Introduction

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, SIRT1–7, 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 specificity2. 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 modification on lysine (K)19 and K20 of tumour necrosis factor α (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 identified 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-β1 (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 modifications 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 modification represents a prominent mechanism by which the nucleosome plasticity is regulated and chromatin configuration is shaped21,22.

A plethora of histone modifications have been described and, in last decade, various histone modifications, including phosphorylation, acetylation, methylation, ubiquitination, sumoylation and ADP-ribosylation have been the subjects of extensive study in the field of epigenetics23. It is believed that factors involved in the deposition (writer), binding (reader) and removal (eraser) of these histone modifications (marks) are at the epicentre of the regulatory circuits controlling the chromatin dynamics22,23,24. It is proposed that various histone modifications in combination constitute distinct histone languages to encode for different chromatin-related events25. In effect, chromatin modifiers (writers or erasers) act in an interdependent manner and coordinated fashion to load or remove histone marks to control the chromatin configuration and to determining the biological consequence22,23,24. Accordingly, identification and functional characterization of these chromatin modifiers have been the major theme in the understanding of epigenetic regulation. Strikingly, recent studies identified a series of new types of histone modifications, including biotinylation, citrullination, crotonylation, glutathionylation, propionylation, malonylation and succinylation9,26,48. Modifications 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 stereospecific blockage than acetylation. In addition, compared with acetylation, succinylation is a more acidic modification 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 efficient DSB repair.

It is documented that chromatin modifiers, 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 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 significance 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 influence 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 reflection 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 modification. 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 influence 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 amplified 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 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 confirmed 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 purified by protein A-conjugated agarose followed by affinity 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), α-tubulin (clone B-5-1-2, T6074) and β-actin (A1978) from Sigma; SIRT7 (sc-365344), Ku80 (sc-5280) and PARP1/2 (sc-7150) from Santa Cruz Biotechnology; γH2AX 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 affinity 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 **ngzhi Xu (Capital Normal University, Bei**g). The cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (FBS). Transfections were carried out using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s 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 manufacturer’s 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). 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, clarified by filtration through 0.45-μm filters and concentrated by ultracentrifugation. The concentrated viruses were used to infect 5 × 105 cells (20–30% confluent) in a 60-mm dish with 8 μg ml−1 polybrene. Infected cells were selected with 1.5 μg 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 Dulbecco’s modified Eagle’s 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 efficiency. After that, the cells were further expanded in SILAC media to desired cell number (∼5 × 108) in 15 × 150-mm2 flasks. 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 manufacturer’s instructions, followed by analysis with LC–MS/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 modification site were specified at 1%.

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-β-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-γH2AX (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 quantified by densitometry with ImageJ software. Uncropped scans of the most important blots are shown in Supplementary Fig. 11.

Protein purification

Protein purification was performed as described previously65 with some optimization. Briefly, 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 affinity gel for 2 h and the beads were washed five times with lysis buffer. The immobilized proteins was eluted with 1 × FLAG peptide and used in desuccinylation assays as described below or resolved on SDS–PAGE followed by Coomassie brilliant blue staining.

Preparation of mononucleosome

Preparation of mononucleosomes was conducted according to the procedure described previously66. Briefly, 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 final 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 3,000–8,000 gel units per ml MNase. The reaction was incubated for 15 min at 37 °C and stopped by adding EDTA to a final concentration of 10 mM. The mononucleosomes were then purified 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 verified by LC–MS. Two micrograms of purified 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 final volume of 30 μl 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 0.25–5 μg 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 final volume of 30 μl for 2 h at 37 °C. The reaction mixture was boiled in SDS sample buffer and subjected to SDS–PAGE analysis and mass spectra analysis.

Immunopurification and mass spectrometry

HEK293T cells transfected with empty vector or FLAG-SIRT7wt for 48 h were lysed in lysis buffer (50 mM Tris·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 five times, 1 × FLAG peptide was used to elute the protein complex from the beads following the manufacturer’s instructions. The eluted protein complex was then resolved on NuPAGE 4–12% Bis-Tris gel (Invitrogen), silver stained and subjected to LC–MS/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 μM of 5-bromo-2′-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 epifluorescence 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 quantification of protein accumulations at laser-generated DSBs, the mean fluorescence 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 immunofluorescence assays, cells were grown on LabTek II chamber slides (Thermo Scientific) in the presence of 10 μM BrdU in phenol red-free medium (Invitrogen) for 24 h before induction of DNA damage by a ultraviolet-A laser (λ=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).

Immunofluorescence

Cells were washed with PBS, fixed in 4% paraformaldehyde for 10 min. Specifically, for H3K122succ stain, before fixed in 4% paraformaldehyde, 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; γH2AX, CST, 9718P, 1:400; H3K122succ, 1:100; γH2AX, 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 final concentration of 0.1 μg ml−1 4,6-diamidino-2-phenylindole dihydrochloride (Sigma) was included in the final 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 final concentration of 125 mM for 5 min. The fixed 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 ∼300 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 specific antibodies (3–5 μg) for 12 h at 4 °C with constant rotation, and 50 μl 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 purified with PCR purification 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. Briefly, cells were collected and washed twice in ice-cold PBS by centrifugation at 500g. Cell pellet was resuspended completely in 500 μl buffer A (20 mM HEPES (pH 7.9), 0.5 mM DTT, 1 mM phenylmethyl sulphonyl fluoride, 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 1–3) 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 verified by PCR and sequencing.

Cell flow cytometry

For measurement of repair efficiency, DR-GFP-U2OS or EJ5-HEK293 cells were trypsinized, washed with PBS and collected with FACSCalibur. The data were analysed by FlowJo. For analysis of BrdU incorporation, cells were pulsed with 10 μM BrdU for 15 min followed by trypsinization, PBS wash and fixation with ice-cold 70% ethanol. Cells were then resupended in 2 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 manufacturer’s instructions and analysed using the FACSCalibur flow 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 μl 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 μl of total reaction volume. The reaction was stopped by adding EDTA at the final concentration of 10 mM. Genomic DNA was purified 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, fixed 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 efficiency 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 Student’s t-test (GraphPad Prism software, version 5.01) and expressed as mean±s.d. unless otherwise indicated. P<0.05 was considered to be statistically significant.

Data availability

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

Additional information

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. 7:12235 doi: 10.1038/ncomms12235 (2016).