were treated with or without IR at the indicated doses and then subjected to clonogenic survival assays. Data were represented as means.d. for triplicate experiments. *Po0.05, **Po0.01 and ***Po0.001 (two-tailed unpaired Students t-test).

condensation of chromatin is required for inhibiting local transcription, compacting the local chromatin structure, rewriting the local epigenetic landscape, and limiting DSB mobility during the initial moments following DSB production53. Clearly, the biological signicance of SIRT7-associated chromatin condensation needs further elucidation.

Recent reports indicate that PAR-dependent accumulation of transcription repression-associated chromatin regulators such as histone variant macroH2A1.1 (ref. 55), the nucleosome remodelling and deacetylase complex36, ALC1 (ref. 57) or SUV39H1 (ref. 53) function to modulate chromatin structures at sites of DNA breaks to facilitate signalling and/or repair of DNA damage. Interestingly, both PARP1 and SIRT7 are enzymes requiring NAD as coenzyme, and, intriguingly, several studies indicate that NAD-consuming enzymes such as PARPs or cADP-ribose synthase inuence sirtuin activity by restricting

NAD availability58. For example, it is reported that PARP1 activation was associated with a depletion of NAD pool thus an inhibition of SIRT1 activity, leading to the death of cells59,60. It is believed that PARP catalysis is the main NAD catabolic source in cells that forces the cell to continuously synthesize NAD from the de novo pathway or recycling pathway in the case of cellular stresses, especially during the DNA repair process61. Whether or not the PARP1-dependent recruitment of SIRT7 during DNA-damage response is a reection of coordinated enzymatic actions between PARP1 and SIRT7 in terms of NAD usage is currently unknown. In light of the reports that several other sirtuins including SIRT1 (ref. 62) and SIRT6 (refs 63,64) are also involved in DNA-damage repair, it will be interesting in future investigations to investigate the importance of metabolic processes in chromatin remodelling during DNA repair. In addition, further studies are needed to elucidate the full spectrum of the regulation of histone desuccinylation and to decipher the histone languages encoded by this modication. Moreover, due to technical limitations, our current study focuses on H3K122; regulation of desuccinylation by SIRT7 on other histone sites cannot be excluded. It is also possible that the reported SIRT7 substrates and SIRT7-associated desuccinylation coordinately inuence chromatin environment during DNA-damage repair. Nevertheless, our study indicates that SIRT7 is a NAD-dependent histone desuccinylase, providing a molecular basis for the understanding of epigenetic regulation by this sirtuin protein. Our experiments revealed that SIRT7-catalysed H3K122 desuccinylation is critically implemented in DNA-damage response and cell survival, providing a mechanistic insight into the cellular function of SIRT7.

Methods

Plasmids. The cDNA for wild-type SIRT5, SIRT6 or SIRT7 was amplied by PCR and ligated into pcDNA3.1( ) plasmid containing a FLAG tag. SIRT7 mutants

including S111A, H187Y and S111A/H187Y were generated by using QuikChange Lightning Site-Directed Mutagenesis Kit. The GFP-SIRT7 was constructed by cloning full-length of SIRT7 into pEGFP-N1 vector. SIRT7 siRNA-1 resistant pcDNA3.1( )-FLAG-SIRT7wt (rSIRT7wt) and SIRT7 siRNA-1 resistant

pcDNA3.1( )-FLAG-SIRT7H187Y (rSIRT7H187Y) were generated by

synonymous mutations (G606A, G609A and C612T). The pLVX-IRES-FLAG-rSIRT7wt and pLVX-IRES-FLAG-rSIRT7H187Y for lentiviral production were subcloned from pcDNA3.1( )-FLAG-rSIRT7wt/H187Y. The PITA-FLAG-H3wt

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Western blotting. Western blotting was performed according to standard procedures. Antibodies used were anti-SIRT7 (Santa Cruz Biotechnology, sc-365344, 1:500), anti-HA (MBL, M180-3, 1:2,000), anti-Flag (Sigma, F3165, 1:10,000), anti-b-actin (Sigma, A1978, 1:10,000), anti-tubulin (Sigma, clone

