SCIENCE ADVANCES | RESEARCH ARTICLE BIOCHEMISTRY Copyright © 2020 The Authors, some SirT7 auto-ADP-ribosylation regulates glucose rights reserved; exclusive licensee starvation response through mH2A1 American Association for the Advancement of Science. No claim to Nicolás G. Simonet1,2, Joshua K. Thackray3, Berta N. Vazquez1,2,3, Alessandro Ianni4, original U.S. Government Maria Espinosa-Alcantud1,2, Julia Morales-Sanfrutos5,6, Sarah Hurtado-Bagès7, Eduard Sabidó5,6, Works. Distributed Marcus Buschbeck7,8, Jay Tischfield3, Carolina De La Torre9,10, Manel Esteller11,12,13,14, under a Creative Thomas Braun4, Mireia Olivella15,16, Lourdes Serrano3,17, Alejandro Vaquero1,2* Commons Attribution NonCommercial Sirtuins are key players of metabolic stress response. Originally described as deacetylases, some sirtuins also exhibit License 4.0 (CC BY-NC). poorly understood mono–adenosine 5′-diphosphate (ADP)–ribosyltransferase (mADPRT) activity. We report that the deacetylase SirT7 is a dual sirtuin, as it also features auto-mADPRT activity. SirT7 mADPRT occurs at a previously undefined active site, and its abrogation alters SirT7 chromatin distribution. We identify an epigenetic pathway by which ADP-ribosyl-SirT7 is recognized by the ADP-ribose reader mH2A1.1 under glucose starvation, inducing SirT7 relocalization to intergenic regions. SirT7 promotes mH2A1 enrichment in a subset of nearby genes, many of them involved in second messenger signaling, resulting in their specific up- or down-regulation. The Downloaded from http://advances.sciencemag.org/ on February 11, 2021 expression profile of these genes under calorie restriction is consistently abrogated in SirT7-deficient mice, resulting in impaired activation of autophagy. Our work provides a novel perspective on sirtuin duality and suggests a role for SirT7/mH2A1.1 axis in glucose homeostasis and aging. INTRODUCTION levels (2). One of the most intriguing features of sirtuin biology is that The members of the Sir2 family, also known as sirtuins, play a key they catalyze two NAD+-dependent enzymatic activities: NAD+-­ role in the response to different types of stress, including metabolic, dependent deacetylation and mono–adenosine 5′-diphosphate (ADP)–­ oxidative, and genotoxic stress. Thus, sirtuins are at the crossroads ribosylation (mADPR), which involves the reversible transfer of one of many pathways that control genome stability, metabolic homeo- ADP-ribose moiety from the coenzyme NAD+ to specific protein stasis, cell cycle control, and apoptosis (1). The family members residues. The former seems to be more prominent among mammalian originated in the prokaryotes, where they participated in the metab- sirtuins since almost all of them are NAD+-dependent protein olism of nicotinamide adenine dinucleotide (NAD+). During evolu- deacetylases, whose activity can, in some cases, target other single tion, sirtuins have expanded and diversified, acquiring a key role in acyl groups such as butyryl or succinyl, or even long-chain fatty acyl the cross-talk between chromatin and environment. Reflecting this, groups (3). The relevance of this duality goes beyond sirtuins, as we four of the seven mammalian sirtuins (SirT1, SirT2, SirT6, and know of just a handful of enzymes, most of them prokaryotic, that SirT7) have been linked to the control of genome stability at different combine two activities in a single protein (4, 5). By far, the best studied ADP-ribosyltransferase (ADPRT) is 1 Chromatin Biology Laboratory, Josep Carreras Leukaemia Research Institute (IJC), poly(ADP-ribose) polymerase 1 (PARP1), which is responsible for Ctra de Can Ruti, Camí de les Escoles s/n, 08916 Badalona, Barcelona, Catalonia, up to about 90% of the cellular poly-ADP-ribosylation (pADPR) Spain. 2Chromatin Biology Laboratory, Cancer Epigenetics and Biology Program generated by DNA damage and is a key pharmacological target for (PEBC), Bellvitge Biomedical Research Institute (IDIBELL), Av. Gran Via de l’Hospitalet, the treatment of DNA repair-deficient cancers. In contrast to the 199-203, 08908 L’Hospitalet de Llobregat, Barcelona, Catalonia, Spain. 3Department of Genetics and the Human Genetics Institute of New Jersey, Rutgers University, relatively well studied pADPR, the biological relevance of mADPR 145 Bevier Road, Piscataway, NJ 08854, USA. 4Max Planck Institute for Heart and is more limited. mADPR has been shown to regulate a wide range Lung Research, Department of Cardiac Development and Remodelling, Bad of functions, from cellular stress response upon DNA damage or Nauheim, Germany. 5Proteomics Unit, Centre de Regulació Genòmica (CRG), Barcelona Institute of Science and Technology (BIST), Barcelona, Catalonia, Spain. 6Universitat metabolic stress to infectious diseases and cancer (6). Reflecting Pompeu Fabra, Barcelona, Spain. 7Cancer and Leukemia Epigenetics and Biology their functional relevance and complexity, there are not only different Program, Josep Carreras Leukaemia Research Institute (IJC), Campus ICO-GTP-UAB, classes of protein-binding modules (readers) that recognize ADP-­ 08916 Badalona, Barcelona, Catalonia, Spain. 8Program for Predictive and Person- alized Medicine of Cancer, Germans Trias i Pujol Research Institute (PMPPC-IGTP), ribosylation but also enzymes involved in the removal of ADP-ribose 08916 Badalona, Barcelona, Catalonia, Spain. 9Proteomics Unit, Josep Carreras called ADP-ribosylhydrolases (erasers) (6). Among the readers, Leukaemia Research Institute (IJC), Ctra de Can Ruti, Camí de les Escoles s/n, 08916 macrodomains are an ancient group of protein domains formed by Badalona, Barcelona, Catalonia, Spain. 10Proteomics Unit, Bellvitge Biomedical Re- 130 to 190 residues present from viruses to humans that bind search Institute (IDIBELL), Av. Gran Via de l’Hospitalet, 199-203, 08908 L’Hospitalet de Llobregat, Barcelona, Catalonia, Spain. 11Cancer Epigenetics Group, Josep Carreras mono(ADP-ribose), poly(ADP-ribose), and NAD+-derived metabolites. Leukaemia Research Institute (IJC), Badalona, Barcelona, Catalonia, Spain. 12Centro de In humans, at least 12 proteins contain macrodomains belonging to Investigación Biomédica en Red Cáncer (CIBERONC), 28029 Madrid, Spain. 13Institucio four of the six known classes of macrodomains. One of the best Catalana de Recerca i Estudis Avançats (ICREA), Barcelona, Catalonia, Spain. 14Physio- logical Sciences Department, School of Medicine and Health Sciences, University of studied macrodomain-containing proteins is the histone H2A variant Barcelona (UB), Barcelona, Catalonia, Spain. 15Bioinformatics Area, School of Inter- macroH2A (mH2A). Of the three mH2A isoforms (mH2A1.1, national Studies, ESCI-UPF, Barcelona, Catalonia, Spain. 16Bioinformatics and Medical mH2A1.2, and mH2A2), only mH2A1.1 has the ability to bind Statistics Group, UST, Universitat de Vic–Universitat Central de Catalunya (UVIC-UCC), ADPR (7). Moreover, mH2A1.1 can also bind free ADP-ribose Barcelona, Catalonia, Spain. 17Department of Science, BMCC, The City University of New York (CUNY), 199 Chambers Street N699P, New York, NY 10007, USA. molecules and the product of sirtuin deacetylation, O-acetyl-ADP- *Corresponding author. Email: avaquero@carrerasresearch.org ribose (8), which suggests that mH2A1.1 may play a role as an Simonet et al., Sci. Adv. 2020; 6 : eaaz2590 24 July 2020 1 of 16 SCIENCE ADVANCES | RESEARCH ARTICLE energetic/metabolic sensor and could be functionally linked to the sought evidence of the key role of the SirT7/mH2A axis in calorie-­ stress response role of sirtuins. mH2A1.1 has been implicated in restricted mice. This axis epigenetically regulates the expression of gene repression and activation (9), in the latter case, especially after genes involved in differentiation, proliferation, metabolism, autophagy, signal-induced gene activation through PARP1 inactivation. and aging pathways. Our functional studies suggest a direct role for Sirtuin-mediated ADP-ribosylation was described in yeast Sir2p SirT7 in the control of the stress response through this newly found before the discovery of the main deacetylation activity (10), and it enzymatic activity. was originally hypothesized that all sirtuins could potentially harbor mono-ADPRT (mADPRT) activity (11), although this activity is, in most cases, either orders of magnitudes slower than deacetylation RESULTS or simply undetectable in in vitro studies (12). Among mammalian SirT7 is an mADPRT sirtuins (SirT1 to SirT7), only two—reported to be weak deacetylase Several lines of evidence suggest that SirT7 may also harbor mAD- enzymes in vitro (13, 14)—are known to ADP-ribosylate other protein PRT activity. SirT7 and SirT6 are close homologs with a common targets. SirT4-dependent ADP-ribosylation of glutamate dehydro- origin in early eukaryotes. They consistently show a high level genase represses its enzymatic activity by limiting the metabolism (42%) of sequence identity and structure in their catalytic core of glutamate and glutamine to produce adenosine 5′-triphosphate domain. SirT7 shares a unique feature with SirT6 in the catalytic (15). SirT6 can mediate the ADP-ribosylation of two targets: PARP1 domain that is different from other mammalian sirtuins (Fig. 1A). under oxidative stress, thereby inducing double-strand break repair Whereas there is a large helix bundle in the NAD+-binding Rossmann after DNA damage (16), and KRAB-associated protein-1 (KAP1), to fold in the catalytic region of most sirtuins that connects this domain Downloaded from http://advances.sciencemag.org/ on February 11, 2021 promote long interspersed nuclear element–1 (LINE-1) silencing and with a small Zn-binding domain, this structure is missing from genomic stability (17). SirT6 was also found to auto–ADP-ribosylate SirT6 and SirT7. This feature was thought to considerably reduce itself (18), although this activity has not been characterized. In vitro the flexibility of the structure, causing the low deacetylase activity kinetic analysis suggests that the deacetylase activity of SirT6 with an detected in SirT6 (14). Consistent with this, the in vitro catalytic acetylated H3K9 peptide is about 1000 times