Articles in PresS. Am J Physiol Regul Integr Comp Physiol (December 17, 2014). doi:10.1152/ajpregu.00281.2014

Evolution of the oxygen sensitivity of cytochrome c oxidase subunit 4 KM Kocha, K Reilly, DSM Porplycia, J McDonald, T Snider, CD Moyes1

Department of Biology, Queen’s University Kingston, Ontario, Canada

Running Head: Evolution of the oxygen sensitivity of COX4 1

Address for Correspondence:

CD Moyes, Biosciences Complex, Queen’s University, Kingston, ON, Canada, Email: [email protected] Phone: 1 613 533 6157

8 Figures, 4 Tables

Copyright © 2014 by the American Physiological Society.

Abstract Vertebrates possess two paralogs of cytochrome oxidase subunit 4: a ubiquitous COX4-1 and a hypoxia-linked COX4-2. Mammalian COX4-2 is thought to have a role in relation to fine-tuning metabolism in low oxygen levels, conferred through both structural differences in the subunit protein structure and regulatory differences in the gene. We sought to elucidate the pervasiveness of this feature across vertebrates. The ratio of COX4-2 / 4-1 mRNA are generally low in mammals, but this ratio was higher in fish and reptiles, particularly turtles. The COX4-2 gene appeared unresponsive to low oxygen in non-mammalian models (zebrafish, goldfish, tilapia, anoles, turtles) and fish cell lines. Reporter genes constructed from the amphibian and reptile homologues of the mammalian oxygen responsive elements and hypoxia responsive elements did not respond to low oxygen. Unlike the rodent ortholog, the promoter of goldfish COX4-2 did not respond to hypoxia or anoxia. The protein sequences of the COX4-2 peptide showed that the disulfide bridge seen in human and rodent orthologs would be precluded in other mammalian lineages and lower vertebrates, all of which lack the requisite pair of cysteines. The coordinating ligands of the ATP binding site are largely conserved across mammals and reptiles, but in Xenopus and fish, sequence variations may disrupt the ability of the protein to bind ATP at this site. Collectively, these results suggest that many of the genetic and structural features of COX4-2 that impart responsiveness and benefits in hypoxia may be restricted to the Euarchontoglires lineage that includes primates, lagomorphs and rodents.

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1

Introduction

2

To survive in variable conditions, organisms must possess the ability to sense changes in

3

the environment, and modulate energy production accordingly. An important point of

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metabolic regulation is thought to occur at cytochrome c oxidase (COX) * (3,13,42,2), the final

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complex of the electron transport system. COX catalyzes the reduction of oxygen to water while

6

translocating protons across the inner mitochondrial membrane, contributing to a proton

7

gradient that drives ATP synthesis. A complete vertebrate COX complex is a dimer composed of

8

two monomers, each of thirteen subunits. The three largest subunits, which form the enzymatic

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core of the complex, are mitochondrially encoded and are highly conserved across eukaryotes

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(36). The remaining ten nuclear-encoded subunits serve structural and regulatory functions.

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Many of these subunits exist as paralogs in vertebrates, some of which are lineage-specific. For

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example, though COX4, 6A, 6B, 6C, 7A, and 7C have equal numbers of paralogs in fish and

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mammals, COX7B and 8 have more in mammals, and COX5A and 5B have more in fish (29).

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COX4 is of particular interest as the only subunit with a paralog pair that responds to

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environmental changes in mammals (22). COX4 is the largest nuclear-encoded subunit and interacts directly with two of the three

16 17

catalytic subunits within the complex (41). The subunit contains two sites which competitively

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bind ATP and ADP, allowing COX to sense cellular energy conditions and modulate turnover

19

rates accordingly (31,32,3). In mammals, COX4 exists as an isoform pair and displays a major-

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minor pattern of isoform transcription, where COX4-1 mRNA is present in vast excess in most

21

tissues, and COX4-2 mRNA is present at detectable levels in brain and in highest amounts

22

(about equal to COX4-1 transcripts) in respiratory tissues (23,22). During exposure to hypoxia,

23

the COX4 isoform pattern has been shown to reverse, with transcription switching from COX4-1

*

Abbreviations: COX, cytochrome c oxidase; CT , cycle threshold value; dNTPs, deoxyribonucleotide triphosphates; EF-1α, elongation factor 1 alpha;HIF-1, Hypoxia inducible factor 1; HRE, hypoxia responsive element; IGFBP-1, insulin-like growth factor binding protein 1; iNOS, nitric oxide synthase; ORE, oxygen responsive element; OXPHOS, oxidative phosphorylation; qPCR, quantitative PCR; ROS, reactive oxygen species; RPL13α, 60S ribosomal protein L13α;TBP, TATA-binding protein; VEGFA, vascular endothelial growth factor A; X-gal, bromo-chloro-indolylgalactopyranoside.

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to COX4-2, while COX4-1 peptides are selectively degraded by the LON protease ( 22,14,25).

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The trigger for the oxygen-dependent switch in transcription has been attributed to either

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conventional hypoxia responsive elements (HRE) (14) or oxygen responsive elements (ORE)

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(25). Fukuda and coworkers (2007) characterized two putative HRE in the human COX4-2 gene,

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one present in the proximal promoter region and one in the first intron. Through transfection

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experiments, the group was able to show that these regions conferred a hypoxic response in

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COX4-2 through transactivation by HIF-1 (14). Conversely, Hüttemann and coworkers (2007)

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noted that the putative HREs present in human COX4-2 were poorly conserved across

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mammals. They identified a 13 bp region of the COX4-2 proximal promoter that is more

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conserved. This ORE conferred hypoxia responsiveness independently of HIF-1 and the HREs

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(25). Three transcription factors have since been identified as ORE binding proteins.

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Recombination signal sequence-binding protein Jκ (RBPJ) and coiled-coil-helix-coiled-coil-helix

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domain 2 increase transcription levels (CHCHD2) and CXXC finger protein 5 acts as an inhibitor

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(CXXC5) (1).

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The COX4 paralog switch is thought to optimize electron transfer reactions in COX in

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response to changing energetic conditions. COX complexes containing COX4-2 have a turnover

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rate approximately twice that of complexes containing COX4-1 (24,25), and are able to optimize

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the electron transfer reactions, resulting in a decrease of the production of reactive oxygen

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species (ROS) under hypoxic conditions (14). Although COX4-2 containing COX shows a normal

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response to ATP and ADP as was shown with purified enzyme from cow lung (24), there is some

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evidence to suggest that under hypoxic conditions, adenylate regulation of COX is lost

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altogether (22). These differences may be caused by a pair of cysteine residues on COX4-2 that

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form a disulfide bond, preventing adenyate binding (23). The COX4 paralogs differ in efficiency

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of electron transfer and proton pumping, and perhaps sensitivity to cytosolic adenylate

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concentrations. Thus, subunit switching may enable a cell to fine tune COX to alter ATP

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production, respiration, and mitochondrial ROS production in response to tissue type, energetic

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status and hypoxia (39). 2

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While it seems clear that the COX4-2 protein and gene adaptations can play a role in the

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regulation of energy production in rodents and humans, as well as some tumors, the

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evolutionary origins and phylogenetic distribution have not been well characterized. Given that

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the COX4 paralog pair is present throughout vertebrates, it is likely that the isoforms arose

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from a whole genome duplication event early in vertebrate evolution, although one paralog has

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been secondarily lost in birds and the available evidence suggests that COX4-2 of fish may have

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a different origin than COX4-2 of tetrapods (29). If this pathway is central to oxygen sensing

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and/or hypoxic metabolism, natural selection may affect the gene both in regulatory and

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structural regions in lineages that have distinctive hypoxic adaptations. For example, it could be

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hypothesized that more hypoxia tolerant species may express COX4-2 constitutively in more

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tissues, or that these species may have a more responsive promoter. In this study we were

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interested in determining if the hypoxia-dependant COX4 paralog swich is present in lower

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vertebrate taxa, and whether COX4-2 appears to be of importance in hypoxa-tolerant species.

64

Interestingly, we find that the familiar features of the COX4-2 gene and protein may be

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restricted to the Euarchontoglires lineage that includes rodents and primates.

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Methods

67 68

COX4-2 sequence analysis

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COX4-2 genes and their promoters were characterized from candidate vertebrate

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species. Sequences were obtained from the online databases Ensembl and NCBI, and the

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presence or absence of the putative elements responsible for the hypoxic response of COX4-2

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was determined by alignments with the human element sequences found by Fukuda et al. (14)

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and Hüttemann et al. (25) using MultAlin (7).

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Translated nucleotide sequences of COX4-2 were also examined from a wide array of

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vertebrate species, focusing on the conservation of amino acids suggested to be involved in the

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COX ATP/ADP binding site (Arg-20, Arg-73, Tyr-75, Glu-77, and Trp-78), and the presence or 3

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absence of two cysteine residues thought to be important in the isoform-specific function of

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human COX4-2 (Cys-16 and Cys-30) (23). Polypeptide sequences were obtained from the online

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database Ensembl for candidate species from several vertebrate classes and aligned using

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ClustalW2 (28,16). Accession numbers for all sequences used can be found in Table 1.

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Animal experiments

82 83

All animal experiments were carried out in accordance with the Queen’s University Animal Care Committee.

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Zebrafish (Danio rerio, Hamilton) were donated from a wild type population at

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Université de Montréal. They were held in a 140 L aquarium with dechlorinated water

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maintained at 25°C for at least six weeks. The fish were kept under a 12:12 light:dark

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photoperiod and fed Omega One Freshwater Flakes (OmegaSea, Painesville, OH, USA) daily ad

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libitum. Groups of 8 fish were transferred to 8 L jars at 25oC with aerated water flowing through

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overnight. After 12 h, the water flowing to the hypoxic fish was bubbled with 4% O2/96% N2 for

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8 h. At the onset of treatment, gas mixtures were bubbled directly into the jar, with no flow

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through. After 2 h, flow-through of gas-equilibrated water began at a rate of 4 ml min-1. Control

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fish were treated in the same manner, but their water was bubbled with air. Immediately

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following the experiment, fish were lethally anaesthetized in a solution of 0.4 g L-1 of tricaine

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methanesulphonate and 0.8 g L-1 sodium bicarbonate. Gills, liver, brain, and white muscle were

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dissected, flash frozen in liquid nitrogen and stored at -80°C.

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Goldfish (Carassius auratus, Linnaeus) were acquired from a local aquarium store. They

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were held in a 750 L tank with dechlorinated water maintained at approximately 15˚C for at

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least six weeks to allow for acclimation. The fish were kept under a 12:12 light:dark

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photoperiod and fed Omega One Freshwater Flakes (OmegaSea, Painesville, OH, USA) daily ad

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libitum. Anoxia experiments were carried out in air-tight 120 L tanks with a flow-through water

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system. Eight fish were exposed to anoxia (100% nitrogen) for 21.5 h. The water was de-

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oxygenated by pumping nitrogen gas into the tank via an air stone while monitoring oxygen

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levels using a FOXY oxygen probe (Ocean Optics, Dunedin, FL, USA). After 3 h, no oxygen was 4

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detected in the tank. Immediately following the experiment, tissues were collected from fish as

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described above for the zebrafish. Control fish were taken from the holding tank at the

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beginning of the anoxia treatment and tissues were collected in the same manner described

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above.

