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Methyltransferase SMYD3 impairs hypoxia tolerance by augmenting hypoxia signaling independent of its enzymatic activity

  • Zixuan Wang
    Affiliations
    State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, China

    University of Chinese Academy of Sciences, Beijing, China
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  • Xiaoyun Chen
    Affiliations
    State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, China

    University of Chinese Academy of Sciences, Beijing, China
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  • Sijia Fan
    Affiliations
    State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, China

    University of Chinese Academy of Sciences, Beijing, China
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  • Chunchun Zhu
    Affiliations
    State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, China

    University of Chinese Academy of Sciences, Beijing, China
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  • Hongyan Deng
    Affiliations
    College of Life Science, Wuhan University, Wuhan, China
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  • Jinhua Tang
    Affiliations
    State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, China

    University of Chinese Academy of Sciences, Beijing, China
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  • Xueyi Sun
    Affiliations
    State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, China

    University of Chinese Academy of Sciences, Beijing, China
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  • Shuke Jia
    Affiliations
    State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, China

    University of Chinese Academy of Sciences, Beijing, China
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  • Qian Liao
    Affiliations
    State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, China

    University of Chinese Academy of Sciences, Beijing, China
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  • Wuhan Xiao
    Correspondence
    For correspondence: Xing Liu; Wuhan Xiao
    Affiliations
    State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, China

    University of Chinese Academy of Sciences, Beijing, China

    The Innovation of Seed Design, Chinese Academy of Sciences, Wuhan, China

    Hubei Hongshan Laboratory, Wuhan, China
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  • Xing Liu
    Correspondence
    For correspondence: Xing Liu; Wuhan Xiao
    Affiliations
    State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, China

    University of Chinese Academy of Sciences, Beijing, China

    The Innovation of Seed Design, Chinese Academy of Sciences, Wuhan, China
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Open AccessPublished:October 20, 2022DOI:https://doi.org/10.1016/j.jbc.2022.102633
      Hypoxia-inducible factor (HIF)1α, a main transcriptional regulator of the cellular response to hypoxia, also plays important roles in oxygen homeostasis of aerobic organisms, which is regulated by multiple mechanisms. However, the full cellular response to hypoxia has not been elucidated. In this study, we found that expression of SMYD3, a methyltransferase, augments hypoxia signaling independent of its enzymatic activity. We demonstrated SMYD3 binds to and stabilizes HIF1α via co-immunoprecipitation and Western blot assays, leading to the enhancement of HIF1α transcriptional activity under hypoxia conditions. In addition, the stabilization of HIF1α by SMYD3 is independent of HIF1α hydroxylation by prolyl hydroxylases and the intactness of the von Hippel-Lindau ubiquitin ligase complex. Furthermore, we showed SMYD3 induces reactive oxygen species accumulation and promotes hypoxia-induced cell apoptosis. Consistent with these results, we found smyd3-null zebrafish exhibit higher hypoxia tolerance compared to their wildtype siblings. Together, these findings define a novel role of SMYD3 in affecting hypoxia signaling and demonstrate that SMYD3-mediated HIF1α stabilization augments hypoxia signaling, leading to the impairment of hypoxia tolerance.

      Keywords

      Abbreviations:

      DFX (deferoxamine mesylate salt), FBS (fetal bovine serum), HIF (hypoxia-inducible factor), MEF (mouse embryonic fibroblast), VHL (von Hippel-Lindau), PHD (prolyl hydroxylase), ROS (reactive oxygen species), SETSu (var) 3–9 (Enhancer-of-zeste, and N-terminal Trithorax)
      It is well-known that oxygen profoundly affects physiology of aerobic organisms through multiple mechanisms. Molecular oxygen not only acts as the terminal electron acceptor at complex IV of the respiratory chain that yields energy during aerobic respiration and builds metabolites but also promotes to change the configuration and function of nucleic acids, sugars, lipids, proteins, and metabolites. Inadequate oxygen availability can lead to cellular dysfunction and even cell death. Under low oxygen (hypoxic) conditions, aerobic organisms utilize their cardiovascular system and respiratory system to ensure adequate oxygen delivery to cells and tissues. In addition, cells undergo adaptive changes to initiate gene expression that either enhance oxygen delivery or promote survival (
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      SMYD3 is a member of the SMYD lysine methylase family containing two conserved structural domains: the catalytic Su (var) 3–9, Enhancer-of-zeste, and N-terminal Trithorax (SET) domain, which is split by a Myeloid-Nervy-DEAF1 domain (
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      Whether or not SMYD3 involved in hypoxia signaling is still not understood. In this study, we show that SMYD3 interacts with HIF1α and stabilizes HIF1α independent of its methyltransferase activity, leading to the augment of the hypoxia signaling, the accumulation of ROS, and the enhancement of hypoxia-induced cell apoptosis. By zebrafish model, we found that disruption of smyd3 facilities zebrafish hypoxia tolerance, which might be resulted from the impact of smyd3 on hypoxia signaling.