weaker than that of other activity of SirT7 with H3K18ac is considerably weaker than that of sirtuins (14). Both enzymatic activities in SirT6 seem to involve specific other sirtuins such as SirT1 or SirT2 (23). Considering all this evi- residues within the same catalytic domain since specific mutations dence, we aimed to determine whether SirT7 was also a dual sirtuin in this domain can selectively abrogate either mADPRT or deacety- and whether it harbored an ADPRT activity. Our first approach was lase without altering the other activity (16). The coexistence of the two to use an in vitro ADP-ribosylation assay, using [32P]-labeled NAD+ activities in the same protein is uncommon and raises many questions to determine whether SirT7 was able to transfer [32P]-ADP-ribose to about the nature of this duality, the cross-talk between the two activ- recombinant core histones. The ADPRT reactions of SirT7 on re- ities, and their specific contribution to the physiological role of sirtuins. combinant and native histones were very weak, but we detected a SirT7 is the closest sirtuin to SirT6 and probably one of the least very efficient incorporation of [32P] in SirT7, strongly suggesting that studied. Its deficiency has been linked to genome instability and SirT7 is itself a major target of this activity (Fig. 1B and fig. S1A). The several pathologies, including cancer and aging (19, 20). SirT7 is absence of a smear in the [32P] signal indicated that, as with SirT6, involved in active ribosomal DNA (rDNA) transcription (21), DNA SirT7 harbors mADPRT activity (Fig. 1C). This auto-ADPRT activity repair (20), metabolic homeostasis (22), and heterochromatin silenc- was not altered by mutations previously described as abrogating ing and active transcriptional elongation through deacetylation of SirT6 ADP-ribosylation activity toward PARP1 and KAP1 (16, 17). histone H3 lysine 18 (H3K18ac) and histone H3 lysine 36 (H3K36ac), Mutation of SirT6 G60 to alanine or its equivalent in SirT7 S115 did respectively (19, 23). Recently, a direct link between SirT7, nuclear not abrogate this auto-ADPRT activity. Moreover, the same result lamina, and LINE-1 has been described, suggesting a role for SirT7 in was obtained upon mutation of SirT6 S56 (fig. S1B), which was pre- genome organization (24). SirT7 knockout (KO) mice show severe viously shown to eliminate both SirT6 activities, or of its equivalent metabolic defects, including resistance to high-fat diet–induced fatty in SirT7 S111 (Fig. 1D). In contrast to the close parallelism observed liver, obesity, glucose intolerance, and diminished levels of hepatic between SirT6 and SirT7 ADP-ribosylation activity, SirT7 S111 or triglycerides and white adipocyte tissue (25, 26). In addition, these S115 mutations did not abrogate the deacetylase activity (fig. S1C). mice exhibit developmental defects and aging-related phenotypes This result, which was previously reported for S111 (28), suggests such as perinatal lethality, degenerative heart hypertrophy, osteopenia, that the deacetylation active site of SirT6 and SirT7 may be not as and kyphosis, and they die significantly younger than their wild- equivalent as had been proposed previously. Nevertheless, our obser- type (Wt) counterparts (20, 27). While there is a large body of evi- vations led us to consider the possibility that sirtuin auto-mADPRT dence on sirtuin function and longevity, the mechanism by which activity is not associated with the previously described active site. SirT7 controls aging and the relationship between caloric restriction response and gene expression programs have not yet been ade- SirT7 auto-mADPRT activity depends on the highly conserved quately addressed. residues, E185 and N189, present in the ELHGN motif Here, we describe how SirT7 harbors an auto-mADPRT activity To define this site, we compared all seven sirtuins at the structural at an alternative catalytic site in addition to its primary catalytic site, level. In contrast to SirT1-5, the lack of the  helix bundle (in cyan; which is responsible for the deacetylation reaction. Auto-mADPRT Fig. 1E, left) in SirT6 and SirT7 (Fig. 1E, right) induced a reorganiza- of SirT7 confers its binding capacity on specific chromatin regions tion in the center of the structure that created a big cavity (Fig. 1E, through the histone variant mH2A1.1, an interaction that is boosted red rectangle), located at the other side of the main catalytic site (in under glucose starvation (GS), which promotes accumulation of red). The cavity contained an ELHGN motif, conserved in the mH2A1 in a SirT7-dependent manner. To advance our under- SirT6/SirT7 lineage, including the common SirT6/SirT7 ortholog standing of SirT7 function under energy-stress conditions, we Caenorhabditis elegans Sir-2.4 (Fig. 1F and fig. S1D). SirT7 H187, Simonet et al., Sci. Adv. 2020; 6 : eaaz2590 24 July 2020 2 of 16 SCIENCE ADVANCES | RESEARCH ARTICLE A B Core histones kDa C Core D E Downloaded from http://advances.sciencemag.org/ on February 11, 2021 F G J Primary Cavity cat. site activity (%) of SirT7 Relative enzymatic H I Fig. 1. SirT7 harbors an mADPRT activity that is catalyzed by an alternative domain. (A) Secondary structure of the conserved domain in mammalian sirtuins. Differ- ences in  helix length between SirT1 to SirT5 and SirT6 to SirT17 are shown in blue. (B) In vitro ADPRT assay of SirT7 purified from human embryonic kidney (HEK) 293F cells ± [32P]-NAD+ with purified HeLa core histones. Autoradiography ([32P]-NAD+) and Coomassie blue staining are shown. CBB, Coomassie Brilliant Blue. (C) SirT7 titration in ADPRT assay as in (B). (D) Top: Schematic representation of SirT7 and SirT6 proteins tested. Bottom: ADPRT assay performed as in (B) and (C) with the indicated proteins. (E) Structural comparison of SirT1 to SirT5 (magenta) and SirT6 and SirT7 (green) conserved domains. The primary catalytic (cat.) site with a bound acetylated peptide is shown in red, while the  helix bundle missing in SirT6 and SirT7 is shown in cyan. Loss of this region induces the formation of a cavity in SirT6 and SirT7 (red square). (F) Alignment of the sequence present in the cavity in the SirT6 and SirT7 lineage. E185, H187, and N189 are shown in green, blue, and red, respectively. (G) Molecular model of the SirT7 catalytic domain based on the crystal structure of SirT6. H187 is oriented toward the NAD+ molecule (orange) and the acetylated substrate (red). E185 and N189 are located in the same loop as H187 although pointing in the opposite direction. (H and I) In vitro auto-ADPRT assay of SirT7 proteins purified from HEK293F cells. (J) Deacetylation and ADPRT activity of the indicated proteins. The mADPRT and deacetylation activities were quantified from assays as in (H) and as in fig. S1H, re- spectively. For the deacetylation reaction, the indicated proteins were incubated with hyperacetylated core histones purified from HeLa cells and the deacetylation of H3K18ac was monitored by Western blot. Quantifications of both activities (n = 3) are shown relative to WT SirT7 activity (*P < 0.05, **P < 0.01, and ***P < 0.005). present in the motif, is a key conserved residue among sirtuins in- action of both residues through their side chain. We paid particular volved in the recognition of acetylated substrate in the deacetylation attention to these two conserved residues because of their possible involve- activity. While H187 was oriented toward the NAD+-binding pocket ment in the ADP-ribosylation reaction: E185 is the only residue in this motif and the main catalytic site, the two flanking residues, E185 and that could initiate the reaction, whereas N189, the only residue in the motif N189, faced in the opposite direction, toward the surface of the cavity conserved exclusively in the whole SirT6/SirT7 lineage (Fig. 1F), could (Fig. 1G and fig. S1, E and F), forming a loop sustained by the inter- act as the first acceptor of the ADP-ribosyl moiety. This possibility Simonet et al., Sci. Adv. 2020; 6 : eaaz2590 24 July 2020 3 of 16 SCIENCE ADVANCES | RESEARCH ARTICLE was confirmed with the finding that the E185N and N189A muta- SirT7 affinity purification in human embryonic kidney (HEK) 293F tions both abrogated SirT7 auto-mADPRT activity (Fig. 1H). cells and identified potential SirT7 interactors by MS analysis. We A highly conservative mutation to glutamine (N189Q) had the identified the histone variant mH2A1 from among the candidates same effect, suggesting that the lack of activity of the N189 mutant (Fig. 3A and table S2). mH2A1.1 was the only one of the three was not due to an indirect structural effect (Fig. 1I). The fact that mH2A isoforms that specifically binds ADP-ribose through its N189Q is structurally very similar to the WT protein (fig. S1G) but macrodomain (Fig. 3B) (7) and interacted specifically with SirT7 cannot replace glutamine as acceptor of ADP-ribose suggests that (Fig. 3C). We next examined whether the SirT7-mH2A1.1 interac- N189 may play an essential role in the mechanism of ADP-ribosylation. tion depends on SirT7 ADP-ribosylation by incubating bacterially N189 mutants were defective in ADPRT activity but still featured expressed recombinant SirT7 (rSirT7) WT or N189A in the presence deacetylase activity, while the H187Y mutant, just two residues or absence of NAD+ and dialyzing the reaction to remove all remnant away, had the completely opposite pattern (Fig. 1J and fig. S1H). NAD+ molecules. We tested its ability to specifically pull down mH2A1.1 Considered together, these observations indicate that SirT7 auto-­ from HEK293F nuclear extracts under stringent conditions (Fig. 3D). mADPRTion is catalyzed at a conserved alternative secondary We found that only rSirT7 WT previously incubated with NAD+ active site located in a previously uncharacterized domain. was able to interact with WT mH2A1.1 under these conditions (Fig. 3E). Consistently, while the ADPRT-defective mutant N189A N189-dependent auto-mADPRT involves several residues was not able to interact in any of these conditions (Fig. 3D), the distributed around the SirT7 surface and regulates SirT7 deacetylase mutant H187Y behaved similarly