B-5-1-2, T6074, 1:50,000), anti-SIRT6 (Abgent, AP-6245a, 1:500), anti-PARP1/2 (Santa Cruz Biotechnology, sc-7150, 1:5,000), anti-Ku80 (Santa Cruz Biotechnology, sc-5280, 1:2,000), anti-BRCA1 (Proteintech, 22362-1-AP, 1:1,000), anti-gH2AX (Millipore, 05-636, 1:2,000), anti-H2AX (Abcam, ab11175, 1:2,000)

anti-pan-acetylation (PTM BioLabs, PTM-105, 1:1,000), anti-pan-succinylation (PTM BioLabs, PTM-401, 1:1,000), anti-H3K122succ (1:4,000), anti-H2BK120succ (1:8,000), anti-H3K122ac (Abcam, ab33309, 1:2,000), anti-H3K18ac (PTM BioLabs, PTM-114, 1:1,000), anti-H3 (Abcam, ab1791, 1:100,000) and anti-rabbit (Jackson ImmunoResearch, 115-035-003, 1:8,000) or anti-mouse (Jackson ImmunoResearch, 111-035-003, 1:8,000) secondary antibodies conjugated to horseradish peroxidase. The bands were quantied by densitometry with ImageJ software. Uncropped scans of the most important blots are shown in Supplementary Fig. 11.

Protein purication. Protein purication was performed as described previously65 with some optimization. Briey, for FLAG-SIRT7wt, FLAG-SIRT7H187Y, FLAG-SIRT5 and FLAG-SIRT6, HEK293T cells expressing full-length FLAG-tagged SIRT7wt, SIRT7H187Y, SIRT5 or SIRT6 were collected and lysed in lysis buffer (50 mM Tris HCl (pH 7.4), 300 mM NaCl, 1% Nonidet P-40, 1 mM

EDTA, 10% (vol/vol) glycerol and 1 mM dithiothreitol (DTT)) supplemented with protease inhibitors (Roche). The resulting lysate was incubated with anti-FLAG M2 afnity gel for 2 h and the beads were washed ve times with lysis buffer.

The immobilized proteins was eluted with 1 FLAG peptide and used in

desuccinylation assays as described below or resolved on SDSPAGE followed by Coomassie brilliant blue staining.

Preparation of mononucleosome. Preparation of mononucleosomes was conducted according to the procedure described previously66. Briey, HeLa cells were collected by ice-cold PBS, resuspended in lysis buffer (10 mM Tris HCl

(pH 7.5), 10 mM NaCl, 3 mM MgCl2 and 0.4% Nonidet P-40) in the presence of protease inhibitors and the nuclei were pelleted. Glycerol buffer (10 mM Tris HCl

(pH 7.4), 0.1 mM EDTA, 5 mM MgAc2 and 25% (vol/vol) glycerol) was add to get a nal concentration of 1 to 2 mg ml 1 nuclei. To generate nucleosomal material, digestions were conducted by adding 1 volume of 2 MNase buffer (50 mM KCl,

8 mM MgCl2, 2 mM CaCl2 and 100 mM Tris HCl (pH 7.4)) and 3,0008,000 gel

units per ml MNase. The reaction was incubated for 15 min at 37 C and stopped by adding EDTA to a nal concentration of 10 mM. The mononucleosomes were then puried by sucrose gradient assay.

Histone desuccinylation assay. The sequence of synthesized H3K122 (117-128) succinyl peptide was IRRYQK(succinyl)STELLI. The identity and purity of the peptides were veried by LCMS. Two micrograms of puried FLAG-SIRT7wt, FLAG-SIRT7H187Y, FLAG-SIRT5 or FLAG-SIRT6 were incubated with 500 ng H3K122succ peptides in desuccinylation assay buffer8 (20 mM Tris HCl (pH 7.5)

and 1 mM DTT) with or without 1.0 mM NAD in a nal volume of 30 ml for 2 h at 37 C. The reaction mixture was boiled and subjected to dot blot analysis. One microgram of calf thymus bulk histones (Sigma) or mononucleosomes isolated from HeLa cells were incubated with 0.255 mg of SIRT7wt, SIRT7H187Y, SIRT5 or SIRT6 in desuccinylation assay buffer in the presence or absence of 1.0 mM NAD and/or 10 mM NAM in a nal volume of 30 ml for 2 h at 37 C. The reaction mixture was boiled in SDS sample buffer and subjected to SDSPAGE analysis and mass spectra analysis.