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Tilapia (Oreochromis niloticus) were received from Red fish Ranch of BC. They were held

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in a 620 L aquarium with dechlorinated water maintained at 28°C for at least six weeks. The fish

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were kept under a 12:12 light:dark photoperiod and fed crushed pellets once daily. Six fish were

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used in each of 4 treatment groups: 21% oxygen (control), 5% oxygen (hypoxia), 3% oxygen

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(hypoxia), and 1.5% oxygen (hypoxia). Each fish was transferred into individual 6L containers at

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28°C with aerated heated water maintained for 18 hours for each specific oxygen treatment.

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Water was bubbled with 5% O2/95% N2 for the 5% hypoxic treatment group, 3% O2/97% N2 for

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the 3% hypoxic treatment group, and 1.5% O2/98.5% N2 for the 1.5% hypoxic group. Water was

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gradually brought from 21% (control) oxygen to the correct percent oxygen level for each

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hypoxia treatment over half an hour to allow the fish to acclimatise. A time course experiment

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was also completed at 5% oxygen where the fish were held for 4, 8, 12, 18, 24, 30, 36, 42, 48,

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54, 60, 66 and 72 hours in the hypoxic treatment. All control fish were taken from the holding

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tank at the beginning of the hypoxia treatment and tissues from all fish were collected in the

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same manner described above.

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Green anoles (Anolis carolinensis, Voigt) were acquired from Boreal Scientific. They were

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kept in an enclosure at ambient temperature (23˚C) and supplied with an infrared heat lamp

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and fresh water supply for 2 weeks. The anoles were held under a 12:12 light:dark photoperiod

125

and fed live crickets every other day ad libitum. Hypoxia experiments were carried out in plastic

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500 ml containers maintained at 23˚C in an incubation chamber. Anoles were exposed to either

127

normoxia or 4% O2/ 96% N2. Immediately following the experiment, animals were euthanized

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using an injection of pentobarbital (150 mg/kg), and heart, brain, and lung tissues were

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collected and frozen as described above.

130 131

Western painted turtle (Chrysemys picta bellii, Schneider) liver and pectoralis muscle samples were generously donated by Dr. Leslie Buck (University of Toronto). Tissues were 5

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dissected from individuals that were from a control group or had been submerged in

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freshwater for 24 h, and immediately frozen in liquid nitrogen, as described in (35).

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Tissue culture experiments

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Two fish cell lines and two mammalian cell lines were employed in this study. Fish cell

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lines were grown in Leibovitz’s L-15 media (Sigma-Aldrich, St. Louis, MO, USA) supplemented

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with 10% fetal bovine serum (FBS, Invitrogen, Carlsbad, CA, USA) under regular atmospheric

138

conditions. ZEB2J cells (Danio rerio epithelium) were maintained at 26°C and RTG-2 cells

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(Oncorhynchus mykiss, Walbaum gonadal fibroblasts) were maintained at 19°C. Mammalian cell

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lines were grown in Dulbecco’s Modified Eagle Medium (DMEM, Invitrogen, Carlsbad, CA, USA)

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supplemented with 10% FBS and held at 5% CO2 and 95% humidity. Sol8 mouse (Mus musculus,

142

Linnaeus) myoblast cells and LNCaP human prostate cancer cells were all maintained at 37°C.

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Hypoxia and anoxia treatments were done using gas mixtures of 2% oxygen/98%

144

nitrogen or 100% nitrogen, respectively and lasted for 8 or 24 h. Control treatments were done

145

under normal atmospheric conditions for 24 h. Cells were grown in 6-well plates until they were

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approximately 90% confluent. The media was then switched to L-15 with 10% FBS that had

147

been pre-equilibrated to the appropriate temperature and oxygen levels. Hypoxia treatment

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plates were kept in an air-tight chamber containing the appropriate gas mixture and control

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treatment plates were left at normal atmospheric conditions. All treatments were carried out at

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the temperatures at which the cells were previously grown. Anoxia caused cell death in LNCaP

151

cells after 24 h and in Sol8 cells after 8 h.

152

Cells were harvested immediately following these treatments by removing the media

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and scraping in RLT buffer from Qiagen’s RNeasy kit (Qiagen, Valencia, CA, USA) containing 1%

154

β-mercaptoethanol. Cell suspensions were lysed by vortexing for approximately 10 s and then

155

stored at -80°C.

6

156 157

RNA extraction and cDNA synthesis RNA was extracted from whole animal tissues with TRIzol reagent (Invitrogen, Carlsbad,

158

CA, USA), using 1 ml of TRIzol per 50 mg of tissue. Samples were homogenized in TRIzol with a

159

tissue homogenizer (Fisher, Ottawa, ON, CAN) and then centrifuged at 12 000 × g for 10 min at

160

4°C. The supernatant of each sample was collected and mixed with 0.2 ml of chloroform per 1

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ml of TRIzol. Samples were incubated at room temperature for approximately 3 min and then

162

centrifuged at 12 000 × g for 15 min at 4°C. The upper aqueous layer was collected and mixed

163

with 0.5 ml of isopropanol per 1 ml of TRIzol. After 5 min of incubation at room temperature,

164

the samples were centrifuged at 12 000 × g for 10 min at 4°C. The supernatant was removed

165

and an equal volume of 75% ethanol was used to wash the pellet. Samples were re-suspended

166

in RNase-free water and quantified using a spectrophotometer reading at 260 nm. Quality of

167

the RNA samples was also assessed during the quantification using the 260/280 ratio.

168 169 170

Extraction of RNA from cell lysates was done using an RNeasy Mini kit (Qiagen, Valencia, CA, USA). Purified RNA samples were quantified by reading absorbance at 260 nm. RNA was reverse-transcribed to cDNA using the QuantiTect Reverse Transcription Kit

171

(Qiagen, Valencia, CA, USA) with 1 μg of RNA in a 20 μl reaction, as per the manufacturer’s

172

instructions.

173

Quantitative PCR

174

Primers for qPCR were designed to amplify 50-200 bp (Table 2) in a manner that was

175

linear with respect to template concentration and produced a single dissociation peak. When

176

possible, primers were designed using sequences available online. For those genes measured in

177

this study that lack a published sequence, primers were designed from consensus sequences of

178

published genes of related species. While consensus primers served for qPCR in most instances,

179

some turtle genes required partial sequencing using consensus primers before qPCR primers

180

could be designed from these resulting partial sequences. Thus, for turtle fragments of the

181

transcripts of COX4-2 and VEGFA were amplified using consensus primers (COX4-2: Forward 5’-

182

CCCTGAGCAGAAAGCCCTGAAA-3’, reverse 5’-CATTCGTTCTTCTATAATCCCACTTG-3’; VEGFA: 7

183

Forward 5’-GCAGCTTCTGCAGGACAATTGA-3’, reverse 5’-GCAAGTGCGCTCGTTTAACTCA-3’).

184

These larger fragments were amplified, cloned and sequenced. To confirm that the amplified

185

fragment was of the desired gene, the obtained sequence was aligned with the consensus

186

sequences used to design the primers. qPCR primers were then designed from the resulting

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sequence fragments.

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Cloning and sequencing was carried out for all primer sets used. They were first tested

189

by PCR using an Eppendorf Mastercycler Gradient thermocycler (Eppendorf, Hamburg,

190

Germany). All reactions were run in 25 μl volumes containing 1× PCR buffer, 1 mM MgCl2, 0.75

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units Taq DNA polymerase (Qiagen, Valencia, CA, USA), 0.4 μM dNTPs (Promega, Madison, WI,

192

USA), 0.3 μM of each primer, 50-100 ng of cDNA template, and the balance of nuclease-free

193

water. PCR reactions involved a 3 min denaturation at 94°C followed by 35 cycles of 15 s at

194

94°C, 30 s at the appropriate annealing temperature, and 30 s at 72°C. There was a final

195

extension period of 10 min at 72°C. PCR products were gel purified, ligated into the pDrive

196

cloning vector (Qiagen, Valencia, CA, USA), transformed into cloning-competent DH5α cells

197

(Invitrogen, Carlsbad, CA, USA), and grown on agar plates containing 0.05 mM IPTG, 0.2 mM X-

198

gal, and 50 µg/ml ampicillin. Colonies testing positive for inserts were grown overnight in

199

Lysogeny Broth with 50 μg/ml ampicillin. Plasmids were purified using the QIAprep Spin

200

Miniprep kit (Qiagen, Valencia, CA, USA), quantified by spectrophotometric readings at 260 nm,

201

and then sequenced (Robarts Research Institute, London, ON, Canada). Samples were

202

sequenced with an Applied Biosystems 3730 Analyzer (Applied Biosystems, Carlsbad, CA, USA)

203

and aligned with published sequences to verify the amplification of the correct gene fragment.

204

Real-time quantitative PCR was performed on an ABI 7500 real-time PCR system

205

(Applied Biosystems, Carlsbad, CA, USA) using GoTaq qPCR Master Mix (Promega, Madison, WI,

206

USA). Reactions were done in 25 μl volumes with 12.5 μl of the GoTaq Master Mix, 25-100 ng of

207

cDNA template, 0.58 μM each of the forward and reverse primer, and the balance of water.

208

qPCR reactions consisted of an initial 15 min denaturation at 95°C followed by 40 cycles of 15 s

209

at 95°C, 15 s at the appropriate annealing temperature, and 36 s at 72°C. All samples were

210

analyzed in duplicate and each primer set was tested with a no-template control. 8

211

For every transcript level measured in qPCR, the threshold cycle (CT) of each cDNA

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sample was normalized to a housekeeping factor obtained from the geometric mean of the Ct

213

recorded for TBP (goldfish only) or β-actin (all other species) and RPL13A (zebrafish only) or EF-

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1α (all other species). Thus changes in CT levels for a particular transcript between samples

215

from different treatments could be corrected for any changes due to the efficiency of cDNA

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synthesis. When comparing the relative amounts of COX4-1 and COX4-2 transcripts within

217

single tissues, a further correction was made to ensure each primer pair was able to detect

218

their target transcripts with a comparable accuracy. The correction factor was determined by

219

comparing the CTs obtained for 1 pg of pDrive plasmid containing the appropriate insert using

220

the target primers and pDrive specific primers (Table 2).

221

Transfection experiments

222

To address the question of the hypoxia-sensitivity of COX4-2 promoter elements across

223

vertebrates, we used two approaches. First, we used plasmid constructs of tandem repeats of

224

the human HRE1, HRE2, or ORE to assess which elements are hypoxia-responsive in lower

225

vertebrate taxa. Unfortunately, this approach limited our investigation to those species where

226

the elements could be identified, notably excluding all teleost species. In order to broaden the

227

investigative range of species, we then measured the ability of ~2 kb of COX4-2 proximal

228

promoters from candidate mammalian and teleost species to respond to hypoxia.