as SirT7 WT (fig. S3A). genomic distribution As further evidence that SirT7 auto-mADPRT mediates this binding, Downloaded from http://advances.sciencemag.org/ on February 11, 2021 We also confirmed the conserved role of N189 since the equivalent no interaction was detected under these conditions when we used mutation in SirT6 N135 also abrogated SirT6 mADPRT activity mH2A1.1 G224E (Fig. 3E, lanes 5 and 6), a mutant unable to bind (Fig. 2A) but did not eliminate its deacetylase activity toward H3K18ac specifically to ADP-ribose (7). This is compelling evidence that (Fig. 2B and fig. S2A). There were two further lines of evidence of SirT7 auto-mADPRT mediates this binding. the importance of N189: First, the N189 mutant was significantly The in vivo interaction between SirT7 and mH2A1.1 was specific less ADP-ribosylated in vivo than was WT SirT7 (Fig. 2C); second, but weak. Considering the direct link between sirtuins and the stress mass spectrometry (MS) analysis of SirT7 auto-mADPRTion identified response, we investigated whether the SirT7-mH2A1.1 interaction eight ADP-ribosylated peptides distributed across the entire surface is boosted under specific stress conditions, such as ultraviolet (UV) of the SirT7 protein (Fig. 2, D and E; fig. S2, B and C; and table S1). or ionizing irradiation (IR), oxidative stress (H2O2), or GS. Under All but one of them were undetected in the N189 mutant (Fig. 2E, in highly stringent conditions, there was a significant increase in the magenta), confirming a key role for N189 in SirT7 ADPRT. Together, interaction between the two factors, specifically upon GS (Fig. 3F), these observations strongly suggest that SirT7 auto-mADPRTion is which was consistent with the established link between both proteins catalyzed at an alternative secondary active site. and glucose homeostasis (8, 25). MS analysis of SirT7-FLAG purified Taking advantage of our ability to specifically inhibit each of the under these stress conditions identified SirT7 ADP-ribosylation two SirT7 enzymatic activities, we next studied the impact of these under UV, oxidative, and GS conditions but not in the absence of mutations on SirT7 distribution. Our first step was to study immuno- stress (C) or under IR [Fig. 3F (bottom), fig. S3, and table S1]. The fluorescence (IF) signal of WT, H187Y, N189A, and N189Q in SirT7−/− ADP-ribosylation detected under GS and IR had been observed to mouse embryonic fibroblasts (MEFs). We observed that while H187Y be N189-dependent in our previous experiment (Fig. 2D). This dis- had an identical distribution to WT SirT7, whereby it was enriched crepancy between the stress-induced SirT7 ADP-ribosylation and in the nucleolus, the patterns of localization of N189A and N189Q binding to mH2A1.1 suggested that while ADP-ribosylation is were radically different. Both N189 mutants showed a more dispersed essential for binding to mH2A1.1, other GS-specific signaling may pattern of distribution that resulted in a loss of nucleolar enrichment be important to drive the interaction. In contrast to normal culture in around 80% of the observed cells (Fig. 2, F and G). Although we conditions [no treatment (NT)], GS consistently induced coelution observed a mild decrease in the expression of N189 mutants compared of both factors in high molecular weight fractions (around 1 MDa) to SirT7 WT and H187Y, these differences could not account for of gel filtration chromatography analysis (Fig. 3G). This observation the marked changes observed in the distribution of these proteins suggests that both factors may be part of a multiprotein complex (Fig. 2H). The altered distribution of these mutants was confirmed formed under these conditions. As further evidence of this link, by biochemical fractionation of cells expressing SirT7-­flagged SirT7 was able to immunoprecipitate endogenous mH2A1.1 specifically mutants into cytoplasm, soluble nuclear fraction (nucleoplasm), and under GS (Fig. 3H, lanes 3 and 4), an interaction that was abrogated chromatin-insoluble fraction (chromatin). As in the IF studies, H187Y by N189 mutation (Fig. 3H, lanes 4 to 6). behaved similarly to WT and was present in similar levels in the nucleoplasm. By contrast, N189A-Q mutants were significantly en- SirT7 binds to distal intergenic regions upon GS riched in the chromatin fractions and depleted in the nucleoplasm in an mH2A1-dependent manner (Fig. 2H). Overall, our results suggest that, in contrast to SirT7 deacetylase While the levels of total mH2A1 and mH2A1.2 decreased upon GS, activity, mADPRT activity, and, in particular, N189, plays a key role in we observed a consistently specific increase of mH2A1.1 under the both SirT7 genomic localization and chromatin-binding dynamics. same conditions, suggesting a direct role for this isoform in the GS response (Fig. 4A). Consistently, biochemical fractionation analysis of SirT7 interacts with the ADP-ribose binding protein SirT7 also showed a progressive accumulation of SirT7 in chromatin mH2A1.1 upon GS fractions upon GS, peaking at 40 hours of treatment (Fig. 4B). We We tried to identify SirT7 binding partners to understand the func- chose this specific timing, as we determined experimentally that the tional link between SirT7 and ADP-ribosylation. We performed GS-dependent enrichment of SirT7 was detectable from 24 hours of Simonet et al., Sci. Adv. 2020; 6 : eaaz2590 24 July 2020 4 of 16 SCIENCE ADVANCES | RESEARCH ARTICLE A D B C F Downloaded from http://advances.sciencemag.org/ on February 11, 2021 E H G Fig. 2. SirT7 N189-dependent auto–ADP-ribosylation regulates SirT7 distribution and chromatin-binding dynamics. (A) In vitro auto-mADPRT assay as in Fig. 1 with bacterially expressed rSirT6 WT or N135Q. (B) Deacetylation reaction with the recombinant bacterial SirT6 WT, N135Q, and H133Y incubated with recombinant mononucleosomes acetylated in H3K18ac (*P < 0.05). Quantification of three experiments similar to the one shown in fig. S2A. The results are represented relative to WT SirT7 activity (100%). (C) ADP-ribosylation levels of SirT7 WT or N189A expressed in HEK293F cells monitored by far Western blot with anti–pan–ADP-ribose binding re- agent. HDAC, histone deacetylase. (D) Analysis of SirT7 auto-mADPRTion identified by MS. ADP-ribosylated peptides identified in SIRT7 WT or N189A after incubation with NAD+ were analyzed using high-energy collisional dissociation (HCD) and electron-transfer/higher-energy collision dissociation fragmentation methods. The most probable ADP-ribosylated sites are highlighted in red, as the modification cannot be localized with 100% confidence. Further information is included in table S1 and in Materials and Methods. (E) Structural model of the SirT7 catalytic domain indicating localization of the ADP-ribosylated peptides identified in (C). The ADP-ribosylation N189-­ dependent (magenta) and N189-independent (orange) residues, the N189/E185 cavity (blue), and the primary catalytic site bound to acetylated peptide (red) are shown. (F) IF assay of the indicated green fluorescent protein (GFP)–tagged SIRT7 proteins expressed in SirT7−/− MEFs. Nucleophosmin (B23) was included as a nucleolar marker. DAPI, 4′,6-diamidino-2-phenylindole. A representative image of the experiment is shown. Scale bar, 5 m. (G) Quantification of IF experiment in (F), indicating the percentage of GFP-positive cells with a regular nucleolar distribution (*P < 0.05). (H) Cellular distribution of SirT7 WT, H187Y (HY), N189A (NA), and N189Q (NQ) in whole-cell extract (WCE), cytoplasm, nucleoplasm, and chromatin in NIH3T3 cells. Controls for the nuclear fraction (fibrillarin), cytoplasm (tubulin), and chromatin (histone H3) are also shown. GS and that 40 hours was the maximum safe time of GS treatment Supporting the biochemical fraction results, SirT7 chromatin before entering apoptosis. To understand the functional relation- immunoprecipitation sequencing (ChIP-seq) analysis showed that ship between the two factors under GS, we next studied the distri- GS boosted SirT7 localization to chromatin (Fig. 4C). In the case bution of SirT7 and mH2A1 under NT and GS conditions in MEFs. of mH2A1, we observed a SirT7-dependent global enrichment of Simonet et al., Sci. Adv. 2020; 6 : eaaz2590 24 July 2020 5 of 16 SCIENCE ADVANCES | RESEARCH ARTICLE A B C Macro Macro Macro D E F Downloaded from http://advances.sciencemag.org/ on February 11, 2021 1–2 MDa 670 kDa 443 kDa 150 kDa 68 kDa G H Fig. 3. SirT7 interacts with mH2A1.1 upon GS in an ADP-ribosylation–dependent manner. (A) Affinity purification of SirT7-binding factors identified the histone H2A variant mH2A1 by MS analysis (table S2). HA, hemagglutinin. (B) Schematic representation of the three mH2A isoforms, of which, only mH2A1.1 binds to ADP-ribose (7). (C) Endogenous SirT7 specifically immunoprecipitates mH2A1.1 in HEK293F cells. IgG, immunoglobulin G. (D and E) mH2A1.1 recognizes and binds ADP-ribosylated SirT7. Immunoprecipitation (IP) of bacterially expressed rSirT7 WT or N189A preincubated ± NAD+ and added to nuclear extracts of HEK293F SirT7 KO cells (CRISPR-Cas9–­ mediated KO of SIRT7) expressing mH2A1.1 WT or G224E, a mutant deficient in ADP-ribose binding. Inputs (I) and elutions (E) are shown. A similar experiment with H187Y is shown in fig. S3A. (F) Top: Interaction between endogenous SirT7 and mH2A1.1 under high-stringency conditions upon different types of stress in HEK293 cells. C, un- treated; IR, 7-gray ionizing irradiation; H2O2, oxidative stress. Western blot of the input and elution of immunoprecipitation experiments with anti-SirT7 antibody under these conditions. Bottom: Summary of the ADP-ribosylation events detected by MS in SirT7-FLAG expressed in the same cells and purified under the same stress condi- tions. Further details are shown in table in fig. S3B and in table S1. (G) Superose 6 gel filtration chromatography of nuclear endogenous proteins from nuclear extracts of HEK293F cells under normal cell growth (NT) or upon GS. Fraction numbers and approximate molecular weights (MW) are indicated. Western blot of SirT7 and mH2A1.1 are shown. (H) High-stringency immunoprecipitation of endogenous mH2A1.1 by