Immunopurication and mass spectrometry. HEK293T cells transfected with empty vector or FLAG-SIRT7wt for 48 h were lysed in lysis buffer (50 mMTris HCl (pH 7.4), 150 mM NaCl, 0.3% Nonidet P-40, 1 mM DTT and 5 mM

EDTA) plus protease inhibitors (Roche) for 30 min at 4 C. This was followed by centrifugation at 14,000g for 15 min at 4 C. Protein supernatant was incubated with anti-FLAG M2 gel for 2 h at 4 C. After washing with lysis buffer for ve times, 1 FLAG peptide was used to elute the protein complex from the beads

following the manufacturers instructions. The eluted protein complex was then resolved on NuPAGE 412% Bis-Tris gel (Invitrogen), silver stained and subjected to LCMS/MS for sequencing and data analysis.

Laser microirradiation and X-ray irradiation. For time-lapse imaging of living cells, cells grown on a dish with thin glass bottom (NEST) in the presence of 10 mM of 5-bromo-20-deoxyuridine (BrdU, Sigma-Aldrich) in phenol red-free medium (Invitrogen) for 24 h were locally irradiated with a 365-nm pulsed nitrogen ultraviolet laser (16 Hz pulse, 45% laser output) generated from the micropoint system (Andor). This system was directly coupled to the epiuorescence path of the Nikon A1 confocal imaging system with time-lapse imaging every 30 s for 15 min. A heated stage with an objective lens heater was used to keep the cells at the appropriate temperature (37 C) and growth conditions during imaging. Images were analysed using ImageJ software. For quantication of protein accumulations

at laser-generated DSBs, the mean uorescence intensity within the regions of interest (ROI) was measured for each time point. The intensity values were background subtracted, and the ratio of intensity within the microirradiated nuclear area to non-microirradiated area was calculated. At least 30 independent cells were scored. For laser microirradiation and immunouorescence assays, cells were grown on LabTek II chamber slides (Thermo Scientic) in the presence of 10 mM BrdU in phenol red-free medium (Invitrogen) for 24 h before induction of

DNA damage by a ultraviolet-A laser (l 355 nm, 40% energy) using a Zeiss

Observer.Z1 inverted microscope with a PALM MicroBeam laser microdissection workstation. After irradiation, the cells were incubated at 37 C for an appropriate time and processed for immunostaining. IR was delivered by an X-ray generator (RS2000 PRO, 160 kV, 25 mA; Radsource Corporation).

Immunouorescence. Cells were washed with PBS, xed in 4% paraformaldehyde for 10 min. Specically, for H3K122succ stain, before xed in 4% paraformalde-hyde, cells were washed once with cold PBS, extracted with CSK buffer (10 mM Pipes (pH 7.0), 100 mM NaCl, 300 mM sucrose, 3 mM MgCl2 and 0.5% Triton

X-100) for 2 min, washed again with cold PBS. Then the cells were permeabilized with 0.2% Triton X-100 and incubated with appropriate primary antibodies (SIRT7, Santa Cruz Biotechnology, sc-365344, 1:100; gH2AX, CST, 9718P, 1:400;

H3K122succ, 1:100; gH2AX, Millipore, 05-636, 1:100) and secondary antibodies coupled to Alexa Fluor 488 (Jackson ImmunoResearch, rabbit, 111-545-003, 1:100) or Alexa Fluor 594 (Jackson ImmunoResearch, mouse, 115-585-003; rabbit, 111-585-003, 1:100). The cells were then washed for four times, and a nal concentration of 0.1 mg ml 1 4,6-diamidino-2-phenylindole dihydrochloride (Sigma) was included in the nal wash to stain nuclei. Images were acquired with a

FluoView FV1000 laser scanning confocal system (Olympus) connected to an inverted microscope (IX-81) equipped with PLAPON 60 oil/numerical aperture

1.42 objective. To avoid bleed-through effects in double-staining experiments, each dye was scanned independently in a multi-tracking mode.