229

Element transfections

230

Plasmid constructs were developed for human (Homo sapiens, Linnaeus), green anole,

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and western clawed frog (Xenopus tropicalis, Wagler), and tested for hypoxic activity in a

232

transfection experiment. The constructs consisted of four tandem repeats of each of the three

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putative elements responsible for the hypoxic response seen in mammalian COX4-2 (HRE1,

234

HRE2, and ORE). Oligonucleotides of the tandem repeats were ordered, along with a universal

235

reverse primer containing a MluI restriction site (Table 3). As a positive control, a tandem

236

repeat of the human iNOS HRE (5’-GTGACTACGTGCTGCCTAG-3’) was also designed, as outlined

237

in Table 3. 9

238

Double-stranded products were obtained through reactions using the Klenow fragment

239

of DNA polymerase I and an Eppendorf Mastercycler Gradient thermocycler (Eppendorf,

240

Hamburg, Germany). Reactions were run in 25 μl volumes containing 1× PCR NEBuffer 2 (50

241

mM NaCl, 10 mM Tris-HCl, 10 mM MgCl2, 1 mM DTT), 12.5 units Klenow (New England Biolabs,

242

Ipswich, MA, USA), 0.4 μM dNTPs (iNtRON Biotechnology, Seongnam-Si, Gyeonggi-do, Korea),

243

20 mM each of an element oligonucleotide and the universal reverse primer, and the balance of

244

nuclease-free water. The oligonucleotides were first allowed to anneal for 10 min before the

245

addition of Klenow by decreasing the thermocycler temperature from 99 ˚C to 4˚C at a rate of

246

0.3˚C/s. The synthesis reaction was carried out at 25 ˚C for 15 min, after which Klenow was

247

inactivated by holding at 75 ˚C for 20 min. To allow for the ligation of these elements into the

248

target plasmid vector, the double-stranded products were phosphorylated by 2 units of T4

249

polynucleotide kinase per µg DNA (New England Biolabs, Ipswich, MA, USA).

250

The plasmid vector used in this experiment was the pGL4.23[luc2/minP] Vector

251

(Promega, Madison, WI, USA), which contains a multiple cloning site upstream of a minimal

252

promoter and the luciferase gene luc2. To prepare the vector for ligation with the element

253

inserts, it was digested by 40 units of EcoRV per µg DNA (New England Biolabs, Ipswich, MA,

254

USA). To prevent self-ligation of the vector, terminal phosphate groups were removed by 4

255

units of calf intestinal phosphatase per µg DNA (New England Biolabs, Ipswich, MA, USA). The

256

resulting cut plasmid was electrophoresed in a 1% agarose gel, excised, and purified.

257

The ligation of the plasmid and inserts was performed with T4 DNA ligase (New England

258

Biolabs, Ipswich, MA, USA). Plasmid constructs were then transformed into cloning-competent

259

DH5α cells (Invitrogen, Carlsbad, CA, USA), and grown on agar plates containing 0.13 mg

260

ampicillin/ l. Multiple colonies for each construct were grown overnight in LB broth with 50 μg

261

ampicillin/ ml . Plasmids were purified using the QIAprep Spin Miniprep kit (Qiagen, Valencia,

262

CA, USA), quantified by spectrophotometric readings at 260 nm, and tested for the presence of

263

inserts by digestion reactions with MluI and XhoI followed by visualization on an agarose gel

264

with ethidium bromide. Those plasmids showing inserts of the correct size were then 10

265

sequenced as outlined in the quantitative PCR section to verify the correct orientation of the

266

insert.

267

Transfection experiments were carried out using the prostate cancer cell line PC3 in 24-

268

well plates. Cells were grown and kept under the conditions outlined for the mammalian cell

269

lines. When cells were confluent, the media was changed and a mixture of the experimental

270

plasmid construct and a control pRL Renilla vector (Promega, Madison WI, USA) was added to

271

each well using the transfection agent FuGENE6.0 (Roche Applied Science, Laval, Quebec,

272

Canada). Transfected cells were kept in an incubator under normal conditions for the control

273

group or under 2% O2, 5% CO2, and the balance nitrogen for the hypoxia treatment. The anoxia

274

treatment plates were incubated in an air-tight chamber containing 5% CO2 and the balance

275

nitrogen. All plates were held at 37 ˚C for the duration of the experiment.

276

Transfected cells were harvested after 24 h of treatment by incubating with 1× passive

277

lysis buffer (Promega, Madison, WI, USA). The resulting cell suspensions were transferred to 1.5

278

ml tubes and frozen at -80 ˚C for at least 12 h. Luciferase assays were carried out using a Dual-

279

Luciferase Reporter Assay System (Promega, Madison, WI, USA) as per the manufacturer’s

280

instructions. Assays were measured using an Lmax Luminescence Microplate Reader (Molecular

281

Devices, Sunnyvale, CA, USA), and resulting values were determined relative to the control

282

Renilla luminescence values for each sample.

283

Promoter fragment transfections

284

Plasmid constructs containing approximately 2 kb of sequence directly upstream of the

285

first exon of COX4-2 were developed for rat (Rattus norvegicus, Fischer de Waldheim), goldfish,

286

and zebrafish, and tested for hypoxic activity in a transfection experiment. We used the human

287

iNOS HRE construct developed for the element transfections as a positive control.

288

DNA was isolated from tissues collected from the hypoxia experiments for the teleost

289

species; rat tissue was previously obtained from sentinel males from the animal care centre at

290

Queen’s University. Samples were digested by proteinase K, (0.2 mg/ml) in 200 mM NaCl, 20

291

mM Tris, 50 mM EDTA, 0.10% SDS (pH = 8.0), while incubating at 55˚C. When tissues were

292

completely dissolved, the samples were treated with RNase A (10 µg/ml). The DNA was 11

293

extracted in a volume of phenol-chloroform-isoamyl alcohol (25:24:1), and centrifuged for 10

294

min at 1700 × g. The aqueous phase was reserved and DNA was precipitated by adding 0.1 ml of

295

ammonium acetate (7.5 M) and 2 ml of 100% ethanol per ml of extraction buffer, followed by

296

centrifugation for 3 min at 1700 × g. The pellet was washed with 70% ethanol, air-dried and

297

dissolved in 250 µl TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH = 8.0). The DNA was quantified

298

and the purity assessed as described in the RNA extraction section.

299

Promoter fragments approximately 2 kb long immediately upstream of the 5’-UTR were

300

amplified using the isolated DNA. Primers were designed from sequences published online,

301

except in the case of goldfish, where the promoter region was amplified using the ‘RAGE’

302

protocol, as described in (5). Primers contained unique restriction site adapters on the outer

303

ends to allow uni-directional cloning into reporter plasmids (Table 4). Initial amplification,

304

cloning into pDRIVE, and verification sequencing was accomplished as described in the

305

quantitative PCR section. The 2 kb fragments were then excised out of the pDRIVE plasmids

306

with the appropriate restriction enzymes (New England Biolabs, Ipswich, MA, USA), ligated into

307

a luciferase reporter plasmid (pGL2 basic, Promega, Madison, WI, USA), and cloned and

308

sequenced as described in the element transfection section. The vector used for this

309

experiment is similar to the one used in our previous transfections, but lacks any native

310

promoters.

311

Transfections were carried out using the human liver hepatocellular carcinoma cell line

312

HepG2 in 12-well plates. The procedures followed for the transfection and luciferase assays

313

were similar to that described in the element transfection section. The only variations were the

314

use of Renilla-TK vector (Promega, Madison WI, USA) as the internal control, and an oxygen

315

level of 4% for the hypoxia condition.

316 317

Statistical analysis All data are presented as means + / - SEM, and are expressed relative to the control

318

group for each treatment. To determine significance between control and treatment groups,

319

Mann-Whitney U tests were used. Differences were considered significant if P < 0.05. 12

320

Results

321 322

Mammalian cells in culture

323

We investigated two mammalian cell lines from two different species (mouse and

324

human) representing two different tissue types (myoblasts and prostate adenocarcinoma cells),

325

yet neither displayed the expected shift from transcription of COX4-1 to COX4-2 during

326

exposure to hypoxia. The results of these experiments are shown in Figure 1. As neither cell

327

type was able to survive exposure to anoxia, only results from exposure to 2% oxygen are

328

displayed.

329

The murine Sol8 cells exposed to hypoxia displayed significant increases of 2 – 2.6-fold

330

in the mRNA for the hypoxia-responsive gene vascular growth factor A (VEGFA) (Fig. 1c). A

331

significant decrease by half in COX4-1 mRNA levels was seen for both hypoxic treatments as

332

compared to the control group. There was a significant 1.4-fold increase in COX4-2 transcripts

333

only after 24 hours of exposure to hypoxia (Fig. 1a), but for all treatments, COX4-2 transcripts

334

accounted for less than 0.1% of all COX4 transcripts (Fig. 1b).

335

As with Sol8 cells, LNCaP cells displayed significant increases of 4.4 – 11-fold in VEGFA

336

mRNA levels when exposed to hypoxia (Fig. 1c). COX4-1 mRNA levels remained steady across

337

treatment groups. Interestingly, although we were able to amplify fragments of the COX4-2

338

gene using LNCaP DNA (data not shown), we could not detect any COX4-2 transcripts under any

339

condition, suggesting the COX4-2 paralog is not transcribed in this cell line (Fig. 1b).

340

Fish tissues

341

Although we examined four species of teleost fish and employed approaches using both

342

whole animals and tissue cultures, we found little indication that transcription switches from

343

COX4-1 to COX4-2 during hypoxia treatments in teleosts (Fig. 2).

344 345

Adult zebrafish exposed to low oxygen levels displayed a hypoxia response at the transcript level, as indicated by the increases in the mRNA levels of the known hypoxia13

346

responsive gene insulin-like growth factor binding protein 1 (IGFBP-1). With regards to the

347

COX4 paralogs, brain, liver and white muscle displayed significant decreases in COX4-1 mRNA

348

levels, and there was a minor (40%) but significant increase in COX4-2 mRNA levels in zebrafish

349

brain (Fig. 2a). When both paralogs are taken into account, however, COX4-1 remained well in

350

excess of COX4-2 in all tissues during hypoxia, although in brain where COX4-2 accounted for

351

almost 40% of the COX4 transcripts (Fig. 2b).

352

The highly hypoxia-tolerant goldfish required exposure to undetectable levels of oxygen

353

to activate hypoxia response pathways, which we measured as significant increases in IGFBP-1

354

transcript levels in all examined tissues. No significant changes were seen for either COX4

355

paralog in any tissue, except for a 2-fold increase in COX4-1 transcripts in hypoxic liver tissues.

356

The relative abundances of each COX4 paralog changed little between normoxia and hypoxia

357

treatment groups. In agreement with transcript patterns seen in zebrafish, the highest

358

abundance of COX4-2 was seen in brain tissues, though it remained between 9 and 13% of total

359

COX4 transcripts (Fig. 2e).

