Deletion of prolyl hydroxylase domain-containing enzyme 3 (phd3) in zebrafish facilitates hypoxia tolerance

Prolyl hydroxylase domain (PHD)-containing enzyme 3 (PHD3) belongs to the Caenorhabditis elegans gene egl-9 family of prolyl hydroxylases. PHD3 catalyzes proline hydroxylation of hypoxia-inducible factor α (HIF-α) and promotes HIF-α proteasomal degradation through coordination with the pVHL complex under normoxic conditions. However, the relationship between PHD3 and the hypoxic response is not well understood. In this study, we used quantitative real-time PCR assay and O-dianisidine staining to characterize the hypoxic response in zebrafish deficient in phd3. We found that the hypoxia-responsive genes are upregulated and the number of erythrocytes was increased in phd3-null zebrafish compared with their wild-type siblings. On the other hand, we show overexpression of phd3 suppresses HIF-transcriptional activation. In addition, we demonstrate phd3 promotes polyubiquitination of zebrafish hif-1/2α proteins, leading to their proteasomal degradation. Finally, we found that compared with wild-type zebrafish, phd3-null zebrafish are more resistant to hypoxia treatment. Therefore, we conclude phd3 has a role in hypoxia tolerance. These results highlight the importance of modulation of the hypoxia signaling pathway by phd3 in hypoxia adaptation.

Prolyl hydroxylase domain (PHD)-containing enzyme 3 (PHD3) belongs to the Caenorhabditis elegans gene egl-9 family of prolyl hydroxylases.PHD3 catalyzes proline hydroxylation of hypoxia-inducible factor α (HIF-α) and promotes HIF-α proteasomal degradation through coordination with the pVHL complex under normoxic conditions.However, the relationship between PHD3 and the hypoxic response is not well understood.In this study, we used quantitative real-time PCR assay and Odianisidine staining to characterize the hypoxic response in zebrafish deficient in phd3.We found that the hypoxiaresponsive genes are upregulated and the number of erythrocytes was increased in phd3-null zebrafish compared with their wild-type siblings.On the other hand, we show overexpression of phd3 suppresses HIF-transcriptional activation.In addition, we demonstrate phd3 promotes polyubiquitination of zebrafish hif-1/2α proteins, leading to their proteasomal degradation.Finally, we found that compared with wild-type zebrafish, phd3null zebrafish are more resistant to hypoxia treatment.Therefore, we conclude phd3 has a role in hypoxia tolerance.These results highlight the importance of modulation of the hypoxia signaling pathway by phd3 in hypoxia adaptation.
Zebrafish have evolutionarily conserved PHD family members, including phd1, phd2a, phd2b, and phd3, whose functions might be similar to those of mammalian PHDs (22,23).Different from terrestrial organisms, fish encounter more frequent fluctuations of oxygen and they are often threatened by low oxygen levels in the water (24,25).Therefore, zebrafish might be a good model for exploring the role of PHDs in hypoxia tolerance as previous similar studies have shown (26)(27)(28)(29).Interesting, the zebrafish embryos with mutated vhl exhibit a systemic hypoxic response with upregulation of the hypoxia-responsive genes and display typical features of the human VHL-associated disorder, indicating a similar function between mammalian VHL and zebrafish vhl (30).Furthermore, phd3 is one of the most highly expressed genes in vhl mutants at various stages compared with that in the wildtype zebrafish (31,32), further suggesting that zebrafish might be an ideal model for investigating the role of phd3 in hypoxia tolerance.
Zebrafish have two orthologous copies of each hif gene, hif1αa and hif1αb, hif2αa and hif2αb (33).In this study, we find that the expression of phd3 can only be induced by hif1αb, hif2αa, and hif2αb.Moreover, hypoxia-responsive genes are upregulated, and the number of erythrocytes is increased in phd3-null zebrafish compared with those in their wildtype siblings, leading to higher hypoxia tolerance in phd3-null zebrafish.Further assays indicate that phd3 suppresses HIF transcriptional activation.In addition, phd3 promotes polyubiquitination of hif-1/2α, thereby inducing proteasomal degradation of hif-1/2α.This study reveals the role of phd3 in hypoxia tolerance, highlighting the importance of modulation of the hypoxia signaling pathway by phd3 in hypoxia adaptation.