WT or N189A SirT7 in HEK293F cells treated under normal conditions or under GS. In- puts (I) and elutions (E) are shown. mH2A1 in Wt cells upon GS compared with NT conditions since SirT7 to the intergenic regions and suggest that SirT7 has a direct this effect was abrogated in SirT7-deficient MEFs (Fig. 4D). In role in auto-mADPRTion. addition, 72.4% (1711 of 2638) of SirT7-associated genes were also enriched by mH2A1. SirT7 accumulated mostly in intergenic regions The SirT7/mH2A1 regulatory axis regulates the expression [transcription start site (TSS) ± 50 to 500 kb] rather than in proximal of a subset of genes upon GS promoters (Fig. 4E). Genomic Regions Enrichment of Annotations To clarify the mode of action of SirT7 in the genes regulated by Tool (GREAT) analysis associated these regions with specific genes SirT7/mH2A1 when they are stressed, we performed RNA sequencing (Fig. 4F and fig. S4), suggesting that the sequences correspond to (RNA-seq) in Wt and Sirt7−/− MEFs under NT and GS as for the gene regulatory regions. The average distance from these sites to the ChIP-seq analyses. Of the 1711 mH2A-containing regions occupied associated genes ranges between 50 and 500 kb upstream or down- by SirT7, GS induced the up-regulation and down-regulation of 257 stream of the coding region. Many of the SirT7-associated genes and 427 nearby genes, respectively, in Wt MEFs (Fig. 5, A and B). mapped by GREAT were associated by Kyoto Encyclopedia of Genes This up- or down-regulation upon GS was SirT7 dependent in the and Genomes (KEGG) analysis with cell signaling, the majority of case of 143 and 277 genes, respectively (Fig. 5B). There was an in- which directly or indirectly participate in regulating metabolic crease in the levels of mH2A1 around these genes upon GS that was homeostasis (Fig. 4F). abrogated in SirT7-deficient cells (Fig. 5C and fig. S5). Among these Confirming our previous results, the recruitment of SirT7 to genes, we identified some that encoded growth factors, signaling these regions upon GS was mH2A1 dependent since its short hairpin transduction mediators involved in metabolism and differentiation, RNA (shRNA)–driven down-regulation abrogated SirT7 enrichment hormone receptors, enzymes, transcription factors, extracellular (Fig. 4G). These results indicate that, upon GS, mH2A1.1 recruits factors, and cytokines. Many of these genes are directly or indirectly Simonet et al., Sci. Adv. 2020; 6 : eaaz2590 24 July 2020 6 of 16 SCIENCE ADVANCES | RESEARCH ARTICLE A B C D Log2 (ChIP/input) Chromatin fraction Nucleoplasmic fraction −5 kb +5 kb −5 kb +5 kb F P value P value Odds ratio Combined score E −5 kb from TSS +5 kb around TSS +5 kb from TSS Downloaded from http://advances.sciencemag.org/ on February 11, 2021 (kb) G 12 kb 166 kb 54 kb 59 kb 73.4 kb Fig. 4. mH2A1 recruits SirT7 to distal intergenic regions associated with metabolic genes upon GS. (A) Levels of three mH2A isoforms in WCE of NIH3T3 cells trans- fected with scramble shRNA or mH2A1 shRNA and cultured under normal or GS conditions. (B) Levels of endogenous SirT7 and histone H3 in chromatin and nucleoplasm fractions purified from Wt and Sirt7−/− MEFs cultured under normal or GS conditions during the indicated times. (C) Venn diagrams showing the intersection of SIRT7-­ associated genes with mH2A1-enriched genes in Wt cells under NT (top) or GS (bottom). SirT7-associated and mH2A1-enriched genes were derived from GREAT analysis (fig. S3B and Materials and Methods). (D) Average enrichment of mH2A1 at all genes in Wt (left) or SirT7−/− MEF (right) cells under NT (black) or GS (red) conditions. Data are expressed as the log2 ratio of reads per kilobase of transcript per million mapped reads–normalized ChIP/input signals. (E) Distribution of sites occupied by SirT7 upon GS around the TSS by GREAT analysis. The values for each bin from the TSS are shown above each bar (TSS: −5 kb, 5 to 50 kb, 50 to 500 kb, and >500 kb). (F) KEGG cell signaling pathways for SirT7-associated genes mapped by GREAT analysis under GS in MEF cells. The signaling pathways were ranked by their combined score provided by Enrichr analysis. cGMP-PKG, guanosine 3′,5′-monophosphate–protein kinase G; cAMP, cyclic adenosine 3′,5′-monophosphate; TCA, tricarboxylic acid. (G) SirT7 ChIP-qPCR (quantitative polymerase chain reaction) analysis of SirT7 binding sites associated with mH2A1 at distal regions upon shRNA-mediated down-regulation of mH2A1 under normal and GS conditions in NIH3T3 cells. The amplified regions (red) and their distance to each gene are indicated in the upper part of each graph. Each SirT7 ChIP was normalized with respect to its own input. SEM from n = 4. Two-tailed t test (*P < 0.05 and ***P < 0.005). associated with the second messenger regulatory system, including the binding protein 1 (NECAB1), and the transcription factors forkhead Ca+2-dependent pathways and the adenosine 3′,5′-monophosphate box protein A2 (FOXA2) and myocyte-specific enhancer factor 2C (cAMP)/adenylate cyclase or guanosine 3′,5′-monophosphate (cGMP)–­ (MEF2C). CTGF was particularly interesting, given its key role in dependent protein kinase signaling pathways. We also identified differentiation, proliferation, glucose homeostasis, and insulin-like many factors associated with heterotrimeric GTP-binding protein growth factor 1 (IGF-1) and transforming growth factor– signal- (G protein) signaling, such as the G protein–coupled receptor ing and its link to calorie restriction (CR) and aging (29). signaling pathways, some of which have been linked to regulation Reconstitution experiments with SirT7 WT, H187Y, and N189A of cAMP signaling in neural systems. Genes down-regulated upon in Sirt7−/− MEFs revealed that both enzymatic activities of SirT7 are GS were involved in cAMP signaling pathway factors, proliferation, and required to produce the GS-associated expression profile of the tested cell differentiation, while up-regulated genes were mainly associated genes, as well as the associated enrichment of mH2A detected in with cGMP-dependent protein kinase and Rap1 signaling, metabolism, these genes upon GS (Fig. 5D). Consistent with a direct role of and, to a much lesser extent, cAMP/adenylate cyclase signaling (fig. S6A). SirT7 in these genes through binding to the distal regulatory re- Among these up- or down-regulated factors that we identified are, gions, we also observed SirT7-dependent H3K36 deacetylation in for instance, the connective tissue growth factor (CTGF), the adenylate these distal sequences, which was dependent on both catalytic activities cyclase regulator adrenoceptor alpha 2A (ADRA2A), the guanylate-­ (fig. S6B). These lines of evidence suggest a link between this binding protein 6 (GBP6), the histone deacetylase 9 (HDAC9), the deacetylation event and mH2A gene enrichment. As mH2A1 was calcium-­binding signaling protein N-terminal EF-hand calcium previously associated with H3K27me3 (30), we also analyzed the Simonet et al., Sci. Adv. 2020; 6 : eaaz2590 24 July 2020 7 of 16 SCIENCE ADVANCES | RESEARCH ARTICLE A B C D Downloaded from http://advances.sciencemag.org/ on February 11, 2021 E Down-regulated Up-regulated Fig. 5. SirT7 dual activity promotes mH2A1 enrichment in the promoters of associated genes, resulting in transcription up-regulation or down-regulation. (A) Heat map showing RNA expression changes relative to NT (as log2 magnitude of difference between GS and NT) conditions in Wt and SirT7-deficient MEFs. (B) Pipeline applied to RNA-seq data to filter genes associated with SirT7/mH2A1 in Wt and Sirt7−/− MEF cells treated under GS or NT. The analysis was restricted to genes that (i) were associated with SirT7 via GREAT and were mH2A1-enriched, (ii) showed a log2 fold change (FC) of expression between WT-GS and WT-NT >0.6, and (iii) showed a differ- ence between WT and KO log2 FC (GS versus NT) of >0.3 (table S3). (C) mH2A1 ChIP-seq signals across ctgf, a gene differentially enriched in mH2A1 upon GS in Wt MEFs compared with NT. (D) Top: Real-time qPCR (RT-qPCR) analysis of genes regulated by SirT7 upon GS and NT. The expression of SirT7 in SirT7−/− MEFs was rescued by retroviral-­ mediated gene transfer of SirT7 WT, H187Y(HY), N189A(NA), and empty vector (−). SEM from n = 4. Two-tailed t tests (*P < 0.05, **P < 0.01, ***P < 0.005, and ****P < 0.001). Bottom: Relative mH2A1 enrichment (GS versus NT) by ChIP-qPCR analysis at specific regions around the TSS of the indicated genes [gbp6, −6 kb; necab1, −3 kb; lair1, −27.5 kb; ctgf, +600 base pairs (bp); adra2a, −5 kb; and nrip3, +7 kb]. SEM from n = 3. One-way analysis of variance (ANOVA) (*P <0.05, **P < 0.01, and ***P < 0.005). a.u., arbitrary units. (E) Chromatin state transitions induced by GS in Wt and SirT7-deficient cells. The colors of the arrows indicate the frequency (%) of the transition as stated in the color scale (right). Bottom right: State map illustrating the specific combination of mH2A1 and/or H3K27me3 in the four chromatin states defined in the analysis. U1, without H3K27me3 or mH2A1; U2, mH2A1; U3, H3K27me3; U4, enriched by H3K27me3 and mH2A1. correlation between this mark and mH2A/SirT7 through a global H3K27me3 in these sequences upon GS. We observed a positive unbiased analysis of chromatin state changes from NT to GS condi- effect of SirT7 on mH2A recruitment upon GS in up-regulated and tions in Wt and Sirt7−/− MEFs. We defined four states (U1 to U4), down-regulated genes when H3K27me3 was not present. In contrast, depending on the appearance or disappearance of mH2A and/or when mH2A1 and H3K27me3 were both present, SirT7 had the Simonet et al., Sci. Adv. 2020; 6 : eaaz2590 24 July 2020 8 of 16 SCIENCE ADVANCES | RESEARCH ARTICLE opposite effect on mH2A enrichment (Fig. 5E). These results because the ADPRT activity of some of the sirtuins tested was con- suggest an antagonistic interplay between SirT7 and H3K27me3 in siderably weaker and less specific than the deacetylase activity and determining mH2A enrichment upon metabolic stress (Fig. 5E). was dependent on the presence of an acetylated peptide (11, 34). Moreover, the dependency of this activity on the deacetylation reac- SirT7 