360

Similar results were obtained for the experiments involving cultured fish tissues, also

361

summarized in Figure 2g-h. ZEB2J cells showed dose- and time-dependent decreases in COX4-1

362

Fig. 2g. COX4-2 mRNA levels remained unchanged except for a 70% increase in cells exposed to

363

2% O2 for 24 h. COX4-1 transcripts account for more than 99% of total COX4 transcripts across

364

all treatment groups. In experiments with rainbow trout gonad (RTG-2) cells, COX4-1 mRNA

365

levels decreased significantly by 40% with exposure to 2% O2, but did not change during

366

anoxia. No significant changes were seen in COX4-2 mRNA levels for any treatment group.

367

When taking both COX4 paralogs into account, COX4-1 accounted for between 88% and 94% of

368

total COX4 transcripts (Fig. 2h).

369

While VEGFA transcripts increased significantly in ZEB2J cells with increasing severity of

370

hypoxia treatment (Fig. 2i), no significant differences were seen VEGFA transcripts in RTG-2

371

cells. Given that the same incubation conditions resulted in a hypoxic response in ZEB2J cells,

372

we believe the lack of response in RTG-2 cells reflects a different VEGF signaling pathway,

373

rather than a failure to impose hypoxia. 14

374

Since goldfish and zebrafish belong to the same family (Cyprinidae), we extended our

375

studies to tilapia. We exposed tilapia to a range of hypoxic conditions, and confirmed that

376

hypoxia response pathways had been activated by measuring increases in IGFBP-1 mRNA levels

377

in at least one tissue under each treatment (Fig. 3c). In hypoxia treated tilapia, COX4 paralogs in

378

liver and gill displayed slight decreases in COX4-1 mRNA levels, and there was a minor but

379

significant increase in COX4-2 mRNA levels (Fig. 3a). COX4-2 increased 90% in the hypoxic liver

380

tissue and 27% in the hypoxic gill tissue as compared to the control treatment (Fig. 3a). White

381

muscle, red muscle, kidney, and brain tissue were also examined and displayed no changes in

382

mRNA transcript levels between COX4-1/COX4-2 during hypoxia. Minimal changes in COX4-1/2

383

were found in any of the tissues examined in the fish treated at 5% hypoxia over the time

384

course of 0 – 72 h (Fig. 4).

385

Reptile tissues

386

We also studied two reptiles, phylogenetically intermediate between mammals and fish.

387

We failed to observe such a switch in either species we studied, though we found a uniquely

388

high abundance of COX4-2 mRNA (Fig. 5).

389

Green anoles exposed to hypoxia for 24 h possessed VEGFA mRNA levels significantly

390

higher than the control group in brain and muscle tissues, suggesting that a hypoxia response

391

was occurring at the level of transcription (Fig. 5c). COX4 paralog mRNA levels remained largely

392

unchanged, except for a significant 30% decrease in COX4-1 mRNA of brain tissues. COX4-2

393

transcript abundance ranged from 15% to 35% in all tissues examined (Fig. 5b).

394

Results of the turtle experiment were in agreement with our other findings. We found

395

that although VEGFA increased significantly in hypoxic muscle tissue by 2-fold, no switch in

396

either COX4 isoform was detected during exposure to hypoxia in either liver or muscle tissues

397

(Fig. 5d and e). As with anoles, turtles showed COX4-2 mRNA was higher than in fish, but in

398

turtles COX4-2 was the dominant isoform under all treatments in both tissues, accounting for

399

60 - 75% of all COX4 transcripts (Fig. 5e).

15

400 401

Structure of the COX4-2 promoter and polypeptide The promoter sequences of COX4-2 genes from a selection of vertebrate species were

402

compiled and the presence or absence of the putative oxygen-responsive elements was

403

determined. The structures of vertebrate COX4-2 promoter regions as well as the results of our

404

investigation are summarized in Figure 6. Though homologous elements were identified in

405

similar positions in the COX4-2 gene of tetrapods, none of the elements were recognized in the

406

COX4-2 genes of any teleost species (Fig 4a). In comparing elements across tetrapods, the ORE

407

is better conserved across mammals than are either of the HREs (Fig. 6b). Single nucleotide

408

differences are seen in the ORE for pika (Ochotona princeps, Link) of the order Lagomorpha and

409

hyrax (Procavia capensis, Pallas) of the order Afrotheria. The HREs are conserved only within

410

the primate family, although there is a single nucleotide substitution from C to G in macaques

411

(Macaca sylvanus, Linnaeus). Outside of Mammalia, the elements are poorly conserved. Of the

412

three elements, HRE2 is the most conserved in Xenopus and anole: 61% and 67% homology,

413

respectively for HRE2 compared to 53% – 59% for the other elements.

414

The polypeptide sequences of COX4-2 from vertebrate species were also examined to

415

look for the conservation of two cysteine residues present in human COX4-2 (Cys-16, Cys-30).

416

These are thought to form a disulfide bond, blocking an ATP binding site seen in COX4-1

417

(23,25). The two Cys residues are seen in the Euarchontoglires lineage that includes rodents,

418

primates, lagomorphs, scandentians, and dermapterans (Fig. 7). Within this lineage, one of the

419

Cys residues is absent in select species of rodents (sciuridae: ground squirrel, hystriocogathia:

420

guinea pig) and primates (marmoset and galago). Outside Euarchontoglires, one of the Cys

421

residues is replaced with either tyrosine or glycine. Likewise, nonmammalian vertebrates show

422

either one or no Cys residue at this location. The amino acids that form the ATP binding site

423

were highly conserved even if the cysteine residues that disrupt it were not. However, in

424

Xenopus, an insertion moves the conserved E-101, W-102 residues, and in fish, the first polar

425

(R) residue shared in tetrapods is nonpolar (L or V).

16

426 427

Reporter analyses of the COX4-2 promoter Element transfection. The three putative elements in the COX4-2 promoter were

428

developed into reporter constructs in four tandem repeats and analyzed for their response to

429

hypoxia in a reporter experiment. The elements from human, anole, and Xenopus were used in

430

the experiment. Relative to the normoxic control, the well-characterized HRE from the human

431

iNOS gene responded to 24 h of hypoxia and anoxia by increasing 11- and 15-fold, respectively.

432

Of the COX4-2 elements, only the human HRE2 was seen to respond to the oxygen treatments:

433

8-fold in hypoxia, 12- fold in anoxia. The other element constructs were unaffected by oxygen

434

(Fig. 8a).

435

Long promoter transfection. We used 2 kb segments of COX4-2 promoters from rat,

436

goldfish, and zebrafish to produce plasmid constructs which we tested in normoxic, hypoxic,

437

and anoxic conditions. The human iNOS HRE construct was used as a positive control and

438

increased by 10-fold under hypoxic treatment and 19-fold under anoxic treatment. Of the long

439

promoters, only the rat construct was activated more than the empty vector, by 6-fold and 21-

440

fold under hypoxia and anoxia, respectively. The effects of hypoxia on the goldfish and

441

zebrafish promoter constructs under hypoxic and anoxic treatments were indistinguishable

442

from the response of the empty vector, and thus may be related to effects on luciferase

443

degradation rather than synthesis (Fig. 8b). Discussion

444 445

COX regulation plays a central role in the OXPHOS regulation, and it is well accepted

446

that the nuclear encoded genes play a major role in the regulation of COX function in mammals.

447

COX4 has been of particular interest because the genetic paralogs of this subunit appear to be

448

oxygen sensitive, and their peptides cause the ATP turnover rates of the COX complex to differ

449

by almost 2-fold (22,14,25).

450 451

COX4-1 is the ubiquitously-transcribed paralog under normoxic conditions, while transcription switches to COX4-2 during bouts of hypoxia. The presence of COX4-2 in the COX 17

452

complex appears to benefit mammalian tissues by allowing the COX complex to function at high

453

turnover rates regardless of the current energy demands of the cell, thus decreasing the

454

production of mitochondrial ROS under low oxygen conditions. These unique functions are

455

thought to be conferred by the two cysteine residues that preclude ATP-mediated inhibition of

456

COX activity at this subunit. The transcriptional regulation of the COX4-2 paralog is believed to

457

be controlled by some combination of three putative oxygen-sensitive elements present in the

458

gene (termed HRE1 and 2, and ORE) (14, 25). Uncertainties remain about the COX4 paralog

459

function and mechanism even in mammals, but we were interested the evolution of the protein

460

and gene regulation more broadly in vertebrates.

461

Sensitivity of COX4 paralogs to hypoxia

462

The hypoxia-responsive switch in COX4 paralogs was first reported in 2007, and several

463

shortfalls in the collective knowledge of this mechanism remain. To date, the transcriptional

464

response of COX4 paralogs to hypoxia has only been reported for one whole animal model

465

(mouse), a select few number of human cancer cell lines, and most recently in human leg

466

muscles (14,22,38,9). Within the mouse hypoxia experiments, a transcriptional response in

467

COX4-2 was seen in only lung and liver out of the five tissues tested, and only one of two types

468

of brain cells when mouse primary cell cultures were examined (14,22). The use of human

469

cancer cell lines, while common in functional studies, allows for the possibility of generating

470

results which are not characteristic of whole organisms, as these cells have been immortalized

471

through some combination of genetic and epigenetic alterations. The small number of models

472

that have been used to measure this mechanism raises questions about the pervasiveness of

473

the COX4 hypoxia response across mammalian taxa and tissues, as well as other vertebrate

474

lineages. To address this, we used both cellular and whole animal approaches to assess the

475

transcript levels of COX4-1 and COX4-2 in candidate species of fish, reptiles, and mammals.

476

Overall, we found little evidence of the hypoxia-induced switch from COX4-1 to COX4-2 in any

477

of the nonmammalian models, suggesting that hypoxia-responsiveness seen in rodents and

478

humans is a derived trait. 18

479

COX4 transcriptional response in mammalian cells. Previous studies by other groups

480

have involved many cell lines that respond to hypoxia with an increase in COX4-2 mRNA,

481

including rat pulmonary smooth muscle cells (1), mouse astrocytes (22), and cell lines including

482

human lung H460 cells (25) , human embryonic kidney 293 cells (1), human cervical cancer HeLa

483

(14), human liver cancer Hep3B (14) human colon cancer Hct116 cells (14), and human lung

484

cancer A594 cells (14). The impression from these studies is that the COX4-2 response is

485

common within cultured cells, though variability in the response between cells lines is reported

486

(1, 22). Of the human cell types previously used in studies on COX4 hypoxia responsiveness, all

487

have displayed a pronounced increase in COX4-2 transcription during hypoxia, and all but one, a

488

primary culture of pancreatic tissue, have been derived from tumors (14,38). However, tumor

489

cells differ in their dependence on hypoxia, and even lineages of tumor cells can evolve in

490

culture (19). For many tumors, evolution in response to hypoxia in vivo can alter their

491

metabolism, and it has been suggested that COX4 paralog switching may play a role in hypoxic

492

metabolism and tumor survival (15,39).