Chromatin immunoprecipitation. ChIP experiments were performed according to the procedure described previously67. About 10 million cells were crosslinked with 1% formaldehyde for 10 min at room temperature and quenched by the addition of glycine to a nal concentration of 125 mM for 5 min. The xed cells were resuspended in SDS lysis buffer (1% SDS, 5 mM EDTA and 50 mM Tris HCl

(pH 8.1)) in the presence of protease inhibitors and 10 mM NAM, then subjected to 3 10 cycles (30 s on and off) of sonication (Bioruptor, Diagenode) to generate

chromatin fragments of B300 bp in length. Lysates were diluted in buffer containing 1% Triton X-100, 2 mM EDTA, 20 mM Tris HCl (pH 8.1), 150 mM

NaCl plus 10 mM NAM and protease inhibitors. For immunoprecipitation, the diluted chromatin was incubated with control or specic antibodies (35 mg) for 12 h at 4 C with constant rotation, and 50 ml of 50% (vol/vol) protein A/G Sepharose beads was then added and the incubation was continued for an additional 2 h. Beads were washed with the following buffers: TSE I (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris HCl (pH 8.1) and 150 mM NaCl); TSE II

(0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris HCl (pH 8.1) and

500 mM NaCl); buffer III (0.25 M LiCl, 1% Nonidet P-40, 1% sodium deoxycholate, 1 mM EDTA and 10 mM Tris HCl (pH 8.1)); and Tris-EDTA

buffer. Between washes, the beads were collected by centrifugation at 4 C. The pulled-down chromatin complex together with input was de-crosslinked at 70 C for 2 h in elution buffer (1% SDS, 5 mM EDTA, 20 mM Tris HCl (pH 8.1), 50 mM

NaCl and 0.1 mg ml 1 proteinase K). Eluted DNA was puried with PCR purication kit (Qiagen) and analysed by quantitative PCR using primers described in Supplementary Table 3.

Nucleosome stability assay. Nucleosome stability assays were performed as described previously44. Briey, cells were collected and washed twice in ice-cold PBS by centrifugation at 500g. Cell pellet was resuspended completely in 500 ml buffer A (20 mM HEPES (pH 7.9), 0.5 mM DTT, 1 mM phenylmethyl sulphonyl uoride, 1.5 mM MgCl2 and 0.1% Triton) containing 1.0 or 1.5 M NaCl. Cells were incubated for 40 min at 4 C with constant agitation. Samples were then centrifuged at 100,000g (Ultracentrifuge; HITACHI) for 20 min, and the supernatant, containing released histones, retained for further analysis.

Generation of SIRT7 knockout cell lines by CRISPR-Cas9. Three single-guide RNAs (sgRNAs 13) that target different regions of the human SIRT7 gene were selected from previously published genome-wide human sgRNA Libraries68. The sequences are given in Supplementary Table 4. Oligos corresponding to the sgRNAs were cloned into the GV392 vector containing the hSPCas9 gene and a puromycin selection marker gene. U2OS cells were transfected with either of the sgRNAs and selected with puromycin 48 h post transfection. Single clones were retrieved after 7 days of puromycin selection, expanded and analysed for abrogation of SIRT7 expression by western blotting. Manifestation of the SIRT7 mutations was veried by PCR and sequencing.

Cell ow cytometry. For measurement of repair efciency, DR-GFP-U2OS or EJ5-HEK293 cells were trypsinized, washed with PBS and collected with

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FACSCalibur. The data were analysed by FlowJo. For analysis of BrdU incorporation, cells were pulsed with 10 mM BrdU for 15 min followed by trypsinization,

PBS wash and xation with ice-cold 70% ethanol. Cells were then resupended in2 N HCl, 0.5% Triton X-100 and incubated for 30 min at room temperature. Next, cells were resuspended in 0.1 M Na2B4O7 (pH 8.5), spin down and resuspended in antibody incubation buffer (1% BSA and 0.5% Tween 20 in PBS) containing anti-BrdU (Abcam, ab8039, 1:1,000) for 30 min before the addition of the secondary antibody (Alexa Fluor 488-conjugated goat anti-mouse IgG (H L), Jackson

ImmunoResearch, 115-545-003, 1:200) for 30 min. Finally, cells were washed and incubated in propidium iodide buffer and analysed by FACS. Acquisition of the data is performed through Cell Quest software, and analysis through FlowJo and ModFit. Apoptosis was measured using Annexin V-FITC Apoptosis Detection Kit (BD Pharmingen) according to the manufacturers instructions and analysed using the FACSCalibur ow cytometer.