      Results

      SMYD3 augments hypoxia signaling

      We have previously identified that the monomethyltransferase, SET7, represses hypoxia signaling by catalyzing HIF-α methylation (
      • Liu X.
      • Chen Z.
      • Xu C.
      • Leng X.
      • Cao H.
      • Ouyang G.
      • et al.
      Repression of hypoxia-inducible factor alpha signaling by Set7-mediated methylation.
      ). To determine whether other methyltransferases also involved in hypoxia signaling, initially, we examined expression of a series of methyltransferases in HEK293T cells under hypoxia. As shown in Fig. 1A, the typical hypoxia responsive genes, including GLUT1, BNIP3, PDK, PGK1, and VEGF (
      • Liu X.
      • Chen Z.
      • Xu C.
      • Leng X.
      • Cao H.
      • Ouyang G.
      • et al.
      Repression of hypoxia-inducible factor alpha signaling by Set7-mediated methylation.
      ,
      • Wang J.
      • Zhang D.
      • Du J.
      • Zhou C.
      • Li Z.
      • Liu X.
      • et al.
      Tet1 facilitates hypoxia tolerance by stabilizing the HIF-alpha proteins independent of its methylcytosine dioxygenase activity.
      ,
      • Chen Z.
      • Liu X.
      • Mei Z.
      • Wang Z.
      • Xiao W.
      EAF2 suppresses hypoxia-induced factor 1alpha transcriptional activity by disrupting its interaction with coactivator CBP/p300.
      ), were greatly induced under hypoxia, suggesting the hypoxic condition was achieved expectedly. Among the methyltransferase genes tested, SMYD2, SMYD4, SETD1A, EZH1, EZH2, and SUV420H1 were upregulated under hypoxia, but only SMYD3 was significantly suppressed (Fig. 1A), which provoked us to further test the impact of SMYD3 in affecting hypoxia signaling. Subsequently, we examined whether the effect of hypoxia on SMYD3 expression is dependent of HIF signaling. In H1299 cells, the expression of SMYD3 was significantly suppressed under hypoxia (Fig. S1A). However, in ARNT-deficient H1299 cells (ARNT−/−) (Fig. S1B), hypoxia failed to induce expression of PGK1, a typical HIF1α target gene (Fig. S1C) but could still suppress expression of SMYD3 (Fig. S1D). In addition, we added PX478 to inhibit HIF1α activity and then checked the effect of hypoxia on SMYD3 expression (
      • Xu C.
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      • Li S.
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      A pathogen-derived effector modulates host glucose metabolism by arginine GlcNAcylation of HIF-1alpha protein.
      ). When PX478 (100 μM) was added, hypoxia failed to induce expression of PGK1 (Fig. S1E) but could still suppress expression of SMYD3 (Fig. S1F). These results suggest that the effect of hypoxia on SMYD3 is independent of HIF signaling.
      Figure thumbnail gr1
      Figure 1SMYD3 augments hypoxia signaling. A, quantitative real-time PCR (qPCR) analysis of mRNA levels of indicated lysine methyltransferase genes and hypoxia signaling target genes in HEK293T cells under normoxia (21% O2) or hypoxia (1% O2) for 24 h. BD, qPCR analysis of GLUT1 (B), PGK1 (C), and VEGF (D) mRNA in HEK293T cells transfected with or without pCMV-SMYD3 under normoxia (21% O2) or hypoxia (1% O2) for 24 h. EG, qPCR analysis of GLUT1 (E), PDK1 (F), and BNIP3 (G) mRNA in HEK293T cells transfected with or without pCMV-SMYD3 and treated with DFX (150 μM) or DMSO as a control for 8 h. H–J, qPCR analysis of GLUT1 (H), PGK1 (I), and BNIP3 (J) mRNA in HEK293T cells transfected with or without pCMV-SMYD3 and treated with or without CoCl2 (200 μM) for 8 h. EV, pCMV empty vector (control). Data show mean ± SD; Student’s two-tailed t test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. Data from three independent experiments.
      To determine the effect of SMYD3 on hypoxia signaling, we overexpressed SMYD3 in HEK293T cells and examined expression of hypoxia responsive genes under normoxia or hypoxia. Ectopic expression of SMYD3 promoted expression of typical hypoxia responsive genes, including GLUT1, PGK1, and VEGF, under hypoxia (Fig. 1, BD). To further confirm these observations, we changed direct-hypoxia treatment to the addition of deferoxamine mesylate salt (DFX) or CoCl2, two widely used hypoxia-mimic conditions (
      • Wang G.L.
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      Desferrioxamine induces erythropoietin gene expression and hypoxia-inducible factor 1 DNA-binding activity: implications for models of hypoxia signal transduction.
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      Mitochondrial reactive oxygen species trigger hypoxia-induced transcription.
      ) and then examined the effect of SMYD3 on hypoxia responsive gene expression. Consistently, overexpression of SMYD3 also enhanced expression of GLUT1, PDK1, PGK1, and BNIP3 (Fig. 1, EJ). SMYD3 is reported to downregulate the protein level of p53 (
      • Zhang L.
      • Jin Y.
      • Yang H.
      • Li Y.
      • Wang C.
      • Shi Y.
      • et al.
      SMYD3 promotes epithelial ovarian cancer metastasis by downregulating p53 protein stability and promoting p53 ubiquitination.
      ), and p53 plays vital roles in hypoxia signaling (
      • Zhang C.
      • Liu J.
      • Wang J.
      • Zhang T.
      • Xu D.
      • Hu W.
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      The interplay between tumor suppressor p53 and hypoxia signaling pathways in cancer.
      ). To exclude whether the effect of SMYD3 on hypoxia signaling was mediated by p53, we examined the effect of SMYD3 on hypoxia signaling in p53-deficient H1299 cells. Similar results were obtained by H1299 cells (Fig. S1, GI). In contrast, knockout of SMYD3 in HEK293T cell resulted in a reduction of expression of GLUT1, PGK1, PDK1, or BNIP3 under hypoxia or CoCl2 treatment (Fig. 2, AF). Moreover, expression of Glut1 and Pgk1 was also reduced in Smyd3-deficient (Smyd3−/−) mouse embryonic fibroblast (MEF) cells compared to the wildtype MEF cells (Smyd3+/+) (Fig. 2, GI). However, reconstitution of Smyd3 by lentivirus infection in Smyd3-/- MEF cells recovered the induction of expression of Pgk1 and Vegf compared to the empty virus control (pHAGE) (Fig. 2, JL). HIF1α expression was confirmed by Western blot analysis (Fig. S2, AD). In addition, knockdown of SMYD3 by shRNAs in HEK293T cell resulted in a reduction of expression of GLUT1, PDK1, or PGK1 under hypoxia (Fig. S2, E–H). Moreover, SMYD3 had similar effect on HIF2α as that on HIF1α in HEK293T cells (Fig. S3, AF). These data suggest that SMYD3 augments hypoxia signaling.
      Figure thumbnail gr2
      Figure 2Loss of SMYD3 diminishes hypoxia signaling. A, immunoblotting of indicated proteins in SMYD3-deficient or wildtype HEK293T cells (SMYD3−/− or SMYD3+/+). B and C, qPCR analysis of GLUT1 (B) and PGK1 (C) mRNA in SMYD3-deficient or wildtype HEK293T cells (SMYD3−/− or SMYD3+/+) under normoxia (21% O2) or hypoxia (1% O2) for 24 h. Data show mean ± SD; Student’s two-tailed t test. ∗p < 0.05, ∗∗p < 0.01. Data from three independent experiments. D–F, qPCR analysis of GLUT1 (D), PDK1 (E), and BNIP3 (F) mRNA in SMYD3-deficient or wildtype HEK293T cells (SMYD3−/− or SMYD3+/+) treated with or without CoCl2 (200 μM) for 8 h. Data show mean ± SD; Student’s two-tailed t test. ∗∗p < 0.01, ∗∗∗∗p < 0.0001. Data from three independent experiments. G, immunoblotting of indicated proteins in Smyd3-deficient or wildtype MEF cells (Smyd3−/− or Smyd3+/+). H and I, qPCR analysis of Glut1 (H) and Pgk1 (I) mRNA in Smyd3-deficient or wildtype MEF cells (Smyd3−/− or Smyd3+/+) under normoxia (21% O2) or hypoxia (1% O2) for 24 h. Data show mean ± SD; Student’s two-tailed t test. ∗∗∗p < 0.001. Data from three independent experiments. J, immunoblotting of indicated proteins in Smyd3-deficient MEF cells reconstituted with or without wildtype Smyd3 by lentivirus. K and L, qPCR analysis of Pgk1 (K) and Vegf (L) mRNA in Smyd3-deficient MEF cells reconstituted with or without wildtype Smyd3 by lentivirus under normoxia (21% O2) or hypoxia (1% O2) for 24 h. Data show mean ± SD; Student’s two-tailed t test. ∗p < 0.05. Data from three independent experiments. qPCR, quantitative RT–PCR; MEF, mouse embryonic fibroblast.