493

In the course of exploring the utility of cell culture models for transfections, we noted

494

that several common models showed no response in COX4-2. Mouse muscle cells (Sol8) cells

495

expressed COX4-2, showed a 40% increase in COX4-2 (Fig. 1a), but the fraction of COX4-2

496

transcripts present in the COX4 pool amounted to less than 0.1%. With human prostate cancer

497

cells (LNCaP), there was no evidence that the gene was expressed at all (Fig. 1a), though the

498

same primers detected the presence of the gene.

499

Another tissue of interest is striated muscle, given the importance of oxygen in

500

metabolism and adaptive remodeling. Fukuda and colleagues (2007) similarly found no increase

501

in COX4-2 mRNA levels in the cardiac muscle of adult mice exposed to hypoxia (14), though

502

whether this achieved tissue hypoxia is unclear. Thoroughbred horses (Equus ferus caballus,

503

Linnaeus) were found to decrease the transcription of COX4-2 in response to bouts of sprint

504

exercise (20). Nonetheless, a recent study by Desplanches and colleagues (2014) reported that

505

COX4-2 transcription increases in the vastus lateralis of adult human males after endurance 19

506

training under hypoxic conditions (9). Whether the differences in response are linked to

507

species, striated muscle type (cardiac versus skeletal), or skeletal fiber type remains unclear but

508

collectively, these findings highlight the variability in the responsiveness of COX4-2. When

509

responses are seen, it is not clear if the driving force is hypoxia directly, or whether the changes

510

are driven by other signals. Furthermore, none of the studies have assessed whether the

511

changes in muscle lead to changes in COX subunit composition or function.

512

COX4 transcriptional response in fish. We were interested in the COX4 paralog hypoxia

513

response in fish for two reasons. First, fish possess a COX4 isoform pair which is orthologous to

514

those seen in mammals; the COX4 paralogs appear to have arisen from an ancient whole

515

genome duplication event which occurred before the divergence of Actinopterygians and

516

tetrapods. We have previously found these genes to possess similar tissue-specific transcription

517

patterns to the mammalian COX4 paralogs, though COX4-2 transcripts generally contributed a

518

greater fraction to the COX4 pool in fish than mammals (29). Second, many fish reside in

519

environments with highly variable oxygen content, making them necessarily more tolerant to

520

hypoxia than most tetrapods. To investigate the pervasiveness of the hypoxic switch in COX4

521

paralogs across teleost fish, we employed species which differ markedly in hypoxia tolerance

522

(the highly intolerant rainbow trout, moderately tolerant zebrafish, and highly tolerant goldfish

523

and tilapia) and used both cellular and whole animal-based approaches. The results suggest

524

that the COX4-1/COX4-2 switch seen during hypoxia in some mammalian tissues is not present

525

in the teleost lineage.

526

We studied 3 species of fish, including both hypoxia tolerant and sensitive species, and

527

subjected them to a range of hypoxia levels (befitting their tolerance) and various times of

528

sufficient duration to capture changes in mRNA levels. For the most part, there were very few

529

instances of a hypoxic response, and where statistically significant responses were seen, the

530

response was rarely more than 50%, and there was no tissue that typically responded. For

531

example, brain showed a 40% increase with hypoxia in zebrafish (Fig. 2a), but no response in

532

tilapia (Fig. 3a) or goldfish (Fig 2d). In liver, tilapia (Fig 3a) responded with a 90% increase in

533

COX4-2 mRNA, but neither goldfish (Fig. 2d) nor zebrafish (Fig. 2a) responded. 20

534

Another feature that differed amongst fish was the abundance of COX4-2 mRNA,

535

expressed relative to COX4-1 mRNA. Mammals generally show relatively low levels of COX4-

536

2/COX4-1, with lung as the only tissue where COX4-2 levels approach COX4-1 levels (23). We

537

had expected gill to have the highest relative expression of COX4-2, given its functional

538

homology with mammalian lung, however this was not found. Zebrafish and goldfish had

539

generally low levels of COX4-2 mRNA in most tissues, but brain stood out with relatively high

540

COX4-2. Furthermore, tilapia was unlike the two minnow species in showing very high levels of

541

COX4-2 in all tissues (Fig 3b). The physiological significance of these differences in tissue

542

abundance between mammals and fish, and among fish species remains to be established.

543

What is clear though is that even in tissues that showed a modest but statistically significant

544

increase in COX4-2 mRNA in hypoxia, the changes are even less impressive when the relative

545

abundance of COX4-2 to COX4-1 is considered.

546

Though our positive controls showed that there was sufficient oxygen stress to initiate a

547

genetic response, the depth of hypoxia in a given tissue is uncertain because of the capacity for

548

physiological changes in oxygen delivery. Thus, we sought to augment these whole animal

549

studies using cultured cells. In zebrafish ZEB2J cells, hypoxia led to an increase (70%) in COX4-2

550

mRNA levels in only 1 of 4 combinations of oxygen and duration (24h at 2% O2) (Fig. 2g).

551

However, this effect is reduced when it is considered that the relative abundance of COX4-2

552

transcripts is extremely low, less than 0.1% of COX4-1 mRNA levels (Fig 2h). In rainbow trout

553

gonad cells, the relative abundance of COX4-2 mRNA was higher than in zebrafish cells but it

554

showed no response to hypoxia or anoxia (Fig.2 g).

555

To better understand the control of the COX4-2 gene in fish, we created promoter

556

constructs under the control of the proximal promoters from the COX4-2 genes from both

557

goldfish and zebrafish, and compared the response to a rat COX4-2 promoter. Though we

558

observed a significant increase in luciferase activity driven by the rate proximal promoter, the

559

response seen for the COX4-2 promoters from fish we similar to the empty vector with minimal

560

promoter (Fig. 8b). Combined with our transcriptional data, there bulk of the evidence suggests

561

that COX4-2 is not hypoxia responsive in teleost fish. 21

562

COX4 transcriptional response in reptile tissues. To bridge the gap in the hypoxia

563

responsiveness of COX4 paralogs between teleost fish and mammals, we looked at tissues of

564

hypoxic and normoxic western painted turtles and green anoles. Reptiles are ectotherms and

565

are able to survive more extreme bouts of hypoxia than other tetrapods through behavioral

566

lowering of body temperatures, resulting in lowered metabolic rates (18,21). This class also

567

possesses the COX4 paralogs which are orthologs of those found in mammals (29).

568

Green anoles have become a popular model for lab-based research since becoming the

569

first reptile species to have its genome published. In our study, we found no transcriptional

570

response in COX4-2 to 24 h of exposure to hypoxia (Fig. 5a). Interestingly, COX4-2 accounted for

571

a higher proportion of COX4 transcripts in anole tissues compared to the mammalian and fish

572

tissues we looked at (15% in brain to 35% in lung, Fig. 5b).

573

The freshwater turtle group to which the western painted turtle belongs is remarkably

574

tolerant to anoxia, being obligate air-breathers that are able to survive anoxia for up to five

575

months, and are often used to study the cellular mechanisms of hypoxia response in higher

576

vertebrates (26). While we failed to see any significant changes in the mRNA levels of the COX4

577

paralogs when exposed to anoxia, we found that COX4-2 was the predominant transcript in

578

both tissues (Fig. 5e). COX4-2 transcripts were 2- to 4-fold more abundant than COX4-1 under

579

both normoxic and hypoxic conditions. Though anoles showed a higher COX4-2 abundance than

580

other species, the trend is more extreme in turtles. With only two reptiles studied, the

581

evolutionary origins of the greater expression in turtles is unclear, but it is consistent with the

582

superior ability to survive hypoxia in this species. Increased constitutive expression of COX4-2 in

583

turtles could create a COX enzyme that is better suited to hypoxic metabolism, a condition that

584

could arise at any point in the daily life of the animal. This argument is predicated on the

585

assumption that COX4-2 confers kinetic advantages to COX in hypoxia, which has not been

586

shown experimentally in nonmammalian vertebrates. The isoform profile would have little

587

consequence to anoxic metabolism given that any kinetic properties would only manifest if

588

there was flux through the enzyme. 22

589

When energetically challenged through hypoxic exposure, reptiles conserve energy via a

590

coordinated down-regulation of both ATP producing and consuming processes within their

591

tissues. In western painted turtles, this results in a decrease in energy metabolism by at least

592

49% (6,10). During the hypoxic stress and contrary to what occurs in mammalian tissues, ROS

593

production in the brains of western painted turtles decreases during bouts of anoxia compared

594

to normoxia (33). While this may be due simply to the global decrease in oxygen availability, it

595

also could be caused in part by the greater reliance on COX4-2, particularly during the

596

transitioning between normoxia and anoxia, when tissues would be experiencing hypoxic

597

conditions. In mammalian tissues, the presence of COX4-2 lowers ROS production during

598

hypoxia, but increases ROS production during normoxia, relative to complexes containing

599

COX4-1 (14). Anoxia-tolerant turtles, and by extension other reptile species, may have made a

600

trade-off between increased ROS production under normoxic conditions and decreased

601

production during hypoxic conditions as a result of their frequent exposure to low levels of

602

oxygen.

603

This is the first study to our knowledge that has measured the normoxic levels of COX4

604

paralogs or the response of COX4 paralogs to hypoxia in reptiles, and we have only examined

605

four tissue types from two species. Further, we were only able to sequence a portion of each

606

COX4 gene in western painted turtle, so it is yet unknown if they possess similar gene and

607

polypeptide structures to their mammalian counterparts, which may give some indication of

608

the functional consequences each isoform has on the COX complex. It cannot be ruled out that

609

the switch in the dominant COX4 paralog to COX4-2 found in turtles may have arisen from an

610

alternative evolutionary subfunctionalization of the COX4 paralogs. That is, the COX4 paralogs

611

in turtles and other reptiles may have evolved to both serve the same function in the COX

612

complex, or they may have evolved to preform complimentary functions which differ from

613

other vertebrates.

614

The finding that COX4-2 replaces COX4-1 transcription during hypoxic stress in

615

mammalian tissues has led to the hypothesis that this occurs as a cellular survival mechanism to

616

combat increased ROS production. We were interested in determining if this mechanism is 23

617

widespread across vertebrates. Our results indicate that this may be a unique mammalian

618

feature, and further, that it may be limited to a select number of species or tissue types within

619

the mammalian lineage. COX4-1 transcript levels remained dominant during hypoxia in all fish

620

tissues we examined, from both whole animals and cultured cells. Similar results were obtained

621

for cultured cells from mouse muscle and human prostate cancer. Additionally, no functional

622

COX4-2 gene is found in the Avian class (29).

623

Evolution of COX4-2 in vertebrates

624

Due to the presence of orthologous COX4 paralogs across most vertebrate lineages, we

625

hypothesized that the hypoxia responsiveness of COX4-2 would be pervasive in vertebrates.

626

However, our transcriptional data suggest that this is not the case. To gain a better

627

understanding of the function and regulation of COX4-2 in mammals and lower vertebrates, we

628

examined the evolution of the COX4-2 gene and polypeptide across vertebrates.

