Disruption of sirtuin 7 in zebrafish facilitates hypoxia tolerance

SIRT7 is a member of the sirtuin family proteins with nicotinamide adenine dinucleotide (NAD+)-dependent histone deacetylase activity, which can inhibit the activity of hypoxia-inducible factors independently of its enzymatic activity. However, the role of SIRT7 in affecting hypoxia signaling in vivo is still elusive. Here, we find that sirt7-null zebrafish are more resistant to hypoxic conditions, along with an increase of hypoxia-responsive gene expression and erythrocyte numbers, compared with their wildtype siblings. Overexpression of sirt7 suppresses the expression of hypoxia-responsive genes. Further assays indicate that sirt7 interacts with zebrafish hif-1αa, hif-1αb, hif-2αa, and hif-2αb to inhibit their transcriptional activity and mediate their protein degradation. In addition, sirt7 not only binds to the hypoxia responsive element of hypoxia-inducible gene promoters but also causes a reduction of H3K18Ac on these promoters. Sirt7 may regulate hypoxia-responsive gene expression through its enzymatic and nonenzymatic activities. This study provides novel insights into sirt7 function and sheds new light on the regulation of hypoxia signaling by sirt7.

SIRT7 is a member of the sirtuin family proteins with nicotinamide adenine dinucleotide (NAD + )-dependent histone deacetylase activity, which can inhibit the activity of hypoxiainducible factors independently of its enzymatic activity. However, the role of SIRT7 in affecting hypoxia signaling in vivo is still elusive. Here, we find that sirt7-null zebrafish are more resistant to hypoxic conditions, along with an increase of hypoxia-responsive gene expression and erythrocyte numbers, compared with their wildtype siblings. Overexpression of sirt7 suppresses the expression of hypoxia-responsive genes. Further assays indicate that sirt7 interacts with zebrafish hif-1αa, hif-1αb, hif-2αa, and hif-2αb to inhibit their transcriptional activity and mediate their protein degradation. In addition, sirt7 not only binds to the hypoxia responsive element of hypoxiainducible gene promoters but also causes a reduction of H3K18Ac on these promoters. Sirt7 may regulate hypoxiaresponsive gene expression through its enzymatic and nonenzymatic activities. This study provides novel insights into sirt7 function and sheds new light on the regulation of hypoxia signaling by sirt7.
As the terminal electron acceptor at complex IV of the respiratory chain, oxygen (O 2 ) is essential for the survival of aerobic organisms (22). Aerobic organisms have evolved sophisticated cellular mechanisms that sense and respond to O 2 gradients, as well as physiological systems that adapt to changes in these gradients (22)(23)(24)(25)(26)(27). Of note, studies of hypoxia adaptation (chronic hypoxia) have received more attention so far (26,28,29). However, in addition to encountering chronic hypoxia, organisms often encounter acute hypoxic conditions, which can somehow determine the survival or death of aerobic organisms (26,(28)(29)(30). Yet organismal mechanisms of adaptation to acute hypoxia (i.e., hypoxia tolerance) are relatively less known.
Compared with the terrestrial aerobic animals, fish live their whole lives in aquatic environments. In fact, oxygen levels in the water change more frequently than they do on land in the same area, which results from multiple causes, such as biological activities, change in temperature and atmospheric pressure, and water velocity and depth. Therefore, fish face the stress of low oxygen (hypoxia) much higher than land animals in life. Consciously, fish might be a good object and model for investigating the mechanisms of acute hypoxia tolerance. On the other hand, for aquaculture industry, to elucidate the genetic basis of fish in tolerating acute hypoxia and then breed fish strains with higher hypoxia tolerance by genetic manipulation techniques will greatly benefit this industry.
In mammalian cells, SIRT7 has been identified to inhibit the activity of hypoxia-inducible factors (41). However, due to the lack of in vivo data obtained by animal model, the physiological role of SIRT7 in affecting hypoxia signaling is still elusive. In our previous work, we generated a sirt7-null zebrafish line (42). In order to determine the role of sirt7 in affecting hypoxia signaling in vivo, we took advantage of this mutant line, as well as another newly generated mutant line in this study, to perform a series of assays and found that loss of sirt7 in zebrafish facilitates hypoxia tolerance.