dependence on the expression of these genes is tion of yeast Sir2 was also reported, suggesting that it may be a side recapitulated in in vivo mouse models of CR effect of primary catalytic deacylation or a technical artifact linked To confirm the contribution of SirT7 to GS response in vivo, we to the ability of sirtuins to hydrolyze NAD+ as an intermediate step generated a CR model in Wt and Sirt7−/− mice (Fig. 6A) and we before deacetylation (35). It is of particular note that the vast majority performed several correlative analyses in the livers of these mice. of this evidence was based on in vitro ADP-ribosylation assays and Several evidences supported our previous studies with GS. First, we was not validated by other techniques. In this sense, we report the studied the expression of the SirT7/mH2A-associated genes that we first MS analysis of sirtuin ADPRTion and describe a consistent and had identified earlier (Figs. 4 and 5). Consistently, the SirT7-dependent reproducible pattern of N189-dependent ADPRTion in class IV sirtuins, expression profile of these genes under CR versus ad libitum (AL) the common lineage of SirT6 and SirT7 from early eukaryotes to fully replicated our results in cell culture with GS versus NT mammals. We demonstrate not only that the role of the ELHGN (Fig. 6B). Second, as in the case of GS, SirT7 was enriched in chromatin motif in this activity is a highly conserved one but also that the of these livers upon CR, in part, because of an up-regulation of the catalytic site is physically separated, being located in a different levels of SirT7 under these conditions (Fig. 6C). Third, we demon- structural domain. Our studies suggest that N189 could play an strated CR-specific interaction between endogenous SirT7 and important role as the first acceptor of the ADP-ribose molecule Downloaded from http://advances.sciencemag.org/ on February 11, 2021 mH2A1.1 in these livers, further supporting the functional parallelism generated by E185 before its transfer to the final target. This is sup- between CR and GS (Fig. 6D). Fourth, trying to understand the ported by the direct interaction with E185 and the different behavior functional implications of the SirT7-mH2A1 axis under CR condi- of N189Q compared with WT, despite its close structural similarity tions, we studied the status of autophagy in these samples, given the (fig. S1E). Given the wide distribution of these modified residues direct involvement of cAMP, cGMP, and Ca+2-dependent signaling across the surface of SirT7, the automodification probably involves in the control of autophagy and its established implication in the the intramodification of SirT7 oligomeric units within the SirT7 response to CR and aging (31–33). For that purpose, we analyzed complex, similar to what has been described for PARP1 (36). the levels of a well-established marker of autophagy, the formation Although auto-ADPRTion had already been described in the early of the form II of microtubule-associated protein light chain 3 studies of sirtuin, its role has never been properly characterized. (LCIII-2). The rate of formation of LCIII-2 directly indicates the Our report suggests a direct role of this modification in SirT7 global rate of autophagosome formation and activity. As expected, in Wt distribution and in its chromatin-binding dynamics. PARP1 auto-­ animals, we observed an increase of LCIII-2 under CR (Fig. 6E), in ADPRTion was also proposed to regulate PARP1 chromatin-binding agreement with an increase in autophagy as a response to CR as has dynamics (37). As in the case of SirT7, mH2A1.1 has been also in- been described (33). In contrast, in SirT7-deficient mice, while we volved in PARP1 recruitment, although in this case, it was linked to observed increased rates of autophagy in AL conditions, these PARP1 inactivation (38). Although our work has focused on auto-­ decreased significantly compared to CR in Wt animals (Fig. 6E). ADPRTion, the high degree of conservation within the class IV Supporting a role for SirT7 in CR-dependent autophagy, we observed sirtuins and the considerable magnitude of the cavity, which makes a similar pattern in Beclin-1 (Fig. 6F), another key marker of auto- it able to accommodate a wide range of proteins (Fig. 1E and fig. S1D), phagy involved in the formation of autophagosomes (33). These suggest that this novel domain may target other substrates. observations suggest that SirT7 plays a key role, at least in the liver, In contrast to what was reported for SirT6-dependent ADPRTion in the control of autophagy in the physiological response to CR con- of PARP1 and KAP1 (16, 17), SirT6 and SirT7 auto-mADPRTion ditions. These results provide further evidence of a key role for the does not depend on any key residues involved in the primary SirT7/mH2A axis in glucose/CR signaling. Overall, our findings not deacetylation domain, such as SirT6-S56/SirT7-S111, or SirT7-S115 only suggest a key role for SirT7 in aging through gene expression (Fig. 1D and fig. S1B). This suggests that there may be two types of regulation of second metabolism signaling but also suggest that this sirtuin-related ADP-ribosylation events, one that takes place at the role is directly related to CR. primary catalytic site and that is probably directly associated with the main deacetylation reaction and another that is dependent on the ELHGN motif including auto–ADP-ribosylation and modifica- DISCUSSION tion of other protein targets. This hypothesis suggests an interesting We report ADPRT activity in SirT7 and identify a novel ADP-­ line of future work to confirm its validity and, thereafter, to define ribosylation domain containing the ELHGN motif. The direct link the extent and the specific range of substrates associated with each between this modification, SirT7 dynamic distribution, and mH2A1 of these ADP-ribosylation events. binding in response to GS provides a functional explanation of Another major finding of our work is the first evidence of a sirtuin enzymatic duality and suggests a key role for SirT7 in CR direct functional relationship between sirtuins and mH2A through signaling and aging (Fig. 6C). this ADPR activity. This link is supported by the ability of mH2A1.1 The catalytic duality of sirtuins has been widely debated since it to bind to the sirtuin product O-acetyl-ADP ribose (8). Although was hypothesized that all sirtuins may have the ability to transfer we have demonstrated that SirT7 auto-mADPRTion is essential for ADP-ribosyl moieties to target proteins (10, 11). Originally described the interaction with mH2A1.1 between factors and the recruitment of as a potential enzymatic alternative to the main deacylation/ SirT7 to these genomics sites, our observations suggest that there are deacetylation activity, it was assumed to act alongside the primary other factors involved. Thus, while we detected auto-mADPRTion activity at the same catalytic site. This perception arose primarily upon different types of stress such as GS, UV, and H2O2, we only Simonet et al., Sci. Adv. 2020; 6 : eaaz2590 24 July 2020 9 of 16 SCIENCE ADVANCES | RESEARCH ARTICLE A B Ad libitum (8 weeks) C D E Ratio he F G Downloaded from http://advances.sciencemag.org/ on February 11, 2021 region cAMP/ad. cyclase Au messenger autophagy Aging Fig. 6. SirT7 and mH2A1 axis plays a key role in CR. (A) Model validation studies in WT and Sirt7−/− mice fed AL or calorie restricted (CR, 30%) for 8 weeks. (B) RT-qPCR analysis of the indicated genes in liver samples from Wt and Sirt7−/− mice fed AL or CR. Three animals were analyzed for each condition. Each quantification was generat- ed from three replicates. Probabilities are those associated with one-way ANOVA (*P < 0.05, **P < 0.01, and ***P < 0.005). (C) Levels of SirT7 in liver samples from Wt and Sirt7−/− mice AL and CR after subcellular fractionation. WCE and chromatin fractions are shown. (D) SirT7 immunoprecipitation of mH2A1 in the same liver samples. Inputs (I) and elutions (E) are shown. (E) Autophagy activity in the Wt and Sirt7−/− livers under AL or CR monitored by levels of formation of LCIII-2. Left: A representative Western blot of n = 5 replicates used in the quantification shown. Right: Quantification of the relative accumulation of LCIII-2 compared to glyceraldehyde-3-phosphate dehydro- genase (GAPDH) analyzed with a two-tailed t test (*P < 0.05). GAPDH was used as a loading control, as we did not detect a significant alteration of the levels of the protein in our conditions (data not shown). (F) Similar analysis (n = 5) of Beclin-1 as in (E). (G) Model proposed for the dual SirT7/mH2A regulatory axis in GS. On the basis of our data, we speculate that this axis is also involved in CR and aging. Nutrient stress induces SirT7 auto-mADPRT, which leads to mH2A-dependent recruitment of SirT7 to distal regulatory regions and subsequent mH2A enrichment around the associated genes. This axis plays a key role in CR in vivo and possibly in aging by modulating key signaling pathways. observed a boost in the interaction between SirT7 and mH2A1 both up- and down-regulation in these genes suggests that its main under GS conditions (Fig. 3F and fig. S3B). This suggested that role in this functional context is to promote mH2A enrichment, there must be other signaling events involved that provide specific which, in turn, may regulate positive or negative gene expression. response under GS. Given the direct implication of stress-dependent The analysis of chromatin states suggests that H3K27me3 is kinases such as AMP kinase in the regulation of SirT7 exit from the antagonistic to the interplay between SirT7 and mH2A. This intrigu- nucleolus under energy deprivation conditions (39, 40), we speculate ing observation deserves further study. The mechanism by which that phosphorylation of SirT7 may be also required to allow the ob- SirT7 promotes this long-range effect on mH2A enrichment around served activation of the SirT7/mH2A1 regulatory axis under these genes is unclear, and it may involve chromatin looping, as it occurs conditions. However, we cannot discard that other reported modi- in enhancer regions. This possible