629

Regulatory elements in COX4-2. The regulation of the COX4 paralogs has proven

630

difficult to ascertain. One study concluded the oxygen sensitivity of COX4-1 and COX4-2 was

631

due to the well-defined HIF-1 pathway. The authors showed that COX4-2 transcription was

632

activated through the binding of HIF-1 to a pair of HREs while COX4-1 proteins were selectively

633

degraded through the action of the protease LON, which is also activated through the HIF-1

634

pathway (14). A second study argued that COX4-2 is regulated by an unknown pathway

635

independently of HIF-1. This group was able to show that the COX4-2 promoter was activated

636

not by its putative HREs, but by an element they termed the ORE (25). Though our studies do

637

not address which of these contradictory conclusions is valid, we were able to explore the

638

evolution of the elements in vertebrates.

639

We compared the promoters of the COX4-2 gene from a wide range of taxa to

640

determine the conservation of the two HREs and the ORE across vertebrates. The first HRE is

641

present in the proximal promoter, the second is present within the first intron of the gene, and

642

the ORE is found slightly downstream of HRE1 within the proximal promoter (Fig. 6a). None of

643

the putative elements were completely conserved across mammals, though the ORE was far

644

more conserved than either HRE (Fig. 6b). This suggests that the ORE is of more regulatory 24

645

importance than the HREs. Outside of the mammalian class, there was little conservation of the

646

three elements. No recognizable sequence homology for any element could be found in any

647

teleost species, and the elements found in amphibian and reptilian species were as low as 41%

648

conserved compared to the human element sequences. In doing the transfection work in

649

mammalian cells, we do assume that the mammalian DNA-binding proteins have the same

650

sequence preference as their nonmammalian homologs (11). To test the ability of the three hypoxia responsive elements to activate COX4-2 during

651 652

exposure to hypoxia, we conducted reporter gene experiments. This allowed for the elimination

653

of the cell type- and species-specific differences discussed above, placing the reporter

654

constructs containing the putative elements in a common context. We first compared the

655

efficacy of the hypoxia response of the two HREs and the ORE from the human, anole, and

656

Xenopus COX4-2 genes to a well-known HRE from the human iNOS gene. Only the human HRE2

657

reporter construct displayed a response to the hypoxic and anoxic treatments comparable to

658

the iNOS HRE response (Fig. 8a). Further, the ORE is the most conserved across mammals while

659

the HRE2 tends to be the least conserved, although HRE2 was more conserved relative to the

660

human sequence in anole and Xenopus than the other elements (61% and 67%, respectively for

661

HRE2 compared to 53% – 59% for the other elements; Figure 6b). Our results support the

662

hypothesis (14) that these HRE elements are responsible for the hypoxia responsiveness of

663

COX4-2. Although the machinery for a HIF-1 response was present in these cells, as indicated by

664

the iNOS and HRE2 responses, we saw no response to similar constructs based on the first HRE

665

site.

666

The possibility of false negative results in our transfection experiments cannot be

667

excluded. The cell lines used may have lacked the necessary factors for the induction of ORE

668

response.

669

Polypeptide structure of COX4-2 in vertebrates. The functional significance of COX4-2 is

670

believed to be an escape from inhibition by ATP at this subunit, and an increase in turnover rate

671

of the enzyme (22). This is thought to be due to the presence of two cysteine residues in the 25

672

peptide sequence of COX4-2. These cysteines may form a disulfide bond in the matrix ATP

673

binding site of the subunit which prevents the binding of adenylates (25). We examined the

674

COX4-2 polypeptide sequences from a selection of lineages to determine the conservation of

675

the cysteine pair and the conservation of the residues that coordinate ATP binding across

676

vertebrates.

677

We found that the two cysteine residues were only found in mammals, and even within

678

mammals they are not universally conserved (Fig. 7). Though mammals outside the

679

rodent/primate lineage appear to lack the capacity to form the disulfide bond that disrupts an

680

otherwise functional ATP binding site, other taxa appear to have mutations in the coordinating

681

ligands, effectively disrupting ATP binding by another mechanism. The Xenopus COX4-2 protein

682

appears to possess an insertion that shifts the conserved EW residues of the ATP-binding site.

683

In fish, the first polar (R) residue shared in tetrapods is nonpolar (L or V). While these changes

684

are likely to influence the ability of the protein to bind ATP at this site, the functional

685

consequences of these structural variants remain to be established experimentally. It is worth

686

noting that it remains possible that the changes in regulation of this subunit in rodents may

687

depend more on reversible phosphorylation and less on the disulphide bridge (17).

688

In summary, although the importance of the COX4-2 gene paralogs is widely accepted in

689

human and rodent models, and likely plays an important role in hypoxic metabolism, the

690

pervasiveness of the genetic and protein adaptations do not seem ubiquitous and therefore the

691

evolutionary origins are uncertain. There is some evidence of coevolution in that rodents and

692

mammals may impair ATP binding via a disulfide bridge, whereas in amphibians and teleosts,

693

mutations may disrupt ATP binding directly. The divergence in the COX4-2 promoter is also

694

striking, with no evidence of hypoxia responsiveness in fish, but in both fish and reptiles’

695

greater constitutive expression of COX4-2, with turtle showing COX4-2 expression to dominate

696

over COX4-1. The transcriptional activation of COX4-2 in hypoxic conditions may be limited to

697

select tissues within select mammalian species.

698

Acknowledgements 26

699

This study was funded by a Discovery Grant (CDM) of the National Sciences and Engineering

700

Research Council (NSERC) Canada. We thank Niels Bols and Lucy Lui for providing the ZEB2J and

701

RTG-2 cell lines, Les Buck for providing turtle tissues and Gary Armstrong and Pierre Drapeau

702

for providing wild-type zebrafish. We also thank Emily Boniferro, Katharina Bremer, and

703

Rhiannon Campden for their assistance with the cloning and sequencing of goldfish genes.

704

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705 706 707

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33. Pamenter ME, Richards MD, and Buck LT. Anoxia-induced changes in reactive oxygen species and cyclic nucleotides in the painted turtle. J Comp Physiol B 177: 473–481, 2007. 29

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812 813

42. Villani G and Attardi G. In vivo control of respiration by cytochrome c oxidase in human cells. Free Radic Biol Med 29: 202–210, 2000.

814

30

815

Figure Legends

816

Figure 1. Transcriptional response to hypoxia in COX4 paralogs in murine myoblast cells

817

(Sol8) and human prostate cancer cells (LNCaP). Cells were exposed to 8 or 24 h of 2% O2, n = 6

818

per treatment. (A) mRNA levels for COX4-1 and COX4-2, relative to control treatments. (B)

819

Proportion of COX4 transcripts present as COX4-1 or COX4-2. (C) Relative mRNA levels for

820

VEGFA, a known hypoxia-responsive gene. Asterisks represent a significant difference from the

821

control treatment as determined by a Mann-Whitney U test. Error bars depict SEM.

822

Figure 2. Transcriptional response of COX4 paralogs to hypoxic treatments in whole

823

zebrafish and goldfish, as well as ZEB2J and RTG-2 cell lines. (A – C) Transcriptional response in

824

gills, brain, liver, and white muscle (WM) of zebrafish exposed to 8 h of 4% O2, n = 8 per

825

treatment. (D – F) Transcriptional response in the corresponding tissues of goldfish exposed to

826

21.5 h of anoxia (undetectable levels of O2). (G – I) Transcriptional response of zebrafish

827

epithelial cells (ZEB2J) and rainbow trout gonadal fibroblasts (RTG-2) exposed to 8 or 24 h of 2%

828

O2 or anoxia, n = 6 per treatment. Panels (A), (D) and (G) display the mRNA levels for COX4-1

829

and COX4-2 relative to control treatments, while panels (B), (E) and (H) display the proportion

830

of COX4 transcripts present as COX4-1 or COX4-2. Panels (C), (F) and (I) display the relative

831

mRNA levels for IGFBP-1 (whole zebrafish and goldfish), or VEGFA (ZEB2J), which are known

832

hypoxia-responsive genes. No response was seen in any hypoxia-responsive gene we measured

833

in RTG-2 cells (data not shown). Asterisks represent a significant difference from the control

834

treatment as determined by a Mann-Whitney U test. Error bars depict SEM.

835

Figure 3: Intertissue comparisons of the transcriptional response of COX4 paralogs to

836

hypoxic treatments in tilapia. Transcriptional response in various tissues of tilapia exposed for

837

18 h to varying degrees of hypoxia. n = 6 per treatment, with exception of treatment group

838

1.5% where there was an n = 5 due to one mortality. (A) mRNA levels for COX4-1 and COX4-2

839

relative to control treatments. (B) Proportion of COX4 transcripts present as COX4-1 or COX4-2.

840

(C) relative mRNA levels for IGFBP-1, a known hypoxia-responsive gene. Asterisks represent a

31

841

significant difference from the control treatment as determined by a Mann-Whitney U test.

842

Error bars depict SEM.

843

Figure 4: Timecourse of transcriptional response of COX4 paralogs to hypoxia in tilapia.

844

Fish were exposed to hypoxia (5% O2) for 72 h, with single fish sampled throughout the

845

timecourse.

846

Figure 5. Transcriptional response of COX4 paralogs to hypoxic treatments in reptilian

847

species. (A – C) Response in muscle, lung and brain tissues of green anole exposed to 4% O2 for

848

24 h. (D – F) Response in muscle and liver tissues of western painted turtles exposed to 24 h or

849

anoxia, n = 6 per treatment. Panels (A) and (D) Display the mRNA levels for COX4-1 and COX4-2

850

relative to control treatments, while panels (B) and (E) display the proportion of COX4

851

transcripts present as COX4-1 or COX4-2. Panels (C) and (F) display the relative mRNA levels for

852

VEGFA, a known hypoxia-responsive gene. Asterisks represent a significant difference from the

853

control treatment as determined by a Mann-Whitney U test. Error bars depict SEM.

854

Figure 6. Comparison of putative hypoxia responsive elements in the COX4-2 gene across

855

vertebrates. (A – B) The human sequences for the two hypoxia responsive elements (HREs) and

856

the oxygen responsive element (ORE) were used to search for orthologous elements in other

857

vertebrate species representing several different lineages. (A) The location of the three

858

elements in relation to the 5’untranslated region (5’UTR) and the ATG start site (ATG) of COX4-2

859

is shown for major classes of vertebrates. (B) The determined elements are presented in

860

relation to the vertebrate phylogeny. Differences from the consensus (human) sequence are

861

indicated by filled boxes containing the alternative nucleotide or deletion (represented by a

862

dash).