      SMYD3 binds to and stabilizes HIF1α, leading to an increase of nuclear HIF1α and enhancement of HIF1α-mediated target genes expression

      Given that HIF1α is one of the master regulators of hypoxia signaling, the enhancement of SMYD3 on hypoxia responsive gene expression promoted us to test whether this effect is mediated by HIF1α. Co-expression of SMYD3 together with HIF1α caused an induction of expression of GLUT1, PGK1, and VEGF mediated by ectopic expression of HIF1α in HEK293T cells (Fig. 3, AC). HIF1α expression was confirmed by Western blot analysis (Fig. S4A).
      Figure thumbnail gr3
      Figure 3SMYD3 binds to and stabilizes HIF1α, leading to an increase of nuclear HIF1α and enhancement of HIF1α-mediated target genes expression. A–C, qPCR analysis of GLUT1 (A), PGK1 (B), and VEGF (C) mRNA in HEK293T cells cotransfected with Myc-HIF1α or Myc empty vector (control) together with pCMV-SMYD3 or pCMV empty vector (EV) (control) for 24 h. Data show mean ± SD; Student’s two-tailed t test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. Data from three independent experiments. D, co-immunoprecipitation of HA-SMYD3 with Myc-HIF1α. HEK293T cells were co-transfected with indicated plasmids for 24 h. Anti-HA antibody-conjugated agarose beads were used for immunoprecipitation, and the interaction was detected by immunoblotting with the indicated antibodies. E, endogenous interaction between Smyd3 and Hif1α. Smyd3-deficient or wildtype MEF cells (Smyd3−/− or Smyd3+/+) under hypoxia for 4 h and anti-HIF1α antibody was used for immunoprecipitation. F, immunoblotting of exogenous Myc-HIF1α expression in H1299 cells transfected with an increasing amount of HA-SMYD3 expression plasmid (HA empty vector [-] was used as a control). G, immunoblotting of endogenous HIF1α expression in Smyd3-deficient or wildtype MEF cells (Smyd3−/− or Smyd3+/+) under normoxia (21% O2) or hypoxia (1% O2) for 4 h. The relative intensities of Hif1α were determined by normalizing the intensities of Hif1α to the intensities of Gapdh. Data show mean ± SD; Student’s two-tailed t test. ∗p < 0.05. Data from three independent experiments. H, immunoblotting of endogenous Hif1α expression in Smyd3-deficient MEF cells reconstituted with or without wildtype Smyd3 by lentivirus under normoxia (21% O2) or hypoxia (1% O2) for 4 h. The relative intensities of Hif1α were determined by normalizing the intensities of Hif1α to the intensities of Gapdh. Data show mean ± SD; Student’s two-tailed t test. ∗∗p < 0.01. Data from three independent experiments. I, Smyd3-deficient or wildtype MEF cells (Smyd3−/− or Smyd3+/+) were cultured under hypoxia for 4 h. Western blot analysis was used to detect Smyd3 and Hif1α in cytosol and nuclear fractions. J, confocal microscopy image of endogenous Hif1α in Smyd3-deficient or wildtype MEF cells (Smyd3−/− or Smyd3+/+) under hypoxia for 4 h. Scale bar = 50 μm. MEF, mouse embryonic fibroblast; qPCR, quantitative RT–PCR; HIF, hypoxia-inducible factor.
      We next examined whether SMYD3 interacted with HIF1α. Co-immunoprecipitation assays indicated that ectopic-expressed HA-SMYD3 interacted with ectopic-expressed Myc-HIF1α (Fig. 3D). Semiendogenous co-immunoprecipitation assays indicated that ectopic-expressed HA-SMYD3 interacted with endogenous HIF1α under hypoxia (Fig. S4B). Endogenous interaction between SMYD3 and HIF1α was further confirmed in HEK293T cells under hypoxia (Fig. S4C). In Smyd3+/+ MEF cells, but not in Smyd3−/− MEF cells, endogenous Smyd3 interacted with endogenous HIF1α (Fig. 3E). Furthermore, we examined whether the protein stability of HIF1α is affected by SMYD3. Co-expression of SMYD3 together with HIF1α caused HIF1α protein level was increased steadily (Fig. 3F). Overexpression of SMYD3 upregulated endogenous HIF1α protein level under hypoxia (Fig. S4D). By contrast, the endogenous Hif1α protein level was lower in Smyd3-null MEF cells (Smyd3−/−) compared to that in Smyd3-intact MEF cells (Smyd3+/+) under hypoxia (Fig. 3G). Consistently, reconstitution of Smyd3 in Smyd3−/− MEF cells caused an increase of Hif1α protein under hypoxia (Fig. 3H).
      Since stabilized HIF1α needs to translocate into the nucleus to function as a transcription factor; therefore, we investigated the effect of SMYD3 on the nuclear HIF1α levels. Notably, overexpression of SMYD3 enhanced HIF1α protein in the nuclei of HEK293T cells (Fig. S4E). In agreement, Hif1α protein level was higher in the nuclei of Smyd3+/+ MEF cells compared to the nuclei of Smyd3−/− MEF cells, which was further confirmed by confocal microscopy (Fig. 3, I and J). Consistently, in cycloheximide pulse chase assay, overexpression of SMYD3 attenuated degradation of co-expressed HIF1α in HEK293T cells (Fig. S4F).
      These data suggest that SMYD3 interacts with and stabilizes HIF1α, leading to an increase of nuclear HIF1α and enhanced HIF1α-mediated expression of target genes.

      The induction of HIF1α target gene expression and stabilization of HIF1α by SMYD3 are independent of HIF1α hydroxylation and pVHL intactness

      Hydroxylation of HIF1α and subsequent proteasomal degradation mediated by pVHL E3 ubiquitin ligase complex plays a central role in HIF1α regulation. We further investigated whether regulation of HIF1α by SMYD3 relies on this way. Ectopic expression of SMYD3 enhanced HIF1α protein level (Fig. S5A) and expression of GLUT1, PGK1, and PDK1 induced by addition of FG4592, a specific inhibitor of PHDs (Fig. 4, AC) (
      • Rabinowitz M.H.
      Inhibition of hypoxia-inducible factor prolyl hydroxylase domain oxygen sensors: tricking the body into mounting orchestrated survival and repair responses.
      ). These data suggest that the induction of HIF1α target genes expression by SMYD3 might not be dependent of HIF1α hydroxylation. Furthermore, we knocked out VHL in HEK293T cells and then examined the effect of SMYD3 on hypoxia signaling (Fig. S5B). As expected, in VHL-/- HEK293T cells, the hypoxia responsive genes, including GLUT1, PGK1, PDK1, LDHA, BNIP3, PHD3, and PKM2, were increased compared to those in VHL+/+ HEK293T cells (Fig. S5C), indicating that VHL was disrupted in HEK293T cells efficiently. Ectopic expression of SMYD3 in VHL-/- HEK293T cells enhanced HIF1α protein level (Fig. S5D) and hypoxia responsive gene expression (Fig. 4, DF) in a dose-dependent manner. These data suggest that the induction of HIF1α target genes expression by SMYD3 is independent of pVHL intactness.
      Figure thumbnail gr4
      Figure 4The induction of HIF1α target genes expression and stabilization of HIF1α by SMYD3 are independent of HIF1α hydroxylation and pVHL intactness. A–C, qPCR analysis of GLUT1 (A), PGK1 (B), and PDK1 (C) mRNA in HEK293T cells transfected with or without pCMV-SMYD3 for 24 h, followed by treatment with DMSO or FG4592 (100 μM) for 8 h. EV, pCMV empty vector (control). Data show mean ± SD; Student’s two-tailed t test. ∗p < 0.05, ∗∗p < 0.01. Data from three independent experiments. D–F, qPCR analysis of GLUT1 (D), PDK1 (E), and VEGF (F) mRNA in VHL-deficient HEK293T cells (VHL−/−) transfected with an increasing amount of pCMV-SMYD3 expression plasmid. pCMV empty vector was used as a control (-). Data show mean ± SD; Student’s two-tailed t test. ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. Data from three independent experiments. G, immunoblotting of endogenous Hif1α expression in Smyd3-deficient or wildtype MEF cells (Smyd3−/− or Smyd3+/+) treated with an increasing amount of FG4592 for 6 h. H, the relative intensities of Hif1α in (G) determined by normalizing the intensities of Hif1α to the intensities of Gapdh. I, immunoblotting of endogenous Hif1α expression in Smyd3-deficient or wildtype MEF cells (Smyd3−/− or Smyd3+/+) treated with an increasing time of FG4592 (100 μM) for 0 to 6 h. J, the relative intensities of Hif1α in (I) determined by normalizing the intensities of Hif1α to the intensities of Gapdh. HIF, hypoxia-inducible factor; MEF, mouse embryonic fibroblast; qPCR, quantitative RT–PCR; VHL, von Hippel-Lindau.
      In addition, co-expression of SMYD3 together with HIF1α caused HIF1α protein level to increase steadily, which was not affected when the two prolyl residues (P402/P564) were mutated (HA-HIF1α-DM) (P402A/P564A) (Fig. S5, EF). Furthermore, when FG4592 was added either in an increase of dose or in an extended time course, the protein level of endogenous Hif1α in Smyd3+/+ MEF cells kept higher than that in Smyd3-/- MEF cells (Fig. 4, GJ).
      Taken together, these data suggest that the induction of HIF1α target gene expression and stabilization of HIF1α by SMYD3 is independent of HIF1α hydroxylation and pVHL intactness.

