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Molecular characterization and expression analysis of three hypoxia induciblefactor alpha subunits, HIF-1α/2α/3α of the hypoxia-sensitive freshwaterspecies, Chinese sucker

Nan Chen, Li Ping Chen, Jie Zhang, Cheng Chen, Xin Lan Wei, Yas-meen Gul, Wei Min Wang, Huan Ling Wang

PII: S0378-1119(12)00149-7DOI: doi: 10.1016/j.gene.2011.12.058Reference: GENE 37304

To appear in: Gene

Accepted date: 15 December 2011

Please cite this article as: Chen, Nan, Chen, Li Ping, Zhang, Jie, Chen, Cheng, Wei,Xin Lan, Gul, Yasmeen, Wang, Wei Min, Wang, Huan Ling, Molecular characteri-zation and expression analysis of three hypoxia inducible factor alpha subunits, HIF-1α/2α/3α of the hypoxia-sensitive freshwater species, Chinese sucker, Gene (2012), doi:10.1016/j.gene.2011.12.058

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Molecular characterization and expression analysis of three hypoxia inducible factor

alpha subunits, HIF-1α/2α/3α of the hypoxia-sensitive freshwater species, Chinese

sucker

Nan Chen1; Li Ping Chen1; Jie Zhang1; Cheng Chen1; Xin Lan Wei1; Yasmeen Gul2;

Wei Min Wang1; Huan Ling Wang1,*

1Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction,

Ministry of Education, College of Fishery, Huazhong Agricultural University, 430070,

Wuhan, P. R. China

2Department of Biosciences, COMSATS Institute of Information Technology,

Sahiwal, Pakistan

*Corresponding author:

Phone: +86 027 87282113; fax: +86 027 87282114

E-mail: hbauwhl@hotmail.com

ABSTRACT

Hypoxia-inducible factors (HIFs) are transcription factors that respond to

changes in oxygen tension in the cellular environment. In this study, we identified full

length cDNAs of HIF-1α, HIF-2α and HIF-3α in an endangered hypoxia-sensitive

fish species, Chinese sucker. The HIF-1α/2α/3α cDNAs are 3890, 3230 and 3374 bp

in length, encoding 780, 782 and 632 amino acid residues, respectively. The real-time

PCR results suggested that HIF-1α and HIF-3α mRNAs were highly expressed in

liver and gonads, followed by spleen and muscle. Moreover, HIF-1α and HIF-3α

transcription factors revealed similar developmental expression patterns, with the

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lowest expression at 48 hours post-fertilization, and reaching the highest expression

level at 360 hours post-fertilization. Short-term hypoxia exposure (2.2, 2.8 and 3.2

mg/L dissolved oxygen for 24 hours) increased mRNA levels of HIF-1α and HIF-3α.

HIF-2α mRNA showed similar expression patterns as that of HIF-1α and HIF-3α,

however, its expression was extremely low in the spatio-temporal expression patterns

and hypoxia treatment. This is the first report describing the potential to identify

hypoxia-sensitive/tolerant fishes according to the number of the serine residues of fish

oxygen-dependent degradation (ODD) domain. It was suggested that Cyprinomorpha

fishes, with less than 40 serine residues in fish ODD domain were hypoxia-sensitive

fishes and more than 40 serine residues in this domain were hypoxia-tolerant fishes.

Keywords: Chinese sucker; Hypoxia-inducible factor alpha; Gene expression

1. INTRODUCTION

Hypoxia-inducible factors (HIFs) have been reported to regulate gene expression

in response to hypoxia and were discovered during the regulation of erythropoietin

(EPO) expression in the mammalian Hep3B cell line (Semenza and Wang, 1992).

HIFs are heterodimers consisting of two subunits: the hypoxia-regulated α subunit,

(HIF-α), and an oxygen-insensitive β subunit (HIF-β) which is also known as

aryl-hydrocarbon receptor nuclear translocator, ARNT (Wenger and Gassmann, 1997;

Semenza, 1998, 2001). There are three isoforms of the HIF-α subunit (HIF-1α,

HIF-2α and HIF-3α), and three paralogues of the HIF-β subunit (Arnt1, Arnt2 and

Arnt3) (Zagorska and Dulak, 2004). Both HIF-α and HIF-β belong to the basic

helix-loop-helix (bHLH)-Per-Arnt-Sim (PAS) family of transcription factors, which

share several conserved structural domains, including a bHLH region for DNA

binding and two PAS domains for target gene specificity and dimerization (Wang et

al., 1995).

HIF-α protein is regulated by several mechanisms, most notably is degradation.