Zebrafish phd3 is induced under hypoxia
Given that phd3 is one of the most highly hypoxia-inducible genes in zebrafish and zebrafish is suitable for investigating hypoxia tolerance (26,30,31,(33)(34)(35), we sought to know whether zebrafish phd3 has an impact on hypoxia tolerance and to understand the underlying mechanisms.Initially, we compared the homology between the human PHD3 protein and the zebrafish phd3 protein.Overall, PHD3 is evolutionarily conserved, and its catalytic domain shares 85.85% identity between human PHD3 and zebrafish phd3 (Fig. 1A).
Phylogenetic analysis indicated that zebrafish phd3 is close to those of teleost fish (Fig. 1B).Structural prediction also showed that the main structure of PHD3 is conserved between human PHD3 and zebrafish phd3 (Fig. 1C) (36).Among 8 zebrafish tissues, phd3 is highly expressed in the liver (Fig. 1D).These data suggest that phd3 might have conserved functions.
Next, we examined the expression of phd3 in response to hypoxia.In zebrafish larvae, upon hypoxia treatment, phd3 was induced at the highest level among 4 phds and typical hypoxiaresponsive genes examined (Fig. 2, A and B).Similar results were obtained in ZFL cells (Fig. 2, C and D).Consistently, in ZFL cells, among four zebrafish hif-1/2α homologous genes, phd3 was transactivated by ectopic expression of hif1αb, hif2αa and hif2αb, but not hif1αa, which is similar but not the same as ldha and vegfaa, two typical hypoxia-responsive genes (Fig. 2, E-G) (33).These data suggest that phd3 is one of the most highly induced genes by hypoxia.
Disruption of phd3 in zebrafish enhances the expression of hypoxia-responsive genes and increases the number of erythrocytes in response to hypoxia We then took advantage of phd3-null zebrafish to determine the effect of phd3 on the hypoxia signaling pathway (22).
Taken together, these data suggest that disruption of phd3 in zebrafish enhances the expression of hypoxia-responsive genes and increases the number of erythrocytes in response to hypoxia.

Disruption of phd3 in zebrafish facilitates hypoxia tolerance
Given that the increase of the erythrocyte numbers in phd3null zebrafish, we sought to know whether phd3 has an impact on hypoxia tolerance.After phd3 +/+ and phd3 −/− zebrafish larvae were simultaneously put into a hypoxia station (2% O 2 ) for 15 h, more phd3 +/+ zebrafish larvae displayed symptoms of death, such as no blood circulation, curvy body, and body degeneration, and so on.(Fig. 5, A and B).Moreover, when phd3 +/+ and phd3 −/− adult zebrafish were put into the hypoxia station (5% O 2 ), no difference in swimming behavior was observed between phd3 +/+ and phd3 −/− zebrafish at the beginning (Fig. 5C, left panel).However, after 2 h, phd3 +/+ zebrafish started to jump out of the water due to the intolerable low oxygen in the water, while phd3 −/− zebrafish still swam normally (Fig. 5C, middle panel).After 4 h, more phd3 +/+ zebrafish died (Fig. 5C, right panel).The survival curve also indicated that phd3 −/− zebrafish were more resistant to hypoxic conditions.
Collectively, these data suggest that disruption of phd3 in zebrafish facilitates hypoxia tolerance.

Zebrafish phd3 induces the degradation of hif-1/2α proteins by promoting the polyubiquitination of hif-1/2α
We subsequently intended to know whether zebrafish phd3 can mediate the degradation of zebrafish hif-1/2α proteins.As shown in Figure 7, overexpression of phd3 clearly induced the downregulation of hif1αa, hif1αb, hif2αa, and hif2αb protein levels.The cycloheximide (CHX) treatment assays indicated that new protein synthesis was not required for phd3 to mediate the downregulation of hif-1/2α protein levels (Fig. 7, A-L), suggesting that phd3 may induce the degradation of hif-1/2α proteins.In addition, we confirmed these results in EPC cells (Fig. S2, A-F).Furthermore, we screened suitable antibodies that could detect endogenous zebrafish hif-α proteins (Fig. S3, A-D).Only two commercially available antibodies were able detect zebrafish hif2αb (Fig. S3D).As expected, hif2αb protein levels were higher in phd3-deficient zebrafish larvae or adult brains, compared with that in wild-type siblings (Fig. S3, E and F).

