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.
ii
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
4
metabolic regulation is thought to occur at cytochrome c oxidase (COX) * (3,13,42,2), the final
5
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
9
core of the complex, are mitochondrially encoded and are highly conserved across eukaryotes
10
(36). The remaining ten nuclear-encoded subunits serve structural and regulatory functions.
11
Many of these subunits exist as paralogs in vertebrates, some of which are lineage-specific. For
12
example, though COX4, 6A, 6B, 6C, 7A, and 7C have equal numbers of paralogs in fish and
13
mammals, COX7B and 8 have more in mammals, and COX5A and 5B have more in fish (29).
14
COX4 is of particular interest as the only subunit with a paralog pair that responds to
15
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
18
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-
20
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.
1
24
to COX4-2, while COX4-1 peptides are selectively degraded by the LON protease ( 22,14,25).
25
The trigger for the oxygen-dependent switch in transcription has been attributed to either
26
conventional hypoxia responsive elements (HRE) (14) or oxygen responsive elements (ORE)
27
(25). Fukuda and coworkers (2007) characterized two putative HRE in the human COX4-2 gene,
28
one present in the proximal promoter region and one in the first intron. Through transfection
29
experiments, the group was able to show that these regions conferred a hypoxic response in
30
COX4-2 through transactivation by HIF-1 (14). Conversely, Hüttemann and coworkers (2007)
31
noted that the putative HREs present in human COX4-2 were poorly conserved across
32
mammals. They identified a 13 bp region of the COX4-2 proximal promoter that is more
33
conserved. This ORE conferred hypoxia responsiveness independently of HIF-1 and the HREs
34
(25). Three transcription factors have since been identified as ORE binding proteins.
35
Recombination signal sequence-binding protein Jκ (RBPJ) and coiled-coil-helix-coiled-coil-helix
36
domain 2 increase transcription levels (CHCHD2) and CXXC finger protein 5 acts as an inhibitor
37
(CXXC5) (1).
38
The COX4 paralog switch is thought to optimize electron transfer reactions in COX in
39
response to changing energetic conditions. COX complexes containing COX4-2 have a turnover
40
rate approximately twice that of complexes containing COX4-1 (24,25), and are able to optimize
41
the electron transfer reactions, resulting in a decrease of the production of reactive oxygen
42
species (ROS) under hypoxic conditions (14). Although COX4-2 containing COX shows a normal
43
response to ATP and ADP as was shown with purified enzyme from cow lung (24), there is some
44
evidence to suggest that under hypoxic conditions, adenylate regulation of COX is lost
45
altogether (22). These differences may be caused by a pair of cysteine residues on COX4-2 that
46
form a disulfide bond, preventing adenyate binding (23). The COX4 paralogs differ in efficiency
47
of electron transfer and proton pumping, and perhaps sensitivity to cytosolic adenylate
48
concentrations. Thus, subunit switching may enable a cell to fine tune COX to alter ATP
49
production, respiration, and mitochondrial ROS production in response to tissue type, energetic
50
status and hypoxia (39). 2
51
While it seems clear that the COX4-2 protein and gene adaptations can play a role in the
52
regulation of energy production in rodents and humans, as well as some tumors, the
53
evolutionary origins and phylogenetic distribution have not been well characterized. Given that
54
the COX4 paralog pair is present throughout vertebrates, it is likely that the isoforms arose
55
from a whole genome duplication event early in vertebrate evolution, although one paralog has
56
been secondarily lost in birds and the available evidence suggests that COX4-2 of fish may have
57
a different origin than COX4-2 of tetrapods (29). If this pathway is central to oxygen sensing
58
and/or hypoxic metabolism, natural selection may affect the gene both in regulatory and
59
structural regions in lineages that have distinctive hypoxic adaptations. For example, it could be
60
hypothesized that more hypoxia tolerant species may express COX4-2 constitutively in more
61
tissues, or that these species may have a more responsive promoter. In this study we were
62
interested in determining if the hypoxia-dependant COX4 paralog swich is present in lower
63
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
65
restricted to the Euarchontoglires lineage that includes rodents and primates.
66
Methods
67 68
COX4-2 sequence analysis
69
COX4-2 genes and their promoters were characterized from candidate vertebrate
70
species. Sequences were obtained from the online databases Ensembl and NCBI, and the
71
presence or absence of the putative elements responsible for the hypoxic response of COX4-2
72
was determined by alignments with the human element sequences found by Fukuda et al. (14)
73
and Hüttemann et al. (25) using MultAlin (7).
74
Translated nucleotide sequences of COX4-2 were also examined from a wide array of
75
vertebrate species, focusing on the conservation of amino acids suggested to be involved in the
76
COX ATP/ADP binding site (Arg-20, Arg-73, Tyr-75, Glu-77, and Trp-78), and the presence or 3
77
absence of two cysteine residues thought to be important in the isoform-specific function of
78
human COX4-2 (Cys-16 and Cys-30) (23). Polypeptide sequences were obtained from the online
79
database Ensembl for candidate species from several vertebrate classes and aligned using
80
ClustalW2 (28,16). Accession numbers for all sequences used can be found in Table 1.