HIF-α undergoes proteasomal degradation via a ubiquitin-dependent pathway, which

involves post-translational hydroxylation of specific proline residues (Pro402/564)

within the ODD domain by prolyl hydroxylases (PHDs), non-heme, oxygen-, Fe(II)-

and 2-oxoglutarate-dependent dioxygenases under aerobic conditions (Loboda et al.,

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2010). In hypoxia, prolyl hydroxylation does not occur and hypoxia essentially leads

to instantaneous stabilization and accumulation of HIF-α (Jewell et al., 2001). HIF-α

then travels to the nucleus, dimerizes with HIF-β, and binds to hypoxia response

elements (HREs) on hypoxia-sensitive genes (such as vascular endothelial growth

factor, VEGF; multidrug resistance 1, MDR1) (Nordal et al., 2004; Luo et al., 2006;

Comerford et al., 2002), resulting in alterations in their transcription rates (Bracken et

al., 2003). HRE has a core five-nucleotide sequence RCGTG (R: A/G), which is well

conserved among numerous hypoxia responsive genes (Semenza et al., 1996). HIF-1α

subunits are expressed in many tissues, including brain, heart, kidney, liver, pancreas

and embryo, however, HIF-2α mRNA is only slightly expressed in certain tissues, in

contrast to widely expressed HIF-1α (Wiesener et al., 2003). Some studies suggest

that the expression of ADRP (autosomal dominant retinitis pigmentosa), NDRG-1

(N-myc downstream regulated gene 1), and VEGF are increased by the activity of

HIF-2α protein when von Hippel-Lindau (VHL)-deficient and VHL-functional cells

are combined (Hu et al., 2003).

Compared to mammals, the information regarding fish HIF is very limited. The

HIF-1α mRNA sequence of the fish was first characterized in rainbow trout cells

(Soitamo et al., 2001), and the HIF sequences of other fish, such as Fundulus

heteroclitus, Gymnocypris przewalskii, Ctenopharyngodon idella, Danio rerio,

Micropogonias undulatus and Dicentrarchus labrax were subsequently identified

(Powell and Hahn, 2002; Cao et al., 2005; Law et al., 2006; Rytkönen et al., 2007;

Rahman and Thomas, 2007; Terova et al., 2008). A series of physiological and

biochemical adaptations in fish are employed to handle hypoxia. These strategies

include decreased metabolic rate (DallaVia et al., 1994), increased ventilation rate,

hematocrit and haemoglobin O2 affinity (Jensen et al., 1993), and increased anaerobic

respiration (Virani and Rees, 2000). Many HIF-regulated genes participate in glucose

transport, glycolysis, erythropoiesis, angiogenesis, vasodilation and respiration rate,

and function together to minimize the effects of oxygen at cellular, tissue and

systemic levels (Semenza, 1998; Wenger, 2002).

Oxygen availability is more crucial for aquatic than terrestrial animals because

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water contains approximately 3% of oxygen in the same volume of air at the same

partial pressure (Rytkönen et al., 2007), and the rate of oxygen diffusion in water is

ten thousand-fold less than in air (Dejours, 1975), due to which, even modest oxygen

consumption by biological or non-biological processes can quickly decrease oxygen

tension in an aquatic environment. Furthermore, as water temperature increases, the

amount of oxygen dissolved in water is also lowered (Rosenberger and Chapman,

1999). In most of these cases, large diurnal fluctuations in oxygen tension occur

(Nikinmaa and Rees, 2005).

Chinese sucker (Myxocyprinus asiaticus), Cyprinomorpha, is the representative

species of genus Myxocyprinus, and its current geographic distribution is only limited

to the Yangtze River basin, China (Yu et al., 2005). It was historically abundant and

also one of the most important commercial freshwater fish due to its high consumer

preferences. However, the number of natural population of Chinese sucker gradually

decreases due to over-fishing, water pollution, dam construction, especially Gezhou

Dam and other anthropogenic effects (Wang, 1998), and it is currently listed as the

second-grade national protected animal in China. Moreover, fish farming of Chinese

sucker is seriously affected by hypoxia with a 1.0738 mg O2/L suffocation point of

oxygen (Pan et al., 2007). In the present study, we identified and characterized three

distinct HIF-α genes (HIF-1α, HIF-2α and HIF-3α) from cultured Chinese sucker and

examined their spatio-temporal expression patterns and responses to short-term

hypoxia exposure.

2. MATERIALS AND METHODS

2.1 Fish material

In order to analyze the temporal expression of the three HIF-α genes, the

fertilized eggs and larval of Chinese sucker at 48, 72, 96, 120, 144, 360 and 768 hours

post fertilization (hpf), cultured in the normoxic condition with 7.2 mg/L of dissolved

oxygen (DO) and 24.6±1°C of water temperature in the recirculation water system,

were collected from a commercial hatchery in Hubei province, China. The adult

Chinese sucker with the length of 10-12 cm (one year old) were fed in the above same

normoxic condition for one week acclimation, and then different tissues (liver, gonads,

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spleen and muscle) were collected for further expression analysis.