Discussion
Hypoxia adaptation represents one of the most important physiological adaptations for organisms during the evolution process, such as for organisms living at high altitudes (37).These organisms have evolved the capability to tolerate much lower oxygen in the air than organisms living at lower altitudes.Although genetic variants associated with high-altitude adaptation in humans vary widely among populations (including Tibetan, Andean, and Ethiopian) (37-39), most terrestrial mammals living at high altitudes exhibit unique variants in the genes encoding HIF-2α and PHD2, two major components of the hypoxia signaling pathway (15,37,(40)(41)(42)(43)(44).In this study, we found that disruption of phd3 in zebrafish enhances the expression of hypoxia-responsive genes including epoa, which triggers an increase of erythrocytes in response to hypoxia.Erythrocytes are known to be responsible for O 2 delivery (45).Thus, increased erythrocytes may enable organs and tissues to receive more oxygen, resulting in increased survival of zebrafish under hypoxia.This may be a possible mechanism accounting for the effect of disruption of phd3 in zebrafish.
Interestingly, we found that overexpression of phd3 strongly suppresses HIF-α transcriptional activation, but disruption of phd3 only leads to moderately increased expression of hypoxia-inducible genes.In fact, HIF induces the expression of a range of downstream genes under hypoxic conditions.Although many of these genes are only moderately induced, this large number of induced genes work together to modulate the hypoxia response of the organism (46)(47)(48).In addition, we observed that phd3 knockout could induce the expression of phd2a, which might compensate for the function of phd3.Therefore, the effect of phd3 overexpression on the hypoxia signaling pathway is much stronger than that of phd3 knockout.However, we identified that phd3-null zebrafish are more resistant to hypoxia treatment, highlighting the critical role of modulation of the hypoxia signaling pathway by phd3 in hypoxia tolerance.To further screen whether genetic variants of PHD3 are presented in hypoxia-tolerant organisms will provide new molecular signatures for hypoxia adaptation.
Being different from terrestrial animals, fish live in water throughout their whole life span.Noteworthy, at the same altitude, compared with oxygen in air, oxygen dissolved in water is very unstable, which varies frequently depending on seasonal variation, the change of day and night, weather change, temperature change, depth of water, mobility of water, and organisms living in water, and so on.Consequently, different hypoxia-tolerant capabilities have been evolved by different fish species (25).This difference might cause some functional ambiguity of the hypoxia signaling pathway in hypoxia adaptation or tolerance between fish and terrestrial animals, such as the behavior of phd3.Phd3 is one of the most highly induced genes by hypoxia in zebrafish (30,31), but it is only mildly induced by hypoxia in mammalian cells (14).In addition, there are conflicting reports on the effect of PHD3 on HIF regulation, and mammalian PHD3 has been reported to be a transcriptional coactivator of HIF-1α instead of repressor (49)(50)(51).Thus, it is worth further characterizing whether Phd3-null mice also display hypoxia tolerance, which will give insight into the mechanistic difference in hypoxia tolerance between fish and terrestrial animals.
In the modern aquaculture industry, high-density aquaculture is becoming one of the major culture models.The oxygen concentration in water becomes a major limiting factor for fish Figure 7. Zebrafish phd3 induces the degradation of hif-1/2α proteins.A, Western blot analysis of hif1αa protein in HEK293T cells transfected with Flag-hif1αa together with Myc-phd3 or Myc empty vector (control) for 24 h.B and C, Western blot analysis of hif1αa protein in HEK293T cells transfected with Flag-hif1αa together with Myc-phd3 or Myc empty vector (control) for 20 h, followed by treatment with cycloheximide (CHX) (50 μg/ml) for the indicated time.The relative intensities of hif1αa in (B) were determined by normalizing the intensities of hif1αa to the intensities of β-actin.D, Western blot analysis of hif1αb protein in HEK293T cells transfected with Flag-hif1αb together with Myc-phd3 or Myc empty vector (control) for 24 h.E and F, Western blot analysis of hif1αb protein in HEK293T cells transfected with Flag-hif1αb together with Myc-phd3 or Myc empty vector (control) for 20 h, followed by treatment with CHX (50 μg/ml) for the indicated time.The relative intensities of hif1αb in (E) were determined by normalizing the intensities of hif1αb to the intensities of β-actin.G, Western blot analysis of hif2αa protein in HEK293T cells transfected with Flag-hif2αa together with Myc-phd3 or Myc empty vector (control) for 24 h.H and I, Western blot analysis of hif2αa protein in HEK293T cells transfected with Flag-hif2αa together with Myc-phd3 or Myc empty vector (control) for live and growth in this kind of culture system (25,52,53).To resolve this issue, we need to cultivate fish strains with higher hypoxia tolerance.In this study, we found that the knockout of phd3 in zebrafish does not affect fish development, growth, and reproduction but facilitates hypoxia tolerance.Our previous work also showed that the knockout of phd3 in zebrafish benefits antiviral ability (22).Therefore, phd3 appears to be an ideal target for cultivating fish strains with hypoxia tolerance as well as antiviral ability by genetic manipulation.Further identifying whether disruption of phd3 in fish can affect other economic traits will be fully considered in order to breed better varieties in the aquaculture industry.