Zebrafish sirt7 is repressed under hypoxia
Before we compared the effect of hypoxia on sirt7-null and wildtype (WT) zebrafish, we performed phylogenetic analysis and alignment of SIRT7 proteins from 13 species (Fig. S1, A  and B). Based on the amino acid sequences of SIRT7, the taxonomic groups of animals can be easily distinguished (Fig. S1A). Notably, SIRT7 is evolutionarily conserved (Fig. S1B). Particularly, its enzymatic-active sites are conserved from Drosophila to human (Fig. S1B).
Taken together, these data suggest that disruption of sirt7 in zebrafish facilitates hypoxia tolerance.

Zebrafish sirt7 suppresses hypoxia-responsive gene expression
To further figure out the role of zebrafish sirt7 in hypoxia signaling, we examined the effect of sirt7 overexpression on the induction of hypoxia response element (HRE) reporter activity and the expression of hypoxia-responsive genes. In EPC cells, overexpression of sirt7 inhibited hypoxia-induced HRE reporter activity (Fig. 4, A and B). In ZFL cells, overexpression of sirt7 suppressed hypoxia-induced expression of phd3, vegfaa, ldha, and cited2 mRNA (Fig. 4, C-G). These data suggest that zebrafish sirt7 suppresses the hypoxia signaling pathway.
Subsequently, we compared their interaction under normoxia and hypoxia, respectively. Of note, the interaction between sirt7 and all of hif-α was enhanced under hypoxia ( Fig. 5, B-E).
Then, we screened suitable antibodies that could detect endogenous zebrafish hif-α proteins (Fig. S4, A-D). Only one commercially available antibody could detect zebrafish hif-2αb (Fig. S4D). As expected, hif-2αb protein was higher in sirt7deficient zebrafish larvae compared with that in wildtype siblings (Fig. 6, E and F).
Zebrafish sirt7 suppresses hif-α transcriptional activity independently of its deacetylase activity Given that sirt7 has deacetylase activity, we sought to determine whether the suppression of hif-α transcriptional activity by sirt7 is dependent on its deacetylase activity. We made use of the enzyme-deficient mutants of sirt7, sirt7-S115A and sirt7-H191Y (corresponding to human SIRT7- S111A and SIRT7-H187Y, respectively) (5,43), and checked their effects on hypoxia signaling. Overexpression of sirt7-S115A or sirt7-H191Y still suppressed hypoxia-induced HRE reporter activity, similar to overexpression of wildtype sirt7 (Fig. 8, A and B). In ZFL cells, overexpression of sirt7-S115A or sirt7-H191Y also attenuated the expression of phd3, cited2, and ldha mRNA, similar to overexpression of wildtype sirt7 (Fig. 8, C-F). These data suggest that zebrafish sirt7 suppresses hif-α transcriptional activity independently of its deacetylase activity, consistently with the role of mammalian SIRT7 (41).
Zebrafish sirt7 binds to the promoter of hypoxia-responsive genes Given than sirt7 has histone deacetylase activity that can catalyze H3K18 deacetylation (5), we further examined whether H3K18Ac was altered between wildtype and sirt7deficient zebrafish and whether sirt7 bound to the promoters of hypoxia-responsive genes. As expected, H3K18Ac was increased in sirt7-deficient zebrafish compared with wildtype zebrafish (Fig. S6, A and B). In contrast, overexpression of sirt7 reduced H3K18Ac (Fig.  S6C). By chromatin immunoprecipitation-qPCR assays, sirt7 could bind to the HRE of hypoxia-responsive gene promoters (Fig. S6, D-F). Furthermore, overexpression of sirt7 significantly reduced H3K18Ac on the HRE of hypoxia-responsive gene promoters (Fig. S6, G-I). These data suggest that sirt7 can also affect hypoxia-responsive gene expression through directly impacting on the promoters of hypoxia-responsive genes, revealing multiple mechanisms of sirt7 in the regulation of hypoxia signaling.