      The stabilization and activation HIF1α by SMYD3 are independent of its methyltransferase activity

      Given that SMYD3 serves as a methyltransferase, we sought to determine whether the modulation of HIF1α by SMYD3 was mediated by the enzymatic activity of SMYD3. Under hypoxia, ectopic expression of enzymatic-inactive mutant of SMYD3 (SMYD3-F183A) still enhanced expression of PGK1 and PDK1 in HEK293T cells, similar to its wildtype form (Fig. 5, A and B).
      Figure thumbnail gr5
      Figure 5SMYD3 stabilizes and activates HIF1α independent of its methyltransferase activity. A and B, qPCR analysis of PGK1 (A) and PDK1 (B) mRNA in HEK293T cells transfected with expression plasmids encoding wildtype SMYD3 or its enzymatically dead mutant SMYD3-F183A (HA empty vector [EV] was used as a control) under normoxia (21% O2) or hypoxia (1% O2) for 24 h. Data show mean ± SD; Student’s two-tailed t test. ns, not significant, ∗p < 0.05, ∗∗p < 0.01. Data from three independent experiments. C, co-immunoprecipitation of HA-SMYD3-F183A with Myc-HIF1α. HEK293T cells were cotransfected with indicated plasmids for 24 h. Anti-HA antibody-conjugated agarose beads were used for immunoprecipitation, and the interaction was detected by immunoblotting with the indicated antibodies. D and E, immunoblotting of exogenous Myc-HIF1α expression in HEK293T (D) or H1299 (E) cells transfected with expression plasmids encoding wildtype SMYD3 or its enzymatically dead mutant SMYD3-F183A (HA empty vector [-] was used as a control). The relative intensities of HIF1α were determined by normalizing the intensities of HIF1α to the intensities of GAPDH. Data show mean ± SD; Student’s two-tailed t test. ns, not significant, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. Data from three independent experiments. qPCR, quantitative RT–PCR; HIF, hypoxia-inducible factor.
      In addition, the enzymatic-inactive mutant of SMYD3 (SMYD3-F183A) still interacted with co-expressed HIF1α under normoxia (Fig. 5C) and endogenous HIF1α under hypoxia (Fig. S6A). Consistently, overexpression of SMYD3-F183A had similar effect on co-expressed HIF1α protein stability as that of wildtype SMYD3 in either HEK293T cells or H1299 cells (Fig. 5, D and E). In addition, overexpression of SMYD3-F183A still enhanced HIF1α protein stability in H1299 cells under hypoxia (Fig. S6B).
      Taken together, these data suggest that SMYD3 stabilizes and activates HIF1α independent of its methyltransferase activity.

      SMYD3 induces ROS accumulation and enhances hypoxia-induced cell apoptosis

      Many studies have reported that reduction of the cytotoxic ROS level is associated with cell survival during hypoxia adaptation (
      • Kim T.H.
      • Hur E.G.
      • Kang S.J.
      • Kim J.A.
      • Thapa D.
      • Lee Y.M.
      • et al.
      NRF2 blockade suppresses colon tumor angiogenesis by inhibiting hypoxia-induced activation of HIF-1alpha.
      ) and that aberrant control of mitochondrial ROS levels is a major factor resulting in cell apoptosis with long-term exposure to hypoxic environments (
      • Lee H.J.
      • Jung Y.H.
      • Choi G.E.
      • Ko S.H.
      • Lee S.J.
      • Lee S.H.
      • et al.
      BNIP3 induction by hypoxia stimulates FASN-dependent free fatty acid production enhancing therapeutic potential of umbilical cord blood-derived human mesenchymal stem cells.
      ). We examined the effect of SMYD3 on ROS accumulation. Hypoxia treatment significantly induced ROS accumulation, while much lower levels of intracellular and mitochondrial ROS were detected in Smyd3−/− MEF cells compared to Smyd3+/+ MEF cells by flow cytometry assay (Fig. 6, AD).
      Figure thumbnail gr6
      Figure 6Deficiency of SMYD3 alleviates ROS accumulation. A and B, intracellular ROS levels in Smyd3-deficient or wildtype MEF cells (Smyd3−/− or Smyd3+/+) under normoxia or hypoxia detected by flow cytometry analysis. Data show mean ± SD; Student’s two-tailed t test. ∗∗∗∗p < 0.0001. Data from three independent experiments. C and D, mitochondrial ROS levels in Smyd3-deficient or wildtype MEF cells (Smyd3−/− or Smyd3+/+) under normoxia or hypoxia detected by flow cytometry analysis. Data show mean + SD; Student’s two tailed t test. ∗∗∗∗p < 0.0001. Data from three independent experiments. HIF, hypoxia-inducible factor; MEF, mouse embryonic fibroblast; ROS, reactive oxygen species.
      To determine the biological consequences of the transcriptional activity enhancement of HIF1α by SMYD3, we compared cell apoptosis between Smyd3+/+ and Smyd3−/− MEF cells under hypoxia. More apoptotic cells were detected in Smyd3+/+ MEF cells compared to Smyd3−/− MEF cells by flow cytometry assay, which was further confirmed by confocal microscopy (Fig. 7, A and B).
      Figure thumbnail gr7
      Figure 7Disruption of SMYD3 protects cells against hypoxia-induced apoptosis. A, apoptotic cells in Smyd3-deficient or wildtype MEF cells (Smyd3−/− or Smyd3+/+) under normoxia or hypoxia detected by flow cytometry analysis. Data show mean ± SD; Student’s two-tailed t test. ∗∗p < 0.01. Data from three independent experiments. B, apoptotic cells in Smyd3-deficient or wildtype MEF cells (Smyd3−/− or Smyd3+/+) under normoxia or hypoxia detected by fluorescence microscopy. Scale bar = 100 μm. MEF, mouse embryonic fibroblast.
      Subsequently, we examined the effect of overexpression of Smyd3 on cell apoptosis. In contrast, overexpression of Smyd3 enhanced cell apoptosis under hypoxia as detected by flow cytometry assay, which was further confirmed by confocal microscopy (Fig. 8, A and B).
      Figure thumbnail gr8
      Figure 8Reconstitution of Smyd3 in Smyd3-deficient cells promotes hypoxia-induced apoptosis. A, apoptotic cells in Smyd3-deficient MEF cells reconstituted with or without wildtype Smyd3 by lentivirus under normoxia or hypoxia detected by flow cytometry analysis. Data show mean ± SD; Student’s two-tailed t test. ∗∗∗p < 0.001. Data from three independent experiments. B, Smyd3-deficient MEF cells were reconstituted with or without wildtype Smyd3 by lentivirus and treated with DFX (150 μM) or DMSO as a control for 24 h. Apoptotic cells were detected by fluorescence microscopy. Scale bar = 100 μm. DFX, deferoxamine mesylate salt; MEF, mouse embryonic fibroblast.
      These data suggest that Smyd3 enhanced hypoxia-induced apoptosis, which might be mediated by HIF1α.