Sequence alignment and phylogenetic analysis
The amino acid sequences of phd3 from 12 indicated species were downloaded from the National Center for Biotechnology Information (NCBI) (https://www.ncbi.nlm.nih.gov) and subjected to alignment using the CLUSTAL W program.The phylogenetic tree based on the neighbor-joining method was constructed using MEGA7 software.

Cell line and zebrafish
Zebrafish liver (ZFL) cells were originally obtained from the American Type Culture Collection (ATCC) and cultured in Ham's F-12 medium (HyClone) supplemented with 10% fetal bovine serum (FBS) (Viva Cell).Epithelioma papulosum cyprini (EPC) cells (originally obtained from ATCC) were cultured in Medium 199 (Earle's Salts Base) (Viva Cell) supplemented with 10% FBS.ZFL and EPC cells were maintained in a humidified incubator containing 5% CO 2 at 28 C. HEK293T cells (originally obtained from ATCC) were cultured in DMEM (VivaCell) supplemented with 10% FBS in a humidified incubator containing 5% CO 2 at 37 C.All cell lines were free from mycoplasma contamination.
Zebrafish (AB strain) were reared 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.

O-dianisidine staining
The phd3-null zebrafish larvae [3 days post-fertilization (dpf); n = 10] and their wildtype siblings (3dpf; n = 10) in disposable 60-mm cell culture dishes filled with 5 ml egg water were incubated under normoxic and hypoxic conditions, or the indicated larvae were treated with FG4592 (20 μM) or DMSO as a control for 12 h.The larvae were then incubated with O-dianisidine solution (Sigma-Aldrich O-dianisidine dissolved in 100% ethanol, 0.1 M sodium acetate, and 30% H 2 O 2 ) in a 12-well plate for 1 h.After incubation, the larvae were washed with ddH 2 O and fixed with 4% paraformaldehyde in PBS overnight at 4 C.The larvae were then incubated in a bleaching solution (0.9% H 2 O 2 , 0.8% KOH, and 0.1% Tween in ddH 2 O) for 30 min to remove their natural pigmentation, and then in 4% paraformaldehyde for further fixation.After these steps, the larvae were immersed in 3% methylcellulose-M450 solution in a 100-mm cell culture dish and imaged using a Nikon TE2000-U microscope.

Quantitative real-time PCR assay
RNAiso Plus (TaKaRa Bio) was used for total RNA extraction according to the manufacturer's instructions.Revert Aid First Strand cDNA Synthesis Kit (Thermo Scientific) was used for cDNA synthesis.MonAmp SYBR Green qPCR Mix (high Rox) (Monad Bio.) was used for quantitative real-time PCR assays.Primers are listed in Table S1.

Luciferase reporter assay
Cells seeded in 24-well plates were transfected with the indicated plasmids together with pCMV-Renilla as an internal control.Twenty-4 h after transfection, luciferase reporter activity was determined using the dual-luciferase reporter assay system (#E1960, Promega).Data were normalized to Renilla luciferase.Data are expressed as mean ± SD of a representative experiment performed in triplicate based on 3 independent experiments.