mechanism is consistent with the fications of Sirt7, such as arginine methylation or acetylation, may recently established link between SirT7 and the nuclear lamina and be involved. Future studies should characterize these other signaling chromatin organization (24). Our results suggest that the role of events involved in SirT7 activation upon GS and determine their SirT7 in these genes requires both enzymatic activities, which fur- contribution to the SirT7/mH2A1 functional relationship. ther highlights the essential role of sirtuin duality in this regulatory Our results demonstrate that GS triggers SirT7 relocalization mechanism. In agreement with this, we detected active deacetylation mainly to distal regulatory regions mediated by mH2A1.1. These of H3K36 in the distal regulatory regions upon the arrival of SirT7, regions do not appear to be canonical enhancer regions because which may favor the mechanism of mH2A enrichment. However, they are not enriched in enhancer-specific histone marks (data not our studies also suggest that in addition to this proposed mechanism, shown). In turn, SirT7 promotes increased mH2A1 occupancy levels SirT7 may also regulate the expression of some of these target genes in the nearby gene, which results in the transcriptional activation or independently of the up-regulation of mH2A1. For instance, reexpres- repression of the gene (Fig. 5). The fact that SirT7 seems to induce sion of H187Y or N189A in SirT7−/− MEFs induced mild significant Simonet et al., Sci. Adv. 2020; 6 : eaaz2590 24 July 2020 10 of 16 SCIENCE ADVANCES | RESEARCH ARTICLE changes of expression in some of these genes upon GS without altering associated to a decreased autophagy activity. Under AL conditions, significantly the levels of mH2A1. Although, in the majority of these loss of SirT7 seems to promote an up-regulation of autophagy cases, this mH2A1-independent effect seems to be minor, this may (Fig. 6, E and F), which may suggest that SirT7 plays an opposite reflect that both SirT7 enzymatic activities target other proteins in these role in autophagy in the absence of stress. Many genomic structural genes. Since SirT7 has been shown to interact with a wide range of alterations caused by aging in mouse livers seem to take place in factors from chromatin enzymes to transcription regulators and target regions 50 to 500 kb upstream and downstream of the TSS of genes, histone and nonhistone proteins, the observed effect may be due to a which resembles the SirT7 binding profile upon GS. Some of the different combination of SirT7-associated factors depending on each factors regulated by SirT7, such as CTGF or FoxA2, have been specific gene context. Future studies should define these mechanisms, directly implicated in CR signaling and aging (46, 47). Further work the factors involved, and their functional link to mH2A1. is required to assess the contribution of these targets to the accelerated This study also suggests that the Sirt7/mH2A axis regulates a wide aging phenotype associated with SirT7. Our work not only offers a range of factors under GS. We were able to correlate the expression new insight into sirtuin duality and highlights the crucial role of profile of these genes between GS stress conditions in primary sirtuins in the epigenetic regulation of metabolism and CR signal- MEFs and CR treatment in the livers of these mice. In these sense, ing but also provides a broader perspective on the dynamics and we observed, as in GS, a SirT7-depenent expression of tested genes regulation of sirtuin function. upon CR, an increased localization of SirT7 in chromatin fractions, and a specific CR-dependent binding of SirT7 and mH2A1 in these livers (Fig. 6, C and D). Moreover, in agreement with previous studies, MATERIALS AND METHODS Downloaded from http://advances.sciencemag.org/ on February 11, 2021 we detected not only gene expression profile differences associated with Animal studies, CR, and studies caloric restriction [e.g., ADRA2A, CTGF, and leukocyte-associated For the CR procedure, 8-week-old male Wt and SirT7 −/− animals immunoglobulin-like receptor 1 (LAIR1)] (41) but also new potential (27) were housed in individual cages and randomly allocated into AL markers involved in obesity [nuclear Receptor Interacting Protein 3 or CR group. The average of food eaten AL for each mouse was (NRIP3)] (42) and immune response [heparan sulfate-glucosamine determined by weighing the remaining food on a daily basis for 3-sulfotransferase 4 (HS3ST4) and GBP6] (43). Their stress-dependent 1 week. The CR regimen was applied by feeding the animals with expression response profile was SirT7 dependent, confirming the 70% of the calculated amount daily for 8 weeks. All animal experiments function of SirT7 as a nutrient sensor in response to calorie-restricted were approved by the institute’s Institutional Animal Care and Use diet. The observed overrepresentation of second messenger signaling Committee and the local authorities (RP Darmstadt, Germany) among these factors, particularly that related to calcium-associated sig- and performed in accordance with the Guide for the Care and Use naling, is quite unexpected because it suggests that this is a major of Laboratory Animals published by the National Institutes of target of the SirT7/mH2A signaling axis. Considering the wide range Health (NIH). of general and tissue-specific functions regulated by these pathways, the functional relevance of this regulatory axis may extend beyond SirT7 computational molecular model glucose metabolism. cAMP/adenylate cyclase signaling was shown For in silico SirT7 structural model, an initial homology model was to regulate SirT6 activity, which suggests that regulatory feedback constructed for human SirT7 using the coordinates of the determined may operate in this context. SirT7 has been shown to regulate several x-ray crystal structure of human SirT6 (Protein Data Bank ID: 5M6F; key stress-dependent pathways such as p53, FOXO, Akt, mitogen-­ 1.87 Å resolution, 42% of sequence identity). Modeller 9.12 was activated protein kinase, or extracellular signal–regulated kinase, used to model the nondetermined regions of the protein. The side among others (27, 44, 45). The fact that the second messenger signal- chain conformations for nonconserved residues were positioned ing plays an essential key role in many of these pathways suggests that according to SCWRL4 (Krivov). The protein was embedded in a SirT7 regulates these pathways through this SirT7/mH2A1 regulatory TIP3P water box. The initial system was energy-minimized and axis. Our studies suggest that the SirT7/mH2A1 signaling axis is in- subjected to 10 ns of molecular dynamics equilibration and, lastly, volved in cellular adaptation to glucose deprivation. The studies with to a production stage extending to 150 ns. All the simulations were the CR model and the correlation that we have been able to establish performed with GROMACS 5.0 simulation package. The molecular between GS model in cell culture and the CR model in mouse lead us model of SirT7 was used as the initial model to introduce H187Y to speculate that the SirT7/mH2A1 interplay may have an important and N189A mutations. For comparison purposes, SirT1 to SirT5 role in physiological short- and long-term adaptation to nutrient depri- structures were superimposed to SirT6 structure and SirT7 structural vation. This hypothesis should be further validated in future studies. model using PyMOL (The PyMOL Molecular Graphics System, Another significant observation of our work is that SirT7 is version 1.8, Schrödinger LLC.). important in the aging-associated changes linked to glucose homeo- stasis regulation. Although SirT7 was previously associated with In vitro enzymatic assays aging at different levels, from genome stability and structure to ADP-ribosylation assays were performed in a total volume of 50 l in ribosomal biogenesis and mitochondrial function (20–22, 24), our 50 mM tris-HCl (pH 7.8), 1 mM MgCl2, 0.3 mM dithiothreitol (DTT), study is the first to establish a link between SirT7 and CR signaling. 1 M unlabeled NAD+, and 8 Ci of [32P]-NAD+ (800 Ci/mmol; This link is supported by the in vivo metabolic alterations detected PerkinElmer) incubated with 3 to 5 g of recombinant protein from in SirT7 KO mice, such as reduced IGF-1 plasma levels and hepatic Escherichia coli (BL21) or purified from HEK293F cells. The reactions steatosis (20, 22). The decreased levels of autophagy observed in were incubated at least for 3 hours at 37°C. Reactions were stopped SirT7-deficient animals upon CR may also point directly to an effect by adding 5× Laemmli sample buffer (supplemented with 10% on aging given the accelerated aging phenotype observed in SirT7−/− 2-mercaptoethanol) to a final concentration of 1× and then separated by mice and that development of mammalian aging has been directly SDS–polyacrylamide gel electrophoresis. Following gel electrophoresis, Simonet et al., Sci. Adv. 2020; 6 : eaaz2590 24 July 2020 11 of 16 SCIENCE ADVANCES | RESEARCH ARTICLE proteins were then transferred from gel to PVDF membrane and irradiation at 254 nm (100 J/m2) in a Stratagene Stratalinker 2400 UV then stained with Coomassie Brilliant Blue (Sigma-Aldrich). The (Agilent Technologies) and (ii) gamma irradiation delivered using an incorporation of ADP-ribosylation into proteins was visualized aluminum filter at 100 kV peak in a Faxitron cabinet x-ray system by autoradiography. (Faxitron Corp.). Deacetylation assays for SirT6 were performed in 50 mM tris- HCl (pH 8.0), 150 mM NaCl, and 1 mM MgCl2 with 2 mM NAD+ Immunoprecipitations and Western blot (Sigma-Aldrich) in the presence of 3 g of rSirT6 and 1 g of semi- Whole-cell proteins and nuclear extracts were prepared according synthetic recombinant mononucleosomes containing acetyl-lysine to the Dignam protocol. Nuclear extracts were treated with Benzonase at position 18 of histone H3 (H3K18ac dNuc, EpiCypher) for (Sigma-Aldrich), cOmplete Protease Inhibitor (Roche), and 2 mM 5 hours at 30°C. In the case of SirT7, reactions were performed in 50 mM MgCl2 for 6 hours at 4°C. The lysate was clarified