      Disruption of smyd3 in zebrafish facilitates hypoxia tolerance

      SMYD3 is evolutionary conserved among human, mouse, and zebrafish (Fig. 9A). In zebrafish liver cells, ectopic expression of zebrafish smyd3 caused an increase of expression of hypoxia responsive genes under hypoxia, including pdk1, vegf, and phd3 (Fig. 9, BD), suggesting that the function of SMYD3 might be conserved between mammals and zebrafish. To determine the physiological role of the transcriptional activity enhancement of HIF1α by SMYD3, we took advantage of zebrafish model. We knocked out smyd3 in zebrafish via CRISPR/Cas9 and obtained one mutant line (Fig. 10A). Heteroduplex mobility assay (HMA) and quantitative RT–PCR (qPCR) assay indicated that smyd3 was disrupted efficiently in zebrafish (Fig. 10, B and C). One predicted peptide with 176 amino acids might be produced in smyd3-null zebrafish (Fig. 10D). By crossing smyd3 +/− (♀) × smyd3 +/− (♂), the offspring with smyd3 +/+, smyd3 +/−, and smyd3 −/−genetic backgrounds were born at a Mendelian ratio (1:2:1), and no obvious defects in growth rate and reproduction capability were detected in smyd3 −/− zebrafish under normal conditions.
      Figure thumbnail gr9
      Figure 9Zebrafish smyd3 augments hypoxia signaling. A, alignment of smyd3 amino acid sequences from human, mouse, and zebrafish, and the consensus sequence is shown below. B–D, qPCR analysis of pdk1 (B), vegf (C), and phd3 (D) mRNA in ZFL cells transfected with or without pCMV-smyd3 and cultured under normoxia (21% O2) or hypoxia (1% O2) for 24 h. EV, pCMV empty vector (control). Data show mean ± SD; Student’s two-tailed t test. ∗p < 0.05, ∗∗p < 0.01. Data from three independent experiments. qPCR, quantitative RT–PCR; ZFL, zebrafish liver.
      Figure thumbnail gr10
      Figure 10Disruption of smyd3 in zebrafish facilitates hypoxia tolerance. A, scheme of the sequence information in smyd3-null zebrafish. Seven–base pair nucleotides (5′-TGCCGTC-3′) were deleted in exon five of smyd3 in the mutant, resulting in a reading frame shift. B, verification of CRISPR/Cas9-mediated zebrafish smyd3 disruption by HMA (heteroduplex mobility assay). C, qPCR analysis of smyd3 mRNA in smyd3-deficient or wildtype zebrafish larvae (smyd3−/− or smyd3+/+) (3dpf). Data show mean ± SD; Student’s two-tailed t test. ∗∗∗∗p < 0.0001. Data from three independent experiments. D, the predicted protein products of smyd3 in the mutants (176 aa) and their wildtype (429 aa) siblings. aa, amino acids. E–G, qPCR analysis of pdk1 (E), vegf (F), and phd3 (G) mRNA in smyd3-deficient or wildtype zebrafish larvae (smyd3−/− or smyd3+/+) (3dpf) under normoxia (21% O2) or hypoxia (2% O2). Data show mean ± SD; Student’s two-tailed t test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. Data from three independent experiments. H, the survival of wildtype (smyd3+/+; left flask) and smyd3-null (smyd3−/−; right flask) adult zebrafish (3mpf) after 2 h, 4 h, and 6 h under hypoxia (5% O2). Red arrows, dying zebrafish. qPCR, quantitative RT–PCR.
      In agreement with the observations by cell culture system, under hypoxia, expression of hypoxia-responsive genes, including pdk1, vegf, and phd3 was significantly lower in smyd3−/− zebrafish compared to those in smyd3 +/+ zebrafish (Fig. 10, EG). We simultaneously put smyd3-null zebrafish (smyd3−/−; KO) and their wildtype siblings (smyd3+/+; WT) into a hypoxia workstation (5%) and compared their hypoxia tolerance. At the beginning (1 h), no difference in behaviors was observed between smyd3−/− and smyd3+/+ zebrafish. However, 2 h later in the hypoxia workstation (5%), smyd3+/+ zebrafish, but not smyd3−/− zebrafish, exhibited abnormal swimming behavior (Video S1 and Fig. 10H, left panel). After 4 h in the hypoxia workstation (5%), smyd3+/+ zebrafish started to die (Fig. 10H, middle panel). After 5 to 6 h in the hypoxia workstation (5%), all of smyd3+/+ zebrafish were dead, but smyd3−/− zebrafish were still alive (Video S2 and Fig. 10H, right panel). It appeared that smyd3−/− zebrafish were more resistant to hypoxic condition.
      These data suggest that smyd3 impairs hypoxia tolerance, which might be mediated by its enhancement role on HIF1α transcriptional activity.