Western blot analysis
Cells were washed with ice-cold PBS and then lysed in RIPA solution containing 150 mM NaCl, 50 mM Tris (pH 7.4), 1 mM NaF, 1 mM EDTA (pH 8.0), 1% Nonidet P-40, 0.25% sodium deoxycholate, 1 mM PMSF, 1 mM Na 3 VO 4 , and a 1:100 dilution of protease inhibitor mixture (Sigma-Aldrich) for 30 min at 4 C. Cell lysates were separated by 10% SDS-PAGE, incubated with the indicated antibodies, and photographed on a Fuji Film LAS4000 mini-luminescence image analyzer using ECL Western 400 blotting detection reagent (Millipore).Image J software (National Institutes of Health) was used to quantify protein levels based on the band density obtained by Western blot analysis.

Statical analysis
GraphPad Prism software (8.3.0) was used for all statistical analyses.Results with error bars represent mean ± SD.Survival data were calculated by the Kaplan-Meier method and analyzed by the log-rank test.Other statistical analyses were performed using unpaired Student's t test or two-way ANOVA analysis.p values less than 0.05 were considered significant.Statistical significance is indicated as follows: *p< 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; data are representative of three independent experiments performed in three technical repeats.

Figure 6 .
Figure 6.Zebrafish phd3 suppresses HIF transcriptional activation.A, luciferase activity of hypoxia-responsive element (HRE) reporter in EPC cells transfected with Myc-phd3 or Myc empty vector (control) under normoxia (21% O 2 ) or hypoxia (1% O 2 ) for 24 h.B, luciferase activity of the HRE reporter in EPC cells co-transfected with Flag-hif1αa or Flag empty vector (control) together with an increasing amount of Myc-phd3 under normoxia (21% O 2 ) for 24 h.C, luciferase activity of the HRE reporter in EPC cells co-transfected with Flag-hif1αb or Flag empty vector (control) together with an increasing amount of Myc-phd3 under normoxia (21% O 2 ) for 24 h.D, luciferase activity of the HRE reporter in EPC cells co-transfected with Flag-hif2αa or Flag empty vector (control) together with an increasing amount of Myc-phd3 under normoxia (21% O 2 ) for 24 h.E, luciferase activity of the HRE reporter in EPC cells cotransfected with Flag-hif2αb or Flag empty vector (control) together with an increasing amount of Myc-phd3 under normoxia (21% O 2 ) for 24 h.ns, not significant; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, using two-way ANOVA analysis; data are representative of three independent experiments (mean ± SD of three technical replicates).

Figure 8 .
Figure 8. Zebrafish phd3 promotes polyubiquitination of hif-1/2α proteins.A, the ubiquitination of hif1αa protein in HEK293T cells transfected with the indicated plasmids for 24 h, followed by MG-132 (20 μM) treatment for 8 h.The relative intensities of hif1αa ubiquitination were determined by normalizing the intensities of poly-ubiquitinated hif1αa in the Ni 2+ -pulldown to the intensities of hif1αa in the total cell lysates (TCL).B, the ubiquitination of hif1αb protein in HEK293T cells transfected with the indicated plasmids for 24 h, followed by MG-132 (20 μM) treatment for 8 h.The relative intensities of hif1αb ubiquitination were determined by normalizing the intensities of poly-ubiquitinated hif1αb in the Ni 2+ -pulldown to the intensities of hif1αb in the TCL.C, the ubiquitination of hif2αa protein in HEK293T cells transfected with the indicated plasmids for 24 h, followed by MG-132 (20 μM) treatment for 8 h.The relative intensities of hif2αa ubiquitination were determined by normalizing the intensities of poly-ubiquitinated hif2αa in the Ni 2+ -pulldown to the intensities of hif2αa in the TCL.D, the ubiquitination of hif2αb protein in HEK293T cells transfected with the indicated plasmids for 24 h, followed by MG-132 (20 μM) treatment for 8 h.The relative intensities of hif2αb ubiquitination were determined by normalizing the intensities of poly-ubiquitinated hif2αb in the Ni 2+ -pulldown to the intensities of hif2αb in the TCL.