MNase sensitivity assay. About 1 million cells were washed with cold PBS, resuspended in ice-cold Nonidet P-40 cell lysis buffer (10 mM Tris HCl (pH 7.5),

10 mM NaCl, 3 mM MgCl2 and 0.4% Nonidet P-40) in the presence of protease inhibitors and incubated on ice for 5 min, The lysate was cleared with centrifugation at 2,000g for 5 min at 4 C. The resulting pellet was collected and washed with lysis buffer twice. The pellet was then resuspended in 50 ml glycerol buffer (10 mM

Tris HCl (pH 7.4), 0.1 mM EDTA, 5 mM MgAc2 and 25% (vol/vol) glycerol), mixed

with equal volume of 2 MNase buffer (50 mM KCl, 8 mM MgCl2, 2 mM CaCl2

and 100 mM Tris HCl, (pH 7.4)), and incubated at 37 C for 5 min with MNase

(NEB) at the indicated amount per 100 ml of total reaction volume. The reaction was stopped by adding EDTA at the nal concentration of 10 mM. Genomic DNA was puried and separated by electrophoresis in 1.2% agarose gel.

Clonogenic survival assay. Cells were plated in 12-well plates in triplicates (400 cells per well) and were subsequently treated with IR and let to grow in colonies for 10 days. After 10 days, the cells were washed with PBS, xed with 4% formaldehyde for 10 min and stained with crystal violet (0.1% wt/vol) for 20 min. The number of colonies per well was counted, and the plating efciency and surviving fraction for given treatments were calculated on the basis of the survival rates of nonirradiated cells.

Statistical analysis. The data were analysed by a two-tailed unpaired Students t-test (GraphPad Prism software, version 5.01) and expressed as means.d. unless otherwise indicated. Po0.05 was considered to be statistically signicant.

Data availability. All data presented is presented in this manuscript or available from the authors on request.

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Acknowledgements

This work was supported by grants 81071677 (to W.Y.), and 91219201, 81530073 and 81130048 (to Y.S.) from the National Natural Science Foundation of China, and a grant (2014CB542004 to J.L.) from the Ministry of Science and Technology of China.

Author contributions

L.L., L.S., W.Y. and Y.S. conceived the project and designed the experiments; L.L., Lan S., W.Y., S.Y., D.Z. and R.Y. performed and interpreted molecular and cell biology experiments; L.L., L.S., W.Y. and Y.S., wrote and revised the manuscript. C.Z., J.Y., L.H. and W.J. designed and performed the quantitative mass spectrometry in Fig. 1c; L.L., L.S.,X.Y., LY.S., J.L., Lei S., Y.S. and W.Y. analysed the data and provided technical assistance;L.L. and L.S. made independent contributions to the work.

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How to cite this article: Li, L. et al. SIRT7 is a histone desuccinylase that functionally links to chromatin compaction and genome stability. Nat. Commun. 7:12235doi: 10.1038/ncomms12235 (2016).

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Although SIRT7 is a member of sirtuin family proteins that are described as NAD⁺ -dependent class III histone deacetylases, the intrinsic enzymatic activity of this sirtuin protein remains to be investigated and the cellular function of SIRT7 remains to be explored. Here we report that SIRT7 is an NAD⁺ -dependent histone desuccinylase. We show that SIRT7 is recruited to DNA double-strand breaks (DSBs) in a PARP1-dependent manner and catalyses desuccinylation of H3K122 therein, thereby promoting chromatin condensation and DSB repair. We demonstrate that depletion of SIRT7 impairs chromatin compaction during DNA-damage response and sensitizes cells to genotoxic stresses. Our study indicates SIRT7 is a histone desuccinylase, providing a molecular basis for the understanding of epigenetic regulation by this sirtuin protein. Our experiments reveal that SIRT7-catalysed H3K122 desuccinylation is critically implemented in DNA-damage response and cell survival, providing a mechanistic insight into the cellular function of SIRT7.

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Title

SIRT7 is a histone desuccinylase that functionally links to chromatin compaction and genome stability

Author

Li, Lei; Shi, Lan; Yang, Shangda; Yan, Ruorong; Zhang, Di; Yang, Jianguo; He, Lin; Li, Wanjin; Yi, Xia; Sun, Luyang; Liang, Jing; Cheng, Zhongyi; Shi, Lei; Shang, Yongfeng; Yu, Wenhua

Pages

12235

Publication year

2016

Publication date

Jul 2016

Publisher

Nature Publishing Group

e-ISSN

20411723

Source type

Scholarly Journal

Language of publication

English

DOI

https://doi.org/10.1038/ncomms12235

ProQuest document ID

1805502779

SIRT7 is a histone desuccinylase that functionally links to chromatin compaction and genome stability

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