      Discussion

      The modulation of HIF1α activity by its binding partners has been widely recognized, particularly, the most of these binding partners with enzymatic activity can regulate HIF1α activity through multiple posttranslational modifications, leading to the impacts on HIF1α activity in hypoxia signaling pathway (
      • Kubaichuk K.
      • Kietzmann T.
      Involvement of E3 ligases and deubiquitinases in the control of HIF-alpha subunit abundance.
      ,
      • Liu X.
      • Chen Z.
      • Xu C.
      • Leng X.
      • Cao H.
      • Ouyang G.
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      Repression of hypoxia-inducible factor alpha signaling by Set7-mediated methylation.
      ,
      • Wang J.
      • Zhang D.
      • Du J.
      • Zhou C.
      • Li Z.
      • Liu X.
      • et al.
      Tet1 facilitates hypoxia tolerance by stabilizing the HIF-alpha proteins independent of its methylcytosine dioxygenase activity.
      ,
      • Chen Z.
      • Liu X.
      • Mei Z.
      • Wang Z.
      • Xiao W.
      EAF2 suppresses hypoxia-induced factor 1alpha transcriptional activity by disrupting its interaction with coactivator CBP/p300.
      ,
      • Goto Y.
      • Zeng L.
      • Yeom C.J.
      • Zhu Y.
      • Morinibu A.
      • Shinomiya K.
      • et al.
      UCHL1 provides diagnostic and antimetastatic strategies due to its deubiquitinating effect on HIF-1alpha.
      ,
      • Hong K.
      • Hu L.
      • Liu X.
      • Simon J.M.
      • Ptacek T.S.
      • Zheng X.
      • et al.
      USP37 promotes deubiquitination of HIF2alpha in kidney cancer.
      ,
      • Troilo A.
      • Alexander I.
      • Muehl S.
      • Jaramillo D.
      • Knobeloch K.P.
      • Krek W.
      HIF1alpha deubiquitination by USP8 is essential for ciliogenesis in normoxia.
      ,
      • Wu H.T.
      • Kuo Y.C.
      • Hung J.J.
      • Huang C.H.
      • Chen W.Y.
      • Chou T.Y.
      • et al.
      K63-polyubiquitinated HAUSP deubiquitinates HIF-1alpha and dictates H3K56 acetylation promoting hypoxia-induced tumour progression.
      ,
      • Shay J.E.S.
      • Simon M.C.
      Hypoxia-inducible factors: crosstalk between inflammation and metabolism.
      ). Among them, lysine methylation of HIF1α have been widely investigated. SET7-mediated monomethylation and LSD1-mediated demethylation of HIF1α at lysine 32 synergistically regulates the stability and activity of HIF1α (
      • Liu X.
      • Chen Z.
      • Xu C.
      • Leng X.
      • Cao H.
      • Ouyang G.
      • et al.
      Repression of hypoxia-inducible factor alpha signaling by Set7-mediated methylation.
      ,
      • Kim Y.
      • Nam H.J.
      • Lee J.
      • Park D.Y.
      • Kim C.
      • Yu Y.S.
      • et al.
      Methylation-dependent regulation of HIF-1alpha stability restricts retinal and tumour angiogenesis.
      ,
      • Lee J.Y.
      • Park J.H.
      • Choi H.J.
      • Won H.Y.
      • Joo H.S.
      • Shin D.H.
      • et al.
      LSD1 demethylates HIF1alpha to inhibit hydroxylation and ubiquitin-mediated degradation in tumor angiogenesis.
      ), while monomethylation and dimethylation of HIF1α at lysine 674 by G9a/GLP inhibits its transcriptional activity and expression of its downstream target genes (
      • Bao L.
      • Chen Y.
      • Lai H.T.
      • Wu S.Y.
      • Wang J.E.
      • Hatanpaa K.J.
      • et al.
      Methylation of hypoxia-inducible factor (HIF)-1alpha by G9a/GLP inhibits HIF-1 transcriptional activity and cell migration.
      ). However, whether other methyltransferases also involved in hypoxia signaling remains largely unknown. SMYD3 is a well-defined methyltransferase (
      • Bottino C.
      • Peserico A.
      • Simone C.
      • Caretti G.
      SMYD3: an oncogenic driver targeting epigenetic regulation and signaling pathways.
      ,
      • Bernard B.J.
      • Nigam N.
      • Burkitt K.
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      SMYD3: A regulator of epigenetic and signaling pathways in cancer.
      ,
      • Hamamoto R.
      • Furukawa Y.
      • Morita M.
      • Iimura Y.
      • Silva F.P.
      • Li M.
      • et al.
      SMYD3 encodes a histone methyltransferase involved in the proliferation of cancer cells.
      ). Here, we identify that SMYD3 binds to and enhances HIF1α activity, leading to the impairment of hypoxia tolerance, which is independent of its enzymatic activity. Of note, some binding partners with enzymatic activity also can affect HIF1α function independent of their enzymatic activity (
      • Wang J.
      • Zhang D.
      • Du J.
      • Zhou C.
      • Li Z.
      • Liu X.
      • et al.
      Tet1 facilitates hypoxia tolerance by stabilizing the HIF-alpha proteins independent of its methylcytosine dioxygenase activity.
      ,
      • Li B.
      • Qiu B.
      • Lee D.S.M.
      • Walton Z.E.
      • Ochocki J.D.
      • Mathew L.K.
      • et al.
      Fructose-1,6-bisphosphatase opposes renal carcinoma progression.
      ,
      • Hubbi M.E.
      • Hu H.X.
      • Kshitiz, Gilkes D.M.
      • Semenza G.L.
      Sirtuin-7 inhibits the activity of hypoxia-inducible factors.
      ,
      • Altun M.
      • Zhao B.
      • Velasco K.
      • Liu H.Y.
      • Hassink G.
      • Paschke J.
      • et al.
      Ubiquitin-specific protease 19 (USP19) regulates hypoxia-inducible factor 1 alpha (HIF-1 alpha) during hypoxia.
      ). Therefore, it might be a common phenomenon that the proteins can affect HIF1α activity only through protein–protein interaction. However, due to the lack of structure data about the interaction between SMYD3 and HIF1α, we cannot provide more information for understanding the process and the underlying mechanisms of HIF1α activity enhancement by SMYD3.
      SMYD3 contains two conserved structural domains: the Myeloid-Nervy-DEAF1 domain and the SET domain; the SET domain is consisted of the S-sequence, the core SET domain, and the C-terminus domain. The S-sequence is responsible for cofactor binding, while the core SET domain is responsible for the catalytic activity of the methyltransferase (
      • Tracy C.
      • Warren J.S.
      • Szulik M.
      • Wang L.
      • Garcia J.
      • Makaju A.
      • et al.
      The smyd family of methyltransferases: role in cardiac and skeletal muscle physiology and pathology.
      ). Here, we find that SMYD3 binds and stabilizes HIF1α, leading to enhanced hypoxic signaling independent of its enzymatic activity. To further identify which structural domain of SMYD3 interacts HIF1α might give insights into the detailed mechanisms of SMYD3 for acting its roles in hypoxic signaling.
      Given an importance of hypoxia signaling in tumor progression and cell metabolism, the present studies are mainly focused on investigating the effects of HIF1α binding partners in affecting these processes (
      • Luo W.
      • Hu H.
      • Chang R.
      • Zhong J.
      • Knabel M.
      • O'Meally R.
      • et al.
      Pyruvate kinase M2 is a PHD3-stimulated coactivator for hypoxia-inducible factor 1.
      ) (
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      Sirtuin 1 modulates cellular responses to hypoxia by deacetylating hypoxia-inducible factor 1alpha.
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      ,
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      ). In fact, the roles of hypoxia signaling in hypoxia adaptation and tolerance have been noticed, particularly for high-altitude adaptation (
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      ,
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      ,
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      ,
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      • et al.
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      ). High altitude is defined as areas over 2500 m above sea level, in which the ambient oxygen is much lower than low altitude area. Humans living in these areas often face great challenges due to low oxygen. Genetic evidences indicate that some human genes have gone through adaptive mutation for high altitude adaptation, and the most of them are the core components of hypoxia signaling pathway (
      • Bigham A.W.
      Genetics of human origin and evolution: high-altitude adaptations.
      ). In this study, by cell culture system and zebrafish model, we found that disruption of Smyd3 impairs hypoxia-induced cell apoptosis, leading to the facilitation of hypoxia tolerance. These observations not only support an important contribution of HIF1α in hypoxia tolerance but also provide a practical research model for testing hypoxia tolerance by zebrafish model. To further use zebrafish as a model to investigate the factors involved in the regulation of hypoxia signaling as well as their impacts on hypoxia tolerance might open a new window for understanding the mechanisms of high-altitude adaptation.
      In this study, we show that SMYD3 enhances hypoxia-induced cell apoptosis, resulting in the impairment of hypoxia tolerance. However, the multiple functions of HIF1α have been identified, and SMYD3 may also affect HIF1α functions other than hypoxia tolerance, such as tumorigenesis, cell metabolism, etc. To further figure out the other effects of SMYD3 mediated through HIF1α will help us to fully understand the physiological role of SMYD3 in hypoxia signaling and the underlying mechanisms.