81
Animal experiments
82 83
All animal experiments were carried out in accordance with the Queen’s University Animal Care Committee.
84
Zebrafish (Danio rerio, Hamilton) were donated from a wild type population at
85
Université de Montréal. They were held in a 140 L aquarium with dechlorinated water
86
maintained at 25°C for at least six weeks. The fish were kept under a 12:12 light:dark
87
photoperiod and fed Omega One Freshwater Flakes (OmegaSea, Painesville, OH, USA) daily ad
88
libitum. Groups of 8 fish were transferred to 8 L jars at 25oC with aerated water flowing through
89
overnight. After 12 h, the water flowing to the hypoxic fish was bubbled with 4% O2/96% N2 for
90
8 h. At the onset of treatment, gas mixtures were bubbled directly into the jar, with no flow
91
through. After 2 h, flow-through of gas-equilibrated water began at a rate of 4 ml min-1. Control
92
fish were treated in the same manner, but their water was bubbled with air. Immediately
93
following the experiment, fish were lethally anaesthetized in a solution of 0.4 g L-1 of tricaine
94
methanesulphonate and 0.8 g L-1 sodium bicarbonate. Gills, liver, brain, and white muscle were
95
dissected, flash frozen in liquid nitrogen and stored at -80°C.
96
Goldfish (Carassius auratus, Linnaeus) were acquired from a local aquarium store. They
97
were held in a 750 L tank with dechlorinated water maintained at approximately 15˚C for at
98
least six weeks to allow for acclimation. The fish were kept under a 12:12 light:dark
99
photoperiod and fed Omega One Freshwater Flakes (OmegaSea, Painesville, OH, USA) daily ad
100
libitum. Anoxia experiments were carried out in air-tight 120 L tanks with a flow-through water
101
system. Eight fish were exposed to anoxia (100% nitrogen) for 21.5 h. The water was de-
102
oxygenated by pumping nitrogen gas into the tank via an air stone while monitoring oxygen
103
levels using a FOXY oxygen probe (Ocean Optics, Dunedin, FL, USA). After 3 h, no oxygen was 4
104
detected in the tank. Immediately following the experiment, tissues were collected from fish as
105
described above for the zebrafish. Control fish were taken from the holding tank at the
106
beginning of the anoxia treatment and tissues were collected in the same manner described
107
above.
108
Tilapia (Oreochromis niloticus) were received from Red fish Ranch of BC. They were held
109
in a 620 L aquarium with dechlorinated water maintained at 28°C for at least six weeks. The fish
110
were kept under a 12:12 light:dark photoperiod and fed crushed pellets once daily. Six fish were
111
used in each of 4 treatment groups: 21% oxygen (control), 5% oxygen (hypoxia), 3% oxygen
112
(hypoxia), and 1.5% oxygen (hypoxia). Each fish was transferred into individual 6L containers at
113
28°C with aerated heated water maintained for 18 hours for each specific oxygen treatment.
114
Water was bubbled with 5% O2/95% N2 for the 5% hypoxic treatment group, 3% O2/97% N2 for
115
the 3% hypoxic treatment group, and 1.5% O2/98.5% N2 for the 1.5% hypoxic group. Water was
116
gradually brought from 21% (control) oxygen to the correct percent oxygen level for each
117
hypoxia treatment over half an hour to allow the fish to acclimatise. A time course experiment
118
was also completed at 5% oxygen where the fish were held for 4, 8, 12, 18, 24, 30, 36, 42, 48,
119
54, 60, 66 and 72 hours in the hypoxic treatment. All control fish were taken from the holding
120
tank at the beginning of the hypoxia treatment and tissues from all fish were collected in the
121
same manner described above.
122
Green anoles (Anolis carolinensis, Voigt) were acquired from Boreal Scientific. They were
123
kept in an enclosure at ambient temperature (23˚C) and supplied with an infrared heat lamp
124
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
126
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
128
using an injection of pentobarbital (150 mg/kg), and heart, brain, and lung tissues were
129
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
132
dissected from individuals that were from a control group or had been submerged in
133
freshwater for 24 h, and immediately frozen in liquid nitrogen, as described in (35).
134
Tissue culture experiments
135
Two fish cell lines and two mammalian cell lines were employed in this study. Fish cell
136
lines were grown in Leibovitz’s L-15 media (Sigma-Aldrich, St. Louis, MO, USA) supplemented
137
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
139
(Oncorhynchus mykiss, Walbaum gonadal fibroblasts) were maintained at 19°C. Mammalian cell
140
lines were grown in Dulbecco’s Modified Eagle Medium (DMEM, Invitrogen, Carlsbad, CA, USA)
141
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.
143
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
146
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
148
plates were kept in an air-tight chamber containing the appropriate gas mixture and control
149
treatment plates were left at normal atmospheric conditions. All treatments were carried out at
150
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
153
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
161
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
187
sequence fragments.
188
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
191
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
212
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-
214
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
216
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,
231
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
233
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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814
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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