Discussion
In this study, using the zebrafish model, we show that disruption of sirt7 facilitates hypoxia tolerance, providing Figure 5. Zebrafish sirt7 binds to hif-1α and hif-2α. A, coimmunoprecipitation analysis of Myc-sirt7 with Flag-hif-1αa, or Flag-hif-1αb, or Flag-hif-2αa, or Flaghif-2αb. HEK293T cells were transfected with Myc-sirt7, together with Flag empty vector, or Flag-hif-1αa, or Flag-hif-1αb, or Flag-hif-2αa, or Flag-hif-2αb. Anti-Flag-conjugated agarose beads were used for coimmunoprecipitation, and the indicated antibodies were used for detection. B, coimmunoprecipitation analysis of Myc-sirt7 with Flag-hif-1αa under normoxia (Nor) or hypoxia (Hyp). HEK293T cells were transfected with Myc-sirt7, together with Flag empty vector or Flag-hif-1αa, and cultured under normoxia (Nor) or hypoxia (Hyp) for 4 h. Anti-Flag-conjugated agarose beads were used for coimmunoprecipitation, and the indicated antibodies were used for detection. The Flag-hif-1αa protein under hypoxia was adjusted to be similar to that under normoxia. The immunoprecipitated Myc-sirt7 protein by Flag-hif-1αa in IP (*) over the Myc-sirt7 protein in TCL (#) was determined (*/#). C, coimmunoprecipitation analysis of Myc-sirt7 with Flag-hif-1αb under normoxia (Nor) or hypoxia (Hyp). HEK293T cells were transfected with Myc-sirt7, together with Flag empty vector or Flag-hif-1αb, and cultured under normoxia (Nor) or hypoxia (Hyp) for 4 h. Anti-Flag-conjugated agarose beads were used for coimmunoprecipitation, and the indicated antibodies were used for detection. The Flag-hif-1αb protein under hypoxia was adjusted to be similar to that under normoxia. The immunoprecipitated Myc-sirt7 protein by Flag-hif-1αb in IP (*) over the Myc-sirt7 protein in TCL (#) was determined (*/#). D, coimmunoprecipitation analysis of Myc-sirt7 with Flag-hif-2αa under normoxia (Nor) or hypoxia (Hyp). HEK293T cells were transfected with Myc-sirt7, together with Flag empty vector or Flag-hif-2αa, and cultured under normoxia (Nor) or hypoxia (Hyp) for 4 h. Anti-Flag-conjugated agarose beads were used for coimmunoprecipitation, and the indicated antibodies were used for detection. The Flag-hif-2αa protein under hypoxia was adjusted to be similar to that under normoxia. The immunoprecipitated Myc-sirt7 protein by Flag-hif-2αa in IP (*) over the Myc-sirt7 protein in TCL (#) was determined (*/#). E, coimmunoprecipitation analysis of Myc-sirt7 with Flag-hif-2αb under normoxia (Nor) or hypoxia (Hyp). HEK293T cells were transfected with Myc-sirt7, together with Flag empty vector or Flag-hif-2αb, and cultured under normoxia (Nor) or hypoxia (Hyp) for 4 h. Anti-Flag-conjugated agarose beads were used for coimmunoprecipitation, and the indicated antibodies were used for detection. The Flag-hif-2αb protein under hypoxia was adjusted to be similar to that under normoxia. The immunoprecipitated Myc-sirt7 protein by Flag-hif-2αb in IP (*) over the Myc-sirt7 protein in TCL (#) was determined (*/#). IP, immunoprecipitation; TCL, total cell lysates.