      Experimental procedures

      Cell line and culture conditions

      HEK293T and H1299 cells originally obtained from American Type Culture Collection were cultured in Dulbeccos’ modified Eagle medium (VivaCell Biosciences) with 10% fetal bovine serum (FBS) at 37 °C in a humidified incubator containing 5% CO2. RCC4 cells were provided by Peter J. Ratcliffe and maintained as described previously (
      • Liu X.
      • Chen Z.
      • Xu C.
      • Leng X.
      • Cao H.
      • Ouyang G.
      • et al.
      Repression of hypoxia-inducible factor alpha signaling by Set7-mediated methylation.
      ). Zebrafish liver cells were provided by Dr Shun Li and maintained as described previously (
      • Wu H.
      • Sun L.
      • Wen Y.
      • Liu Y.
      • Yu J.
      • Mao F.
      • et al.
      Major spliceosome defects cause male infertility and are associated with nonobstructive azoospermia in humans.
      ). Smyd3-deficient or wildtype MEF cells (Smyd3−/− or Smyd3+/+) were established as described previously (
      • Liu X.
      • Zhu C.
      • Zha H.
      • Tang J.
      • Rong F.
      • Chen X.
      • et al.
      SIRT5 impairs aggregation and activation of the signaling adaptor MAVS through catalyzing lysine desuccinylation.
      ) and cultured in Dulbeccos’ modified Eagle medium supplemented with sodium pyruvate (110 mg/L), 10% FBS, 1× nonessential amino acids (Sigma), and 1% penicillin–streptomycin at 37 °C in a humidified incubator containing 5% CO2. During hypoxia treatment, the cells were cultured under hypoxic condition (1% O2, 5% CO2, and balanced with N2) by using the NBS Galaxy 48R incubator. The cells were transfected with various amounts of plasmids as indicated by VigoFect (Vigorous Biotech).

      Quantitative real-time PCR assay

      Total RNAs were extracted using RNAiso Plus (TaKaRa Bio). cDNAs were synthesized using the Revert Aid First Strand cDNA Synthesis Kit (Thermo Scientific). qPCR assays were conducted with MonAmp SYBR Green qPCR Mix (high Rox) (Monad Bio.). The procedure was done according to the protocol provided by the manufacturer. The primers for quantitative RT-PCR assays are listed in Table S1.

      Antibodies and chemical reagents

      Anti-SMYD3 (#ab199361) antibody was purchased from Abcam. Antibodies including anti-HIF1α (#36169), anti-VHL (#68547), anti-Histone H3 (#4499), anti-HIF2α (#7096), anti-ARNT (#5537), and normal rabbit IgG (#2729) were purchased from Cell Signaling Technology. Anti-ACTB (#AC026) antibody was purchased from ABclonal. Anti-HA (#901515) antibody was purchased from Covance. Anti-Myc (#SC-40) and anti-GAPDH (#SC-477242) antibodies were purchased from Santa Cruz Biotechnology. Anti-α-tubulin (#62204), Alexa Fluor 488 goat anti-rabbit IgG (#A11008), Alexa Fluor 594 goat anti-mouse IgG (#A11005), CM-H2DCFDA (#C6827), and MitoSOX Red (#M36008) were purchased from Thermo Fisher Scientific. CoCl2 (#C8661) and deferoxamine mesylate salt (#D9533) were purchased from Sigma. FG4592 (#S1007) and PX478 (#S7612) were purchased from Selleck. Cycloheximide (#HY-12320) was purchased from MCE.

      Immunoprecipitation and Western blot

      Co-immunoprecipitation and Western blot analysis were performed as described previously (
      • Chen Z.
      • Liu X.
      • Mei Z.
      • Wang Z.
      • Xiao W.
      EAF2 suppresses hypoxia-induced factor 1alpha transcriptional activity by disrupting its interaction with coactivator CBP/p300.
      ). Anti-HA antibody-conjugated agarose beads (#A2095) were purchased from Sigma. Protein G Sepharose (#17–0618–01) was purchased from GE HealthCare Company. The blots were photographed with the Fuji Film LAS4000 mini-luminescent image analyzer. The protein levels were quantified with Image J software (National Institutes of Health) based on the band density obtained by Western blot analysis.

      CRISPR-Cas9 knockout cell lines

      To generate HEK293T knocked-out cell lines of indicated genes, sgRNA sequence were ligated into Lenti-CRISPRv2 plasmid and then co-transfected with viral packaging plasmids (psPAX2 and pMD2.G) into HEK293T cells. Six hours after transfection, medium was changed, and viral supernatant was collected and filtered through 0.45-μm strainer. Targeted cells were infected by viral supernatant and selected by 1 μg/ml puromycin for 2 weeks. The sgRNA sequence targeting VHL was described as previously (
      • Zhang J.
      • Wu T.
      • Simon J.
      • Takada M.
      • Saito R.
      • Fan C.
      • et al.
      VHL substrate transcription factor ZHX2 as an oncogenic driver in clear cell renal cell carcinoma.
      ). The sgRNA sequence targeting SMYD3 is 5′-CCAAGAAGTCGAACGGAGTC-3′. The sgRNA sequence targeting ARNT is GTCGCCGCTTAATAGCCCTC.

      Lentivirus-mediated gene transfer

      HEK293T cells were transfected with pHAGE-Smyd3 or pHAGE empty vector with the packaging vectors psPAX2 and pMD2.G. Eight hours later, the medium was changed with fresh medium containing 10% FBS, 1% streptomycin–penicillin, and 10 μM β-mercaptoethanol. Forty hours later, supernatants were harvested and filtered through 0.45-μm strainer and then used to infect Smyd3-deficient MEF cells (Smyd3−/−).

      Immunofluorescence confocal microscopy

      Immunofluorescence staining was conducted as previously described (
      • Liu X.
      • Zhu C.
      • Zha H.
      • Tang J.
      • Rong F.
      • Chen X.
      • et al.
      SIRT5 impairs aggregation and activation of the signaling adaptor MAVS through catalyzing lysine desuccinylation.
      ). Cells were seeded on glass coverslips and cultured as indicated. Then, the cells were fixed in 4% paraformaldehyde in PBS for 30 min at 25 °C. After washing three times by PBS, the slides were blocked in the blocking buffer (5% goat serum, 2 mg/ml BSA, 0.1%Triton X-100 in PBS) for 1 h and incubated with primary antibodies overnight at 4 °C, followed by incubation with Alexa Fluor 488 goat anti-rabbit IgG or Alexa Fluor 594 goat anti-mouse IgG for 1 h at 25 °C. Subsequently, the slides were mounted with VECTASHIELD mounting medium containing DAPI and photographed with Leica SP8 laser scanning confocal fluorescence microscope.