863

Figure 7. Conservation of features in vertebrate COX4-2 peptides. (A) Alignment of human

864

COX4-1 and COX4-2 amino acid sequences. The COX4 amino acids suggested to be involved in

865

the ATP/ADP binding site are denoted by arrows (Arg-20, Arg-73, Tyr-75, Glu-77, and Trp-78),

866

and the COX4-2 cysteines which may disrupt the site are denoted by asterisks (Cys-16 and Cys-

867

30). (B) Conservation of COX4-2 adenylate-coordinating amino acids and cysteines across 32

868

vertebrates. Species names and accession numbers for each polypeptide sequence used can be

869

found in Table 1.

870

Figure 8. Reporter gene activities for luciferase (pGL4.23) under control of the COX4-2

871

promoter and hypoxia responsive elements from candidate vertebrate species. (A) PC3 cells

872

were transfected with constructs containing four tandem repeats of each hypoxia responsive

873

element (HRE1 and 2) and the oxygen responsive element (ORE) from human (Hu), anole (An)

874

and Xenopus (Xe), and were exposed to normoxia, 2% O2 (hypoxia), or anoxia for 24 h. (B)

875

HepG2 cells were transfected with constructs containing approximately 2 kb of promoter

876

sequence directly upstream of the COX4-2 5’-UTR from rat, goldfish, and zebrafish, and were

877

treated in the same way as the PC3 cells for (A). Also included in both experiments were

878

constructs containing four tandem repeats of a known hypoxia-responsive element from the

879

human iNOS gene and the baseline activities for the empty pGL4.23 vector (EV). Asterisks

880

represent a significant difference from the control treatment as determined by a Mann-

881

Whitney U test. Results were normalized to co-transfected Renilla reporter activity levels and

882

are presented relative to normoxic activities, +SEM.

883 884

33

Tables

885 886 887 888

Table 1. Species used for assessment of conservation of COX4-2 promoter elements (DNA sequences) and cysteine residues (amino acid sequences, AA). Common name Species name

Ensembl accession no.

Use (DNA, AA)

Anole

Anolis carolinensis

ENSACAG00000011459

D, A

Cat

Felis catus

ENSFCAG00000014664

D

Coelacanth

Latimeria chalumnae

ENSLACG00000009141

A

Cow

Bos taurus

ENSBTAG00000016171

D, A

Dog

Canis lupus familiaris

ENSCAFG00000007049

D, A

Dolphin

Tursiops truncatus

ENSTTRG00000007924

D, A

Gorilla

Gorilla gorilla

ENSGGOG00000009052

D

Hedgehog

Erinaceus europaeus

ENSEEUG00000009547

D, A

Human

Homo sapiens

ENSG00000131055

D, A

Hyrax

Procavia capensis

ENSPCAG00000011518

D, A

Macaque

Macaca mulatta

ENSMMUG00000007383 D

Microbat

Myotis lucifugus

ENSMLUG00000004800

D, A

Mouse

Mus musculus

ENSMUSG00000009876

D

Pika

Ochotona princeps

ENSOPRG00000004657

D, A

Rat

Rattus norvegicus

ENSSARG00000003313

D, A

Tilapia

Oreochromis niloticus ENSONIG00000001633

A

Xenopus

Xenopus tropicalis

ENSXETG00000027592

D, A

Zebrafish

Danio rerio

ENSDARG00000022509

A

889 890 891 892 893

34

894

Table 2. Primers used for qPCR quantification of target mRNAs and for pDrive plasmids. Species

Forward (5’-3’)

COX4-1

CAAGTTTGTGCAGCAGCTG

CAAAGAAGAAGATTCCTGCAA

63

29

COX4-2

CGACAGACCTTACAAAGACATCC

GAAGAATAAGATCCCAGCCGT

63

29

IGFBP-1

ACGCAAGAAACTGGTGGAAC

GGGCTGTCTGGAGTTCGATA

59

30

VegFA

AGTATCCCGATGAGATCGAGCACA

CACCTCCATAGTGACGTTTCGTGT

59

-

† β-actin

TCCAGGCTGTGCTGTCCCTGTA

GTCAGGATCTTCATGAGGTAGTC

59

12

RPL13A

TCTGGAGGACTGTAAGAGGTATGC

AGACGCACAATCTTGAGAGCAG

59

40

COX4-1

AGCTTCGCTGAAATGGAAA

CCTCATGTCCAGCATCCTCT

59

14

Goldfish

COX4-2

AAACTTGCCTTGTACAGGCTTACG

AAGAAGAAGATGCCGCCCAC

56

-

(Carassius

IGFBP-1

TGGTTGGGCAGGAACCAATC

TCTCGAAGCACCTCCTCACA

59

-

EF1α

CAGGTCATCATCCTGAACCAC

AACGACGGTCGATCTTCTC

59

5

TBP

AGTGGTGCAGAAGTTGGGTTTTCC

ATGTGSSGCACCAGGCCCTCTAA

59

12

COX4-1*

CACATGGTGTTGCCAAGGTAGAC

GCGCTCAGCTCTGTCACAAACT

63

-

COX4-2*

ACCACGAGGTGTCCGACTCA

AGGACGTCCTTCCATGGTCTGT

63

-

VegFA

AGACAGCCCACATACCCAAG

AAGACGTCCACCAGCATCTC

59

-

EF-1α

TCCTCTTGGTCGTTTCGCTG

ACCCGAGGGACATCCTGTG

59

-

Tilapia

COX4-1

GCAACCAGAGCCCTAAGCCTTATT

CTCTGCACTCAGGTTCTGCACA

67

-

(Oreochromis

COX4-2

GCTGAGGTAGCACAGTCTGGA

TTCCCTAGAGAGGCTCCATCTTTC

67

-

niloticus )

IGFBP-1

AGGAGAGCATGAAAGCCAAA

TCCGTCCAGAGAGGATTCAC

59

-

COX4-1

GAGCAATTTCCACCTCTGT

CAGGAGGCCTTCTCCTTCTC

59

14

Human

COX4-2

ATTTCCTCCAAAGCCGATCAC

GAGACAGCTGGGGATGCAAGTCA

65

-

(Homo sapiens)

VegFA

AGCTACTGCCATCCAATCGAGA

TACACGTCTGCATGGTGATGTTGG

59

-

‡ EF-1α

ATTGATGCTCCTGGACACAGA

CAGCAACAATCAGGACAGCACAGT

59

-

COX4-1

GCAGCCTTTCCAGGGATG

GCTCTTCTCCCAAATCAGA

59

-

Mouse

COX4-2

AGAACTAGAGGGACAGGGACACA

TCGCTGAGCTCTGTGCAGAA

59

-

(Mus musculus)

VegFA

CCAAGGCCAGCAAATCCCTGTGGGCC

CCGCCTCGGCTTGTCACA

59

8

β-actin

AGCACCATGAAGATCAAGATCAT

ATCCACATCTGCTGGAAGGT

59

27

COX4-1

ATGGAGTGCTCTTTCGCTGG

AAAGCCACTGAGGCCAATGA

59

-

COX4-2

AGTTATCCGCTGCCAGACAC

ACGGTCTTCCATTCGTTGCT

59

-

TTCACAGACTCACGTTGCAAGTCG

GCAAGTGCGCTCGTTTAACTCAAG

59

-

COX4-1*

CCAGAGAATGCTGGATATGA

CCATTCCTTCTTTTCGTAGTCCCA

59

-

COX4-2

AAGAGAAGGAGAAGGGCTCATGGAAG

TTCGTTGGATGGCCTGTTCATCTC

59

-

CCGGGTTGGACTCAAGACGATAGTTA

ACGCTGTAGGTATCTCAGTTCGGT

59

-

Zebrafish (Danio rerio)

auratus)

Rainbow Trout

Reverse (5’-3’)

Tann.

Gene

(˚C)

Source

(Oncorhynchus mykiss)

Anole (Anolis carolinensis)

VegFA Turtle

§

(Chrysemys picta bellii)

pDrive-specific

* Primers were designed from consensus sequences from closely related species † Primer set was used for all other species listed except mouse ‡ Primer set was used for mouse, anole, and turtle as well. § Primer set was used for anole and turtle.

895 35

896 897 898

Table 3. Element sequences used in transfection experiment. The plasmid construct for each element contained 4 tandem repeats of the element followed by a 12 bp sequence containing a MluI restriction site (5’- ACGCGTATCGAT -3’). Species

HRE1 (5’-3’)

Human (Homo sapiens) Anole (Anolis carolinensis) Xenopus (Xenopus tropicalis)

HRE2 (5’-3’)

ORE (5’-3’)

Accession no.

CTGTGCGCTCCCACGCC

CAGGCCTGTGTGCACGTA

GGACGTTCCCACGC

NG_012180.1

GTTTGCACTCTTTGGCC

CAGGCATGGGCCAACTTG

AGGCTATCACTCGC

NW_003339315.1

ATTTTCGATACCACATA

CGGGTCTCTGGGCAGTAG

GGAGTTGCTCTAAT

NW_003171408.1

899 900 901 902 903 904

Table 4. Primers used for long promoter transfection experiment. Accession numbers for rat and zebrafish are the same as those listed in Table 1. The promoter sequence for goldfish was generated using a ‘RAGE’ protocol as described in the methods section. Restriction sites are shown in bold. Species Rat (Rattus norvegicus) Goldfish (Carassius auratus) Zebrafish (Danio rerio)

Forward (5’-3’)

Reverse (5’-3’)

ACTGCAACGCGTGGCCTTGAACTCAGT

TGCAGTCTCGAGGTCTAGTTCAGCACC

ACTGCAACGCGTGCATTAGCTTTTGG

TGCAGTCTCGAGGGAGAGACTGTGCA

ACTGCAACGCGTGGTTGCCACAGCGGA

TGCAGTCTCGAGAAACTGTGAGAGACTGTG

905 906

36

1.4

2% O2 8 h

1.2

2% O2 24 h

1.0 0.8 0.6

*

*

0.4 0.2 0

N.D.