in vivo data to support the role of sirt7 in hypoxia signaling. By taking advantage of the ideal model for monitoring hypoxia tolerance, the zebrafish, we have revealed that several genes involved in hypoxia signaling can influence fish hypoxia tolerance (32)(33)(34). Tet1 enhances HIF-α transcriptional activity, while disruption of tet1 in zebrafish reduces hypoxia tolerance (32). Fih inhibits HIF-α transcriptional activity, while deletion of fih in zebrafish facilitates hypoxia tolerance (33). However, smyd3 augments hypoxia signaling, while smyd3null zebrafish exhibit increased hypoxia tolerance (44). Therefore, it appears that modulating HIF-α activity to a certain level may greatly benefit organisms for hypoxia tolerance, but either a too high or too low level of activity could reduce hypoxia tolerance. Understanding this principle will help to select targets for breeding fish strains with hypoxia tolerance by CRISPR/cas9 technique in aquaculture industry.
Notably, SIRT7 exhibits an evolutionarily conserved function on HIF-α activity between fish and mammalian, with a suppressive role independently of its enzymatic activity (41). Even though, compared with other sirtuins, SIRT7 displays low enzymatic activity in vitro, it still mainly affects the function of its targets as a deacetylase (7)(8)(9). So, it is enigmatic how SIRT7 suppresses HIF-α activation independently of its deacetylase activity. In fact, the nondeacetylase activity of SIRT7 has been recognized (17,18). Further figuring out the nonenzymatic role or nondeacetylase role of SIRT7 on HIF-α activation and the underlying mechanisms will not only give insights into the full picture of SIRT7 function but also expand our knowledge about the regulation of hypoxia signaling in affecting acute hypoxia adaptation.
In addition, we also observed that sirt7 not only binds to the HRE of hypoxia-responsive gene promoters but also impairs H3K18Ac on these promoters, revealing another mechanism of sirt7 in the regulation of hypoxia-responsive genes. It appears that sirt7 affects hypoxia-responsive gene expression by two regulatory ways: one is to regulate hif-α transcriptional activity independently of its deacetylase activity; the other is to act as a deacetylase to catalyze the deacetylation of H3K18 binding to the promoters of hypoxia-responsive genes, which is dependent on its deacetylase activity. However, it is still unclear how much each regulatory way contributes to the modulation of hypoxia-responsive gene expression. Further investigation of this question will help to fully understand the function of sirt7 in the regulation of hypoxia signaling.
Disruption of sirt7 facilitates hypoxia tolerance downloaded from NCBI (https://www.ncbi.nlm.nih.gov/) and aligned using CLUSTAL W, and the Neighbor-joining tree was constructed using Bootstrap method with the number of Bootstrap replication set to 1000.