by centrifugation, tris-HCl (pH 7.8), 1 mM MgCl2, and 0.3 mM DTT with 5 mM NAD+ and protein supernatant was incubated with the respective cross-­ with 3 to 5 g of SirT7 and ~0.5 g of hyperacetylated histone sub- linking primary antibody to Protein G Agarose resin (EMD Millipore). strates for 3 hours at 37°C. The acetylation status was determined The cross-linking was performed in 20 mM dimethyl pimelimidate by Western blot analysis using specific antibodies. (DMP) (Pierce, Thermo Fisher Scientific, MA, USA) dissolved in 0.2 M sodium borate (pH 9.0). After quenching with 0.2 M ethanol- Recombinant protein purification amine (pH 8.0), the cross-linked beads with the antibody were stored Human cDNAs of SirT6 or SirT7 were cloned into the pET-30b at 4°C in phosphate-buffered saline (PBS) until use. The affinity-­ expression vector (Novagen, Germany), containing a hexahistidine purified protein complexes from HEK293F cells were gently washed Downloaded from http://advances.sciencemag.org/ on February 11, 2021 tag at the C terminus. The constructs were expressed in E. coli BL21 at least three time with buffer BC300 [20 mM tris-HCl (pH 8.0) (DE3) grown at 37°C unit optical density at 600 nm of 0.5 and then 300 mM KCl, 10% glycerol, 1 mM EDTA, 1 mM DTT, 0.05% Nonidet induced with 1 mM isopropyl--d-thiogalactopyranoside for 4 hours. P-40, and 0.1 mM PMSF]. Purification of tagged proteins was done Harvested cells were resuspended in lysis buffer [20 mM Hepes (pH 7.9), at least five times with BC500 [20 mM tris-HCl (pH 8.0) 500 mM 500 mM NaCl, 10% glycerol, 1 mM EDTA (pH 8.0), 0.1% NP-40, 20 KCl, 10% glycerol, 1 mM EDTA, 1 mM DTT, 0.1% Nonidet P-40, mM 2-mercaptoetanol, and 1 mM phenylmethylsulfonyl fluoride and 0.1 mM PMSF] and then eluted either with synthetic peptide (PMSF)] and lysed by sonication. After centrifugation at 12,000g at (0.4 g/ml) from GenScript (Piscataway, NJ, USA) or by acidifica- 4°C for 1 hour, the cleared lysates were loaded onto Ni–nitrilotriacetic tion using a buffer containing 0.2 M glycine (pH 2.3). acid agarose resin (Qiagen) by gravity flow. After several washes Endogenous SirT7 immunoprecipitated from liver tissues were with lysis buffer and BC500 [20 mM tris-HCl (pH 8.0), 500 mM performed by using a Polytron homogenizer in ice-cold radio- KCl, 10% glycerol, 1 mM EDTA, 1 mM DTT, 0.1% Nonidet P-40, immunoprecipitation assay buffer [50 mM tris-HCl (pH 8.0), 150 mM and 0.2 mM PMSF], rSirT7 was eluted using 100 mM imidazole and NaCl, 1% NP-40, 0.25% sodium deoxycholate, and cOmplete Protease subsequently dialyzed in BC100. Recombinant proteins were then Inhibitor (Roche)]. Whole-cell extract was treated with Benzonase kept at −80°C. for 6 hours in 1 mM MgCl2 at 4°C and then sonicated on ice. After Preparation of recombinant histones were performed from E. coli centrifugation at 17,000g for 5 min at 4°C, protein extracts were in- Bl21 (DE3) cells as described elsewhere (48). Histone octamers were cubated with anti-SirT7 cross-linked beads by gently rotating at 4°C concentrated to 3 to 15 mg/ml, adjusted to 50% (v/v) glycerol, and overnight. After three washes with ice-cold BC350 and 0.075% NP- stored at −80°C. 40, SirT7 and Sirt7-interacting proteins were eluted from the resin with 0.2 M glycine (pH 2.3). Coimmunoprecipitation experiments Cells transfections and treatments between rSirT7 WT and N189A (preincubated ±NAD+) with nuclear MEFs derived from Wt and SirT7 KO mice were generated from extracts from HEK293F SirT7 KO cells (CRISPR-Cas9–mediated day 13.5 embryos by standard methods. For retroviral infection of KO of SIRT7) expressing hemagglutinin (HA)–mH2A1.1 WT or primary MEFs and NIH3T3 cells, Platinum-A cells were transiently G224E were performed in BC300. After three washes with ice-cold transfected using polyethylenimine. Cells were incubated with BC350 and 0.075% NP-40, proteins were eluted from the resin with virus-containing supernatant in the presence of polybrene (8 g/ml). 0.2 M glycine (pH 2.3). Densitometric analysis of the Western blots In the case of shRNA in NIH3T3 cells, after 48 hours of infection, was performed with ImageJ software. infected cells were selected for 72 hours with puromycin (2.0 g/ml). The following antibodies were used for Western blotting: anti-Flag Plasmids encoding mouse shRNAs of mH2A isoforms were obtained M2 Antibody (F1804, Sigma-Aldrich), anti-fibrillarin (B-1, Sc-166001, from Addgene (Cambridge, MA, USA): pSUPER retro puro mH2A1 Santa Cruz Biotechnology), anti-H3 (#9715, Cell Signaling Technology), shRNA (#30517) and pSUPER retro puro Scr shRNA (#30520). anti-tubulin (ab-6160, Abcam), anti-glyceraldehyde-3-phosphate HEK293F cells were transiently transfected with plasmids using 3 l of dehydrogenase (#2118, Cell Signaling Technology), anti-SirT7 (#5360, polyethylenimine (1 mg/ml) per g of DNA. The following plasmids Cell Signaling Technology), anti-SirT7 (sc-365344, Santa Cruz Bio- were used for transfection: pcDEF-Flag-SirT7 (WT, H187Y, N189A, technology), anti-mH2A1.1 (D5F6N, Cell Signaling Technology), and N189Q) and pCMV6-SirT7–green fluorescent protein (GFP) anti-mH2A1.2 (#4827, Cell Signaling Technology), anti-mH2A1 (OriGene, RG205658). (ab37264, Abcam), anti-mH2A.2 (gift from M.B.), anti-histone For GS treatments, MEFs, NIH3T3, and HEK293F cells were H2A (#12349, Cell Signaling Technology), anti-HA (H6908, Sigma-­ treated 24 to 40, 12, and 4 hours, respectively, before harvesting Aldrich), anti-myc (#2276, Cell Signaling Technology), anti-H3K18ac with glucose-free Dulbecco’s modified Eagle’s medium (Gibco) (#9675, Cell Signaling Technology), anti-H3K27me3 (#9733, Cell containing 10% dialyzed fetal bovine serum. For oxidative stress, Signaling Technology), anti-LC3B (L7543, Sigma-Aldrich); anti– cells were treated with 1 mM H2O2 for 1 hour before harvesting. Beclin-1 (#3738, Cell Signaling Technology), and anti–pan–ADP-­ Cells were irradiated by exposure in the following conditions: (i) UV ribose binding reagent (MABE1016, Millipore). Simonet et al., Sci. Adv. 2020; 6 : eaaz2590 24 July 2020 12 of 16 SCIENCE ADVANCES | RESEARCH ARTICLE Subcellular fractionation resolution of 120,000; AGC was set to 5 × 105; isolation window of To determine the amount of SirT7 proteins in the cytoplasm, nucleo- 2 m/z and maximum injection time of 240 ms were used). Precursors plasm, and tight chromatin fraction, NIH3T3 cells were transfected masses selected in previous cycle fragmentation were excluded for with SirT7-HA WT, H187Y, N189Q, and N189A. Cytoplasmic and further selection for 60 s, and the exclusion window was set at 10 parts nucleoplasm fraction were obtained with the Dignam protocol ex- per million (ppm). Analysis of SirT6WT/S56A in vitro assays and of traction (fractions A and C), and the remaining chromatin fraction SirT7 under stress conditions were performed using an Eclipse Orbitrap was then solubilized in 1× Laemmli buffer and sonicated with Branson mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA) 250 sonicator. coupled to an EASY-nLC 1200 [Thermo Fisher Scientific (Proxeon), Preparation of subcellular fractions from the mouse liver tissue Odense, Denmark]. See more details in the Supplementary Materials was prepared by using a Polytron homogenizer in buffer H [20 mM related to the chromatography parameters and MS/MS acquisition tris-HCl (pH 7.4), 0.25 mM sucrose, 3 mM CaCl2, and cOmplete method (Supplementary Materials and Methods). Protease Inhibitor]. The nuclei were washed three times by centrifuga- Acquired spectra were analyzed using the PEAKS X+ software, tion at 300g at 4°C for 5 min. The nuclear pellet was resuspended by which, as a default, performs de novo peptide sequencing before data- vortexing for 5 s in buffer C and incubated for 10 min on ice and base searches, to improve the accuracy of the results. The data were then centrifuged at 17,000g at 4°C for 10 min. The resultant pellet searched against a Swiss-Prot human database (as of October 2019). considered as the chromatin fraction was solubilized in 1× Laemmli Trypsin (specific, up to three missed cleavages allowed) was selected for buffer and then sonicated, whereas the supernatant was considered database searches, and no enzyme was chosen in de novo searches as the nucleoplasmic fraction. (up to five candidates per spectrum reported). The maximal mass Downloaded from http://advances.sciencemag.org/ on February 11, 2021 error was set to 10 ppm for precursor ions and 0.05 Da for product Mass spectrometric analysis ions. Carbamidomethylation was selected as a fixed modification, Protein extracts were precipitated with cold acetone (o/n, −20°C), and methionine oxidation and ADP-ribose (+541.0611 Da at C, D, and the protein pellets were resuspended in 20 l of 200 mM E, H, K, N, Q, R, S, T, and Y) (50) were set as variable modifications. ammonium bicarbonate with 6 M urea solution. Samples were reduced The maximal number of modifications per peptide was set to three. with DTT (10 mM, 30°C, 60 min) and alkylated with chloroacetamide The false discovery rate was set to 0.05 for peptides. (20 mM, 25°C, 30 min). The samples were diluted with NH4HCO3 to reduce urea concentration below 1 M and digested with trypsin RNA isolation, cDNA synthesis, and real-time quantitative (1:10 enzyme:protein, 30°C, 16 hours; Promega). After digestion, polymerase chain reactions the peptide mix was acidified with formic acid and then desalted Total RNA was purified from MEFs and liver using TRIzol reagent with a MicroSpin C18 column (The Nest Group) before liquid (Invitrogen, Carlsbad, CA, USA). The cDNA was synthesized from chromatography–tandem MS (LC-MS/MS) analysis. 