COX4-1

COX4-2

Sol8

COX4-1

COX4-2

LNCaP

1.2

14

COX4-2

1.0

Relative mRNA levels

Control (21% O2)

0.8 0.6 0.4 0.2

12

Control (21% O2) * 2% O2 8 h 2% O2 24 h

10 8 6

*

4 2

*

*

0

0

Co n 2% tro l O 2% 2 8 O h 2 2 4h

*

C VEGFA

COX4-1

Co n 2% tro O l 2% 2 8 O h 2 2 4h

Relative mRNA levels

1.6

B Paralog mRNA abundance Relative mRNA abundance

A Relative paralog mRNA levels

Sol8

LNCaP

Sol8

LNCaP

A Zebrafish relative paralog mRNA levels 1.6 * Control (21% O2) *

1.2

1.2

*

*

1.0 0.8 0.6 0.4 0.2

0.5

Co nt ro Hy l po xic

Co nt ro Hy l po xic

Co nt ro Hy l po xic

Gills

Brain

Liver

WM

COX4-1

8 6 4

*

*

Gills

Brain

Brain

Liver

25

0.8

20

0.6 0.4 0.2

0% O2 24 h

*

**

* *

0.4

*

0.2 0

COX4-2 ZEB2J

COX4-1

COX4-2 RTG-2

10

ic ox

nt Co

0.4 0.2

C

h

24

h

8

h

24

O O2 O O2 2% 2% 0% 2%

ZEB2J

2

**

12 10 8 6 4

0

8

2

Brain

14

2

l

Gills

16

0.6

ro

*

h

l

ro

C

t on

8

h

24

h

8

h

24

O O2 O O2 2% 2% 0% 2% 2

*

5

Liver

WM

I VEGFA in ZEB2J cells

COX4-2

0.8

t on

*

An

ro

l

ic

l Co

An

nt

ox

ro

ic

ox

l Co

An

ro nt

ox

COX4-1

Anoxic

WM

1.0

0

COX4-1

Liver

Relative mRNA levels

0% O2 8 h

ic

l ro nt Co 1.2

Relative mRNA abundance

2% O2 8 h

*

2% O2 24 h

Brain

H Fish cell lines paralog mRNA abundance

G Fish cell lines relative paralog mRNA levels Control (21% O2)

An

X4 CO

Gills

Control (21% O2)

*

15

0

WM

WM

F IGFBP-1 in goldfish

1.0

-2

-1 CO

X4

-2 X4 CO

X4

-1

-2 CO

CO

CO

X4

-1 X4

-2 CO

X4

-1 X4 CO

Gills

Liver

COX4-2

0

0

Relative mRNA levels

10

Relative mRNA levels

Relative mRNA abundance

Relative mRNA levels

1.0

0.6

*

12

0

1.2

1.5

0.8

0.2

Hypoxic (4% O2)

16

E Goldfish paralog mRNA abundance

Anoxic

2.0

1.0

0.4

WM

2

1.2

0.6

Co nt ro Hy l po xic

Liver

D Goldfish relative paralog mRNA levels * Control (21% O )

1.4

Control (21% O2)

14 0.8

CO X4 -2

CO X4 -1

CO X4 -1 CO X4 -2

CO X4 -1 CO X4 -2

CO X4 -1

CO X4 -2

Brain

2.5

1.6

18

0

Gills

1.8

COX4-2

2

0

2.0

COX4-1

1.0

Relative mRNA levels

Hypoxic (4% O2)

Relative mRNA abundance

Relative mRNA levels

1.4

C IGFBP-1 in zebrafish

B Zebrafish paralog mRNA abundance

RTG-2

2

h

* *

Control (21% O2) 2% O2 8 h 2% O2 24 h 0% O2 8 h 0% O2 24 h

A Tilapia relative paralog mRNA levels

Relative mRNA levels

*

Control (21% O2) 5% O2 3% O2 1.5% O2

2.0

1.5

* **

*

* 1.0

** *

0.5

Gills

RM

WM

COX4-1

-2 X4

X4 CO

CO

Kidney

C IGFBP-1 in tilapia

B Tilapia paralog mRNA abundance 1.2

-1

-2 X4 CO

-1 X4 CO

-2 X4

-1 CO

Brain

CO

X4

-2 X4

-1

Liver

CO

CO

X4

-2 X4 CO

-1 X4 CO

X4 CO

CO

X4

-2

-1

0

20

COX4-2

*

Relative mRNA levels

Relative mRNA abundance

18 1.0 0.8 0.6 0.4

16 14

*

12

*

10 8

**

6

*

4

0.2 0

l l l l l l ro O 2 O 2 O 2 ro O 2 O 2 O 2 ro O 2 O 2 O 2 ro O 2 O 2 O 2 ro O 2 O 2 O 2 tro % O 2 % O 2 % O 2 nt % % % nt % % % nt % % % nt % % % nt % % % 5 3 1.5 Co 5 3 1.5 Co 5 3 1.5 Co 5 3 1.5 Co 5 3 1.5 Co 5 3 1.5

n Co

Gills

Liver

Brain

RM

WM

Kidney

0

*

*

2

Gills

Liver

Brain

RM

WM

Kidney

5

A. Gill

4

4

3

3

2

2

1

1

0

0

B. Liver

D. Red muscle

COX 4-2 mRNA

C. Brain 2

2

1

1

0

0

COX 4-2 mRNA

COX 4-2 mRNA

5

2

2

E. White muscle

1

1 0

F. Kidney

0 0

24

48 Time (h)

72

0

24

48 Time (h)

72

B Anole paralog mRNA abundance

A Anole relative paralog mRNA levels

C Anole VEGFA

Control (21% O2)

1.4

Hypoxic (4% O2)

1.2

*

1.0 0.8 0.6 0.4 0.2

COX4-1

1.2

COX4-1 COX4-2

COX4-1 COX4-2

COX4-1 COX4-2

Muscle

Lung

Brain

0.8 0.6 0.4 0.2

l

ro nt

Co

l

ic

x no

A

ro nt

Co

Muscle

D Turtle relative paralog mRNA levels

7

1.0

0

0

COX4-2 Relative mRNA abundance

1.6

Relative mRNA abundance

Relative mRNA levels

1.8

A

Lung

Co

ro nt

Hypoxic (4% O2)

5 4 3

*

2 1 0

l

ic

x no

Control (21% O2)

*

6

ic

Muscle

x no

Lung

Brain

A

Brain

E Turtle paralog mRNA abundance

F Turtle VEGFA

2.0

1.4 1.2 1.0 0.8 0.6 0.4 0.2

COX4-1

1.0

COX4-2 0.8 0.6 0.4 0.2 0

0

COX4-1 COX4-2

COX4-1 COX4-2

Muscle

Liver

l

ro nt

Co

A

Muscle

Co

ro nt

2.5

Control *

Anoxic

2.0 1.5 1.0 0.5 0

l

ic

x no

Relative mRNA abundance

Anoxic

3.0

1.2

Control

1.6

Relative mRNA abundance

Relative mRNA levels

1.8

ic

x no

A

Liver

Muscle

Liver

A COX4-2 hypoxia-responsive element distributions HRE -1

ORE

HRE -2 5’UTR

ATG

Mammalia Aves (none) Reptilia Amphibia Actinopterygii

B Conservation of elements across vertebrates

Cetacea

Dolphin

Artiodactyla

Cow

HRE1

ORE

HRE2

CT GTG CG C TCCCAC GC C

GGTTCCCACGG GGAT T

CAGGCCTGTGTGCACGTA

AG

T

G

AA

Cat

G

Dog

G

A

T T

T T

Microbat

C

Eulipotyphia Hedgehog

Rat

AC T C T ^ T

Carnivora Chiroptera

CCA

^ T

AG

A

G G A GT C

A

^ T TCTCC

T T

A

A

TCC

ACAC G

A- A C

AC

A- A C

AC

C

Rodentia Mouse

T

Lagomorpha Pika

AA

G A

GA

AG

CTGC

Human Primates

Gorilla Macaque

G

Afrotheria

Hyrax

Squamata

Anole

G T

Anura

Xenopus

A T T

C

G

A

TTTG A A

A

A TCTCCA

A G TA AT A

GT G

A T T AA

A G

T

G CCA CA G

T G G

A Alignment of human COX4-1 and COX4-2 peptide sequences 1 86 COX4-1 MLATRVFSLVGKRAISTSVCVRAHESVVKSEDFSLPAY--MDRRDHPLPEVAHVKHLSASQKALKEKEKASWSSLSMDEKVELYRI COX4-2 MLPRAAWSLVLRKGGGGRRGMHSSEGTTRGGGKMSPYTNCYAQRYYPMPEEPFCTELNAEEQALKEKEKGSWTQLTHAEKVALYRL

*

*

87 171 COX4-1 KFKESFAEMNRGSNEWKTVVGGAMFFIGFTALVIMWQKHYVYGPLPQSFDKEWVAKQTKRMLDMKVNPIQGLASKWDYEKNEWKK COX4-2 QFNETFAEMNRRSNEWKTVMGCVFFFIGFAALVIWWQRVYVFPPKPITLTDERKAQQLQRMLDMKVNPVQGLASRWDYEKKQWKK

B Conservation of adenylate-coordinating residues and cysteines across vertebrates

Rat

PYVDCYAQRSY ... DEPYCTELS ... FAEMNHRSNEWKTVMG

Pika

PYTNCYAQRSY ... DEPFCTELS ... FTEMNRRSNEWKTVMG

Human Dolphin

PYTNCYAQRYY ... EEPFCTELN ... FAEMNRRSNEWKTVMG

Cow

PYTNYHAQRSY ... EEPFCMELN ... FAEMNRRSNEWKTVMG

Dog

PYTNYHAQRSY ... DEPFCTELN ... FAEMNRRSNEWKTVMG

Microbat

PYTNYHAQRSY ... DEPFCTELN ... FAEMNRRSNEWKTVMG

PYTNYHAQRSY ... DEPFCTELN ... FTEMNRRSNEWKTVMG

Hedgehog PYTNYHAQRSY ... EAPFCTELN ... FAEMNHRSNEWKTVMG Hyrax PYTNYHAQRSY ... DEPFCTELN ... FAEMNRRSNEWKTVMG Anole

SVPLYYDKRSY ... DTPFCKDLD ... FSEMNKPSNEWKTVLG

Xenopus

TKPMYCERREY ... DVPFVSELN ... YAEMHSGSKSEWKTIV

Coelocanth SVPKYYDRRDH ... DVPYQQELS ... YAEMKKPSNEWKTVLG Gar

SLPMYWDRRDT ... DVPYQRELS ... YAEMKKPSNEWKTVVG

Zebrafish

SRPMYSDRLDT ... DRPYKDILS ... FAEMKKPTGEWKTVTA

Cave fish

SLP-YFDRLDT ... DRPYQDSLS ... YAEMKRPTGEWKTVVA

Cod

SRPLYWNRLDT ... DRPFKDVLN ... YPEMKQSTGEWKTVVG

Tilapia

SQPMYWDRVDI ... DRPYKDVLT ... YPEMKQQTAEWKSVMG

Medaka

SRPMYYDRADV ... DKPYKDLLT ... YPEMKQKTSEWKTVLG

Platyfish

SRPTYYDRVDM ... DKPYKDVLS ... YAEMKAPTGEWKTVLG

Stickleback SRPMYWDRLDI ... DKLYKDELS ... YAEMNQPKSEWKTVVG

*

*

A COX4-2 promoter element transfection 32.0

Luciferase units (rel. to normoxia)

16.0

Hypoxia

*

*

*

8.0

*

4.0 2.0 1.0 0.5

S E1 E2 E1 E2 RE E2 E1 RE RE iNO HuO AnO XeO uHR nHR eHR uHR nHR eHR X X A A H H

B COX4-2 long promoter transfection 25

Luciferase units (rel. to normoxia)

Anoxia

20 15 10 5 0

S

iNO

t Ra

Go

ldfi

sh

h fis bra Ze

EV

EV

Evolution of the oxygen sensitivity of cytochrome c oxidase subunit 4.

Vertebrates possess two paralogs of cytochrome c oxidase (COX) subunit 4: a ubiquitous COX4-1 and a hypoxia-linked COX4-2. Mammalian COX4-2 is thought...
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