Cells and zebrafish
Zebrafish liver (ZFL) cells (originally obtained from the American Type Culture Collection) were cultured in Ham's F-12 medium (HyClone) supplemented with 10% fetal bovine serum (FBS) (Biological Industries). Epithelioma papulosum cyprini (EPC) cells (originally obtained from the American Type Culture Collection) were cultured in Medium 199 Earle's Salts Base (Biological Industries) supplemented with 10% FBS. ZFL and EPC cells were maintained at 28 C in a humidified incubator containing 5% carbon dioxide (CO 2 ). Human embryonic kidney cell line (HEK293T) cells (originally obtained from the American Type Culture Collection) were grown in Dulbecco's modified Eagle's medium (Biological Industries) supplemented with 10% FBS at 37 C in a humidified incubator containing 5% CO 2 .
Zebrafish (AB strain) were raised and maintained in a recirculating water system according to standard protocols. All zebrafish experiments were approved by the Institutional Animal Care and Use Committee of the Institute of Hydrobiology, Chinese Academy of Sciences.

Hypoxia treatment
The Ruskinn INVIVO2 I-400 workstation was used for hypoxia treatment of cells and zebrafish. The O 2 concentration and temperature were adjusted to the corresponding value (1% O 2 , 28 C for cells and 2% O 2 , 28 C for zebrafish larvae) ahead of time. For cells, ZFL or EPC cells were cultured in the hypoxia workstation for 20 to 24 h. For zebrafish larvae under hypoxia, sirt7-null zebrafish larvae (3 dpf; n = 30) and their wildtype siblings (3 dpf, n = 30) in disposable 60-mm cell culture dishes filled with 5 ml egg water were plated in the hypoxia workstation simultaneously. Then, the behavior of zebrafish was closely monitored, recorded, and photographed. For obtaining the survival curve of zebrafish under hypoxia, mortality was monitored every 3 h after the first death. Figure 8. Zebrafish sirt7 attenuates hypoxia signaling independent of its deacetylase activity. A, Western blot analysis of indicated protein levels in EPC cells transfected with empty vector, wildtype sirt7, or the enzyme-deficient mutants sirt7-S115A and sirt7-H191Y. B, luciferase activity of HRE-luciferase reporter in EPC cells transfected with empty vector, wildtype sirt7, or the enzyme-deficient mutants sirt7-S115A and sirt7-H191Y under normoxia (Nor) or hypoxia (Hyp). C, Western blot analysis of indicated protein levels in ZFL cells transfected with empty vector, wildtype sirt7, or the enzyme-deficient mutants sirt7-S115A and sirt7-H191Y. D, quantitative real-time PCR (qPCR) analysis of phd3 in ZFL cells transfected with empty vector, wildtype sirt7, or the enzymedeficient mutants sirt7-S115A and sirt7-H191Y under normoxia (Nor) or hypoxia (Hyp). E, qPCR analysis of cited2 in ZFL cells transfected with empty vector, wildtype sirt7, or the enzyme-deficient mutants sirt7-S115A and sirt7-H191Y under normoxia (Nor) or hypoxia (Hyp). F, qPCR analysis of ldha in ZFL cells transfected with empty vector, wildtype sirt7, or the enzyme-deficient mutants sirt7-S115A and sirt7-H191Y under normoxia (Nor) or hypoxia (Hyp). p Values were calculated by two-way ANOVA analysis (B, D, E, and F); **p < 0.01, ***p < 0.001 and ****p < 0.0001; data based on one representative experiment performed in three biological replicates from at least three independent experiments (mean ± SD).
RNA extraction, reverse transcription, and quantitative realtime PCR assay Briefly, cells or zebrafish larvae were homogenized by adding appropriate amounts of RNAiso Plus (Takara Biomedical Technology) and total RNA was extracted according to the manufacturer's instructions. Then, equivalent amounts of total RNA (2 μg each) were used for cDNA synthesis using the Revert Aid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific) in a 20-μl reaction volume. Subsequently, the synthesized cDNAs were used as templates for quantitative realtime PCR (qPCR) analysis. The analysis was performed using the CFX Connect Real-Time PCR System (Bio-Rad Laboratories) with MonAmp SYBR Green qPCR Mix (High Rox; Monad Biotech Co) under the following conditions: at 95 C for 5 min, followed by 50 cycles at 95 C for 3 s and at 60 C for 15 s. The standard curve acquisition program of the instrument was used to draw the cycle threshold. The changes in gene expression were calculated as the relative fold changes by the comparative cycle threshold method, and the corresponding species β-actin was used as an internal control gene for normalization. Results were obtained from three independent experiments, each performed in triplicate. The primers used are listed in Table S1.

Western blot analysis
HEK293T cells were transfected with the indicated plasmids. After 24 h, the cells were washed with ice-cold PBS buffer and then lysed in RIPA buffer (containing 50 mM Tris [pH 7.4], 1% Nonidet P-40, 0.25% sodium deoxycholate, 1 mM EDTA [pH 8], 150 mM NaCl, 1 mM NaF, 1 mM PMSF, 1 mM Na3VO4, and a 1:100 dilution of protease inhibitor mixture [Sigma-Aldrich]) for 0.5 to 1 h at 4 C. The supernatant was collected by centrifugation at 12,000g for 15 min at 4 C, transferred into a new tube, and resuspended with 2 × SDS sample loading buffer. Samples were boiled for 5 to 10 min, separated on SDS-PAGE, and then transferred to polyvinylidene difluoride membrane (Millipore). The membrane was blocked with 5% (w/v) nonfat milk, probed with the indicated primary antibodies and corresponding secondary antibodies, visualized with ECL Western Blotting Detection Reagent (Millipore), and photographed with a Fuji Film LAS4000 mini-luminescent image analyzer.