5 g of total RNA with a Transcriptor First Strand cDNA Synthesis The peptide samples were analyzed using a Lumos Orbitrap Kit (Roche) according to the manufacturer’s instructions. Real-time mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA) quantitative polymerase chain reaction (RT-qPCR) was performed coupled to an EASY-nLC 1000 [Thermo Fisher Scientific (Proxeon), using the QuantStudio 5 Real-Time PCR System (Thermo Fisher Odense, Denmark]. Afterward, peptides were loaded directly onto Scientific) with SYBR Green PCR Master Mix of Applied Biosystems. the analytical column and were separated by reversed-phase chroma- Relative gene expression was analyzed in QuantStudio 5 software tography using a 50-cm column with an inner diameter of 75 m, (Thermo Fisher Scientific), and values were normalized to the ex- packed with a 2-m C18 particle spectrometer (Thermo Fisher pression of RNF219 in the case of CR and GS treatments. Details of Scientific, San Jose, CA, USA) and a binary solvent system of 0.1% oligonucleotides are shown in table S4. formic acid in H2O (solvent A) and 0.1% formic acid in acetonitrile (solvent B). Chromatographic gradients started at 7% B with a flow Site-directed mutagenesis rate of 300 nl/min and gradually increased to 22% B in 52 min, then The different constructs harboring point mutations were cloned by to 32% B in 8 min, and to 95% B in 10 min. After each analysis, the site-directed mutagenesis on the basis of the QuikChange protocol column was washed for 10 min with 95% B. (Stratagene). The positive clones selected were then verified by The acquisition was performed in data-dependent acquisition sequencing using a 3730 DNA Analyzer (Applied Biosystems, mode, and full MS scans with 1 microscan at a resolution of 120,000 Foster City, CA, USA). were used over a mass range of m/z 300 to 1500. Precursor automated gain control (AGC) was set to 2 × 105. Charge-state screening was Immunofluorescence enabled, and precursors with +2 to +5 charge states and intensities SirT7 −/− cells were fixed with 4% paraformaldehyde for 7 min at >5 × 104 were selected for MS/MS and fragmented as previously room temperature and permeabilized for 5 min with buffer A described (49).Briefly, precursors that exhibited more than two [1% bovine serum albumin (BSA) and 0.5% Triton in PBS]. Cells ADP-ribose fragment peak (136,0623, 250,0940, 348,0709, and 428,0372) were blocked with 1% BSA-PBS solution for 1 hour and further in- after an initial high-energy collisional dissociation (HCD) fragmen- cubated with a rabbit anti-B23 antibody (1:100; Sc-6013-R, Santa tation were submitted to a high-quality HCD fragmentation event Cruz Biotechnology) for 1 hour in buffer B (1% BSA and 0.2% Triton (acquired in the Orbitrap with collision energy of 35% and a resolu- in PBS). Cells were washed three times 15 min each with PBS and tion of 120,000; AGC was set to 5 × 105 isolation window of 2 m/z stained with an Alexa Fluor 555 fluorophore–conjugated secondary and maximum injection time of 240 ms was used) and an electron-­ antibody (1:200; A27039, Life Technologies). Fluorescence images transfer/higher-energy collision dissociation MS/MS (the “use-calibrated were acquired with a Zeiss 710 confocal module coupled to an Axio charge-dependent parameter” option was selected, and it was acquired Observer Z1 equipped with a 63× oil immersion objective. Image in the Orbitrap with a supplementary activation (SA) of 25% and a analysis was performed using ImageJ (NIH). Simonet et al., Sci. Adv. 2020; 6 : eaaz2590 24 July 2020 13 of 16 SCIENCE ADVANCES | RESEARCH ARTICLE ChIP-seq and RNA-seq LearnModel program and allowed to learn four states. The learned For RNA-seq, total RNA from 10-cm dishes containing Wt and model showed posterior enrichment for the following states: U1, no SirT7 − /− exponentially growing MEFs was harvested using an RNeasy enrichment; U2, mH2A1 enrichment; U3, H3K27me3 enrichment; miniprep kit (Qiagen) according to the manufacturer’s instructions. and U4, both mH2A1 and H3K27me3 enrichment. mH2A1-enriched Samples were sent to the RUCDR, Infinite Biologics sequencing regions were considered as the union of regions identified as states center (Rutgers University, NJ, USA) for RNA extraction, library U2 and U4. mH2A1-­enriched genes were called using the GREAT preparation (Illumina TruSeq chemistry), and sequencing [2 × 100–base tool with mH2A1 regions as input with the association rule of “single pair (bp) paired-end reads, >60 × 106 fragments per sample, Illumi- nearest gene” within 10 kb, including curated regulatory domains. na HiSeq in rapid mode]. Reads were demultiplexed, concatenated, Chromatin state transitions induced by GS treatment were computed and delivered as FASTQ files. Raw reads were assessed for quality relative to NT for each genotype. Briefly, we identified 200-bp bins with FastQC. Raw reads were trimmed with Trimmomatic, aligned in GS with differing state calls than NT and recorded the initial state to the University of California Santa Cruz (UCSC) mm9 reference (that occurring in the NT) and the final state (that occurring in the genome with HISAT2 (options: --dta), and converted to sorted and GS). We then took the subset of transition sites at the intersection indexed BAM format with SAMtools. StringTie (in –eB mode) was of gene lists (±3 kb upstream and downstream of each gene). We used to estimate transcript abundance using the UCSC mm9 refer- quantified transition occupancy by counting the number of base ence GTF file (obtained from the Illumina iGenomes UCSC mm9 pairs occupied by each transition type and normalized this by the bundle). Differential expression was analyzed with DESeq2, allow- total number of base pairs that underwent any transition. To ascertain ing modeling terms for genotype, treatment, and batch. the genomic context of these transitions, we submitted each transi- Downloaded from http://advances.sciencemag.org/ on February 11, 2021 ChIP was conducted as described before (20). Briefly, nuclear tion type to the CEAS tool. extracts from Wt and SirT7 − /− cells were first obtained, and chromatin was prepared by sonication to an average fragment size of 300 bp. Statistical analysis For SirT7 ChIP, chromatin was diluted eight times in dilution buffer, The represented values show means of at least three or more indepen- and for macro-H2A and H3K27K3m3 ChIP, chromatin was diluted dent experiments (n ≥ 3 with error bars representing SEM unless five times. Antibodies were conjugated to beads, and ChIP was con- otherwise specified). Data were analyzed using a two-tailed Student’s ducted overnight at 4°C on a rotator. After washes and reversal of t test (ADP-ribosylation and deacetylation assays and RT-qPCRs cross-links, ChIP libraries were prepared using a ThruPLEX DNA- from rescue experiments in MEFs), and for multiple comparisons seq library preparation kit (Rubicon Genomics) following the (RT-qPCRs in mice under caloric restriction), the P value was manufacturer’s protocol. ChIP libraries were then size-selected with calculated using Microsoft Excel software by two-way analysis of Pippin Prep (Sage Science) for a final library fragment size of 350 to variance (ANOVA). 400 bp and quantified with KAPA qPCR. ChIP libraries were then sequenced on an Illumina HiSeq 2500 with 2 × 100 or 2 × 75 bp SUPPLEMENTARY MATERIALS paired-end reads to a depth of >20 × 106 fragments per sample at the Supplementary material for this article is available at http://advances.sciencemag.org/cgi/ content/full/6/30/eaaz2590/DC1 RUCDR, Infinite Biologics sequencing center (Rutgers University, NJ, USA). Reads were demultiplexed, concatenated, and delivered as View/request a protocol for this paper from Bio-protocol. FASTQ files. Raw read quality was first assessed with FastQC version 0.11.3, REFERENCES AND NOTES followed by adapter clipping and quality trimming with Trimmomatic 1. L. Bosch-Presegué, A. Vaquero, Sirtuins in stress response: Guardians of the genome. version 0.36. 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All authors Simonet et al., Sci. Adv. 2020; 6 : eaaz2590 24 July 2020 15 of 16 SCIENCE ADVANCES | RESEARCH ARTICLE participated in the discussions leading to the production of this paper. Competing Submitted 30 August 2019 interests: The authors declare that they have no competing interests. Data and Accepted 4 June 2020 materials availability: All data needed to evaluate the conclusions in the paper are Published 24 July 2020 present in the paper and/or the Supplementary Materials. All sequencing data for 10.1126/sciadv.aaz2590 ChIP-seq and RNA-seq that support the findings of this study have been deposited in the National Center for Biotechnology Information Gene Expression Omnibus (GEO) Citation: N. G. Simonet, J. K. Thackray, B. N. Vazquez, A. Ianni, M. Espinosa-Alcantud, and are accessible through the GEO Series accession number GSE128894. Proteomics J. Morales-Sanfrutos, S. Hurtado-Bagès, E. Sabidó, M. Buschbeck, J. Tischfield, C. De La Torre, data are available in Pride with the dataset identifier PXD018344 and in table S1. M. Esteller, T. Braun, M. Olivella, L. Serrano, A. Vaquero, SirT7 auto-ADP-ribosylation regulates Additional data related to this paper may be requested from the authors. glucose starvation response through mH2A1. Sci. Adv. 6, eaaz2590 (2020). Downloaded from http://advances.sciencemag.org/ on February 11, 2021 Simonet et al., Sci. Adv. 2020; 6 : eaaz2590 24 July 2020 16 of 16 SirT7 auto-ADP-ribosylation regulates glucose starvation response through mH2A1 Nicolás G. Simonet, Joshua K. Thackray, Berta N. Vazquez, Alessandro Ianni, Maria Espinosa-Alcantud, Julia Morales-Sanfrutos, Sarah Hurtado-Bagès, Eduard Sabidó, Marcus Buschbeck, Jay Tischfield, Carolina De La Torre, Manel Esteller, Thomas Braun, Mireia Olivella, Lourdes Serrano and Alejandro Vaquero Sci Adv 6 (30), eaaz2590. DOI: 10.1126/sciadv.aaz2590 Downloaded from http://advances.sciencemag.org/ on February 11, 2021 ARTICLE TOOLS http://advances.sciencemag.org/content/6/30/eaaz2590 SUPPLEMENTARY http://advances.sciencemag.org/content/suppl/2020/07/20/6.30.eaaz2590.DC1 MATERIALS REFERENCES This article cites 50 articles, 12 of which you can access for free http://advances.sciencemag.org/content/6/30/eaaz2590#BIBL PERMISSIONS http://www.sciencemag.org/help/reprints-and-permissions Use of this article is subject to the Terms of Service Science Advances (ISSN 2375-2548) is published by the American Association for the Advancement of Science, 1200 New York Avenue NW, Washington, DC 20005. The title Science Advances is a registered trademark of AAAS. Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).