      Nucleus and cytoplasm separation

      Nucleus and cytoplasm separation was conducted with the Nuclear and Cytoplasmic Extraction Kit (#78833, Thermo Scientific) according to the protocol provided by the manufacturer. The extracts were analyzed by Western blot analysis. To ensure the efficiency of fraction separation, anti-α-tubulin antibody was employed to monitor cytoplasmic proteins, and anti-Histone H3 antibody was used to monitor nuclear proteins.

      Measurement of intracellular ROS level

      MEF cells were cultured under hypoxia as indicated. After treatment, MEF cells were collected and counted. Cells (1 × 106) were incubated in PBS solution containing 1 μM of CM-H2DCFDA (#C6827, Thermo Fisher) at 37 °C for 60 min and then washed with PBS three times, followed by flow-cytometric analysis.

      Measurement of mitochondrial ROS level

      MEF cells were cultured under hypoxia as indicated. After treatment, MEF cells were collected and washed with PBS. Then, the cells were incubated in PBS solution containing 5 μM of MitoSOX Red (# M36008, Thermo Fisher) for 10 min at 37 °C and then washed gently three times with PBS, followed by flow-cytometric analysis.

      Detection of apoptotic cells

      MEF cells were cultured under hypoxia or treated with DFX as indicated. For flow cytometry analysis, the cells were harvested and stained with FITC-Annexin V and PI with FITC Annexin V Apoptosis Detection Kit I (#556547, BD Pharmingen) according to the manufacturer’s instructions. Apoptotic cells were detected using Beckman CytoFLEXS, and the data were analyzed with CytExpert software. Besides, the cells were stained with Annexin V-FITC Apoptosis Detection Kit (#C1062, Beyotime) according to the manufacturer’s instructions in 6-well plate and imaged under a florescent microscope Nikon TE2000-U.

      Generation of smyd3-null zebrafish

      Disruption of smyd3 in zebrafish was accomplished via CRISPR/Cas9 technology. Zebrafish smyd3 sgRNA was designed using the tools provided in the CRISPR Design web site (http://crispr.mit.edu). The sgRNA sequence targeting smyd3 is 5′-TCTGCCGTCCGGCCTCGAC-3′ and sgRNA was synthesized using the Transcript Aid T7 High Yield Transcription Kit (Fermentas). Cas9 RNA and sgRNA were prepared as described previously (
      • Wu H.
      • Sun L.
      • Wen Y.
      • Liu Y.
      • Yu J.
      • Mao F.
      • et al.
      Major spliceosome defects cause male infertility and are associated with nonobstructive azoospermia in humans.
      ) and then mixed and injected into embryos at the one-cell stage. Mutant detection was followed by HMA as described previously (
      • Liu X.
      • Chen Z.
      • Xu C.
      • Leng X.
      • Cao H.
      • Ouyang G.
      • et al.
      Repression of hypoxia-inducible factor alpha signaling by Set7-mediated methylation.
      ). If the results were positive, the remaining embryos were raised to adulthood and treated as F0. The F0 zebrafish were backcrossed with the wildtype zebrafish to generate F1, which were genotyped by HMA and then confirmed by sequencing of target sites. The F1 zebrafish harboring the mutations were backcrossed with the wild-type zebrafish to obtain F2. The F2 adult zebrafish with the same genotype (+/−) were intercrossed to generate F3 offspring, which should contain wild-type (+/+), heterozygous (+/−), and homozygous (−/−) offspring. The primers for detecting mutants were 5′-ATCTCGCAGACATGAGTGAG-3’ (forward) and 5′-CACCGGTCTGACAGCAGCAG-3’ (reverse). The zebrafish smyd3 mutant was named smyd3ihbsm3/ihbsm3 (https://zfin.org/ZDB-ALT-220302-1) following zebrafish nomenclature guidelines (zfin.atlassian.net/wiki/spaces/general/pages/1818394635/ZFIN+Zebrafish+Nomenclature+Conventions).

      Hypoxia treatments of zebrafish

      Hypoxia treatments of zebrafish were conducted in the hypoxia workstation (Ruskinn INVIVO2 I-400) as described previously (
      • Liu X.
      • Cai X.
      • Hu B.
      • Mei Z.
      • Zhang D.
      • Ouyang G.
      • et al.
      Forkhead transcription factor 3a (FOXO3a) modulates hypoxia signaling via up-regulation of the von Hippel-lindau gene (VHL).
      ). For zebrafish larvae (3 days postfertilization [dpf]) experiment, two dish were filled with 10 ml of water. Smyd3-null larvae (3 dpf, n = 30) (smyd3−/−) were put into one dish, and their wildtype siblings (3 dpf, n = 30) (smyd3+/+) were put in the second dish. The oxygen concentration in the hypoxia workstation was adjusted to 2% ahead of time. Then, two dishes were put into the hypoxia workstation simultaneously. Four hours later, the samples were harvested for qPCR analysis. This experiment was repeated three times. For the adult zebrafish (3-months postfertilization [mpf]) experiment, zebrafish of similar weight were chosen for further experiments. Two flasks were filled with 200 ml of water. Three smyd3-null zebrafish (smyd3−/−) were put into one flask, and three wildtype siblings (smyd3+/+) were put into the second flask. The oxygen concentration in the hypoxia workstation was adjusted to 5% ahead of time. After putting the flasks containing zebrafish into the hypoxia workstation, the behavior of the zebrafish was closely monitored. All animal protocols were approved by the Institutional Animal Care and Use Committee at Institute of Hydrobiology, Chinese Academy of Science.

      Statical analysis

      GraphPad Prism software (7.0) was used for all statistical analysis. Results with error bars express mean ± SD. Statistical analysis was performed by using Student’s two-tailed t test. A p value less than 0.05 was considered significant. Statistical significance is represented as follows: ∗p< 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

      Data availability

      Further information and requests for resources and reagents should be directed to and will be fulfilled by Xing Liu and Wuhan Xiao.

      Supporting information

      This article contains supporting information.

      Conflict of interest

      The authors declare that they have no conflicts of interest with the contents of this article.

      Acknowledgments

      We are grateful to Drs Peter J. Ratcliffe, William Kaelin, Amato Giaccia, Eric Huang, and Navdeep Chandel for the generous gifts of reagents.

      Author contributions

      Z. W., X. L., and W. X. conceptualization; Z. W. and X. L. methodology; Z. W., X. C., S. F., C. Z., H. D., J. T., X. S., S. J., and Q. L. visualization; Z. W., X. C., S. F., and X. L. investigation; X. L. data curation; X. L. writing-original draft; W. X. supervision, W. X. writing-reviewing and editing.

      Funding and additional information

      This study was funded by NSFC [31830101 and 31721005 to W. X.]; The Strategic Priority Research Program of the Chinese Academy of Sciences [XDA24010308 to W. X.]; and the National Key Research and Development Program of China [2018YFD0900602, to W. X.]. Funding for open access charge: NSFC [31,830,101 to W. X.]. Supplementary information is available at Journal of Biological Chemistry’s website.

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