Antibodies and chemical reagents
The following antibodies were used as indicated: anti-sirt7 (#S7612) was purchased from Selleck. MG-132 (#474790) was purchased from Sigma.

Coimmunoprecipitation assay
HEK293T cells were seeded overnight into 100-mm cell culture dishes and transfected with a total of 10 μg of the indicated plasmids per dish. After 24 h, the cells were washed with ice-cold PBS buffer and lysed in 1 ml RIPA buffer. The supernatant was transferred into a new tube, and anti-Flag antibody-conjugated agarose beads (Sigma-Aldrich) were used for immunoprecipitation. Total cell lysates and immunoprecipitates were subjected to Western blot analysis.

Ubiquitination assay
Ubiquitination assays were performed according to the protocol described in (42). Briefly, HEK293T cells were transfected with the indicated plasmids for 24 h and then lysed with denatured buffer (6 M guanidine-HCl, 0.1 M Na 2 HPO 4 / NaH 2 PO 4 , 10 mM imidazole), followed by nickel bead purification and immunoblotting with the indicated antibodies.

Luciferase reporter assay
EPC cells seeded in 24-well plates were transfected with the indicated amounts of plasmids, including pTK-Renilla (Promega) as an internal control. The hypoxia response element luciferase reporter (HRE-luc) plasmid was kindly provided by Navdeep Chandel, and the pFR-luc plasmid was purchased from Stratagene. Luciferase activity in cell extracts was determined using a luminometer (Sirius; Zylux Corp) with a Dual-luciferase Reporter Assay System (Promega) according to the manufacturer's protocol. Data were normalized to Renilla luciferase.

o-Dianisidine staining
Sirt7-null zebrafish larvae (3 dpf; n = 10) and their wildtype siblings (3 dpf, n = 10) in disposable 60-mm cell culture dishes filled with 5 ml egg water were incubated in the hypoxia workstation simultaneously (10% O 2 , 28 C) for 12 h. Zebrafish larvae were then incubated in a 12-well plate with o-dianisidine solution (Sigma-Aldrich o-dianisidine in 100% ethanol with 0.1 M sodium acetate and 30% H 2 O 2 in ddH 2 O) for1 h. The embryos were then washed with ddH 2 O and fixed with 4% paraformaldehyde in PBS overnight at 4 C. A bleaching solution (0.8% KOH, 0.9% H 2 O 2 , and 0.1% Tween in ddH 2 O) was added to the embryos for 30 min to remove their natural pigmentation. After another fixation step with 4% paraformaldehyde overnight, larvae were immersed in 3% methylcellulose-M450 solution in a 100-mm cell culture dish and imaged on a Nikon TE2000-U microscope with a × 30 objective. Experiments were performed in biological triplicates.

Chromatin immunoprecipitation assay
Chromatin immunoprecipitation assay was performed according to the protocol with some modifications (34). Briefly, H1299 cells were transfected with empty vector control (Flag empty) or Flag-tagged zebrafish sirt7 (Flag-sirt7) for 24 h and then cultured under hypoxia for 24 h. Then, the cells were incubated in culture medium containing 1% formaldehyde with gentle shaking for 10 min at room temperature, and cross-linking was stopped by adding 2.5 M glycine to a final concentration of 0.125 M. The procedure was then performed according to the protocol of the SimpleChIP Enzymatic Chromatin IP Kit. The purified DNA was analyzed by qPCR, and the primers are listed in Table S2.

Statical analysis
Survival data were calculated by the Kaplan-Meier method and analyzed by the log-rank test using GraphPad Prism 9.3.1 (GraphPad Software). Other statistical analyses were performed using an unpaired t test or two-way ANOVA analysis in GraphPad Prism 9.3.1 (GraphPad Software). All data are representative of at least three independent experiments, and error bars indicate mean ± SD. A p value <0.05 was considered significant. Statistical significance is represented as follows: ns, not significant, *p < 0.05, **p < 0.01, ***p < 0.001, and **** p < 0.0001.

Data availability
Further information and requests for resources and reagents should be directed to and will be fulfilled by X. L. and W. X.
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