2.2 Experimental material

The larval fish (one month old) were exposed to different DO levels (7.2, 3.2, 2.8

and 2.2 mg/L) for 24 hours after one week acclimation with 7.2 mg/L of DO and

24.6±1°C of water temperature Dissolved oxygen was monitored continuously using

the YSI Model 600R dissolved oxygen meter (Geo Scientific Ltd, USA). The fish

were then recovered at normal oxygen saturation (DO: 7.2 mg/L, temperature:

24.6±1°C) for 24 hours. The whole fish (each group: N = 5-6) were respectively

collected after hypoxia and re-oxygenation treatment, and larval fish were not fed

during the experimental period.

2.3 RNA isolation and cDNA synthesis

Total RNA was extracted from different tissues and larval fish at different

development stages using TRIzol reagent (Promega) according to the manufacturer’s

instruction. RNA concentration was measured using the NanoDrop 2000 (Thermo

Fisher Scientific, USA). First-strand cDNA was synthesized by reverse transcriptase

kit (Promega) as follows: 2 µg of total RNA was reverse transcribed into cDNA in a

volume of 25 µL, containing 1 µL of oligo (dT)16 primer (50 pmol) and 2 µL of 10

mM dNTPs at 42°C for 60 min. In order to isolate HIF-α cDNA from Chinese sucker,

universal amplified primers (UAP) (Table 1) were designed with references for the

conserved sequences identified in the multiple alignment of HIF-α sequences from

other species, followed by PCR amplification of the partial sequence of each gene.

The PCR amplification was conducted in a total volume of 15 µL containing 1 × PCR

buffer, 0.30 µM of each primer, 0.25 mM of dNTPs, 0.75 units of Taq DNA

polymerase (Takara) and about 50 ng cDNA template under the following conditions:

one cycle of denaturation at 94˚C for 3 min; 30 cycles of 30 s at 94˚C, 60 s at 58˚C,

and 60 s at 72˚C with a final extension of 10 min at 72˚C. The PCR products were

cloned into pGEM-T Easy vector (Promega) and subsequently sequenced.

2.4 Rapid amplification of 5′and 3′cDNA ends (RACE)

The sequences obtained with the UAP primers were used to design gene-specific

nested primers (GSP) in order to get full-length cDNA sequences using 5′-RACE and

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3′-RACE methods (Table 1). The reactions were performed for the outer and inner

PCR according to the manufacturer′s instructions (Takara). 5′-RACE and 3′-RACE

products were cloned into pGEM-T Easy vector and sequenced. The full length

cDNA sequences of HIF-α genes were assembled by the DNAStar software.

2.5 Sequence analysis

The amino acid sequences of Chinese sucker HIF-1α/2α/3α genes were predicted

using a translator program at open reading frame finder on NCBI

(http://www.ncbi.nlm.nih.gov/gorf/gorf.html). The protein domains were marked

according to UniProt (http://www.uniprot.org/) and SMART

(http://smart.embl-heidelberg.de/) database.

2.6 Quantitative real-time PCR

Expression patterns of the Chinese sucker HIF-1α/2α/3α genes were analyzed

using quantitative real-time PCR (qRT-PCR). Three reference genes, GAPDH

(glyceraldehyde-3-phosphate dehydrogenase), ACTB (β-actin) and EF1α (elongation

factor 1, alpha), were selected based on expression stability, and all primer sequences

are described in Table 1. The following three-step qRT-PCR reaction was performed:

incubation at 95°C for 30 s, followed by 40 cycles at 95°C for 5 s, 60°C for 20 s and

72°C for 30 s. The total 20 uL reaction volume contained 10 uL of 2 × SYBR Green

PCR Master Mix (Takara), 0.4 µM of each primer, and 1.6 uL of cDNA template. The

relative quantification of the target and reference genes was evaluated using standard

curves. The amplification efficiency and threshold were automatically generated by

standard curves, as well as the formula for transcriptional levels of HIF-αs.

2.7 Data analysis

One-way ANOVA was used to examine the differential expression of HIF-α

isoforms in every condition. Probability of p<0.05 is considered statistically

significant, and p<0.01 is considered highly significant.

To determine stability of the reference genes, the gene stability measure (M) was

calculated using the geNorm program (http://medgen.ugent.be/genorm/). Each

reference gene underwent a pair-wise variation with all other reference genes as the

standard deviation of the logarithmically transformed expression ratios. Gene with the

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lowest M values had the most stable expression and was chosen as reference gene.

3. RESULTS

3.1 Identification and characterization of Chinese sucker HIF-α genes

These studies revealed that the full-length HIF-1α cDNA of Chinese sucker

consisted of 3890 bp, which contained a 5′-untranslated region (5′-UTR, 217 bp), an

open reading frame (ORF) of 2340 bp, encoding a polypeptide of 780 amino acid

residues, and a 3′-untranslated region (3′-UTR, 1333 bp) including a polyA signal

sequence [GenBank: HQ432957]. The deduced amino acid sequence of Chinese

sucker HIF-1α showed high identities with that of Ctenopharyngodon idella (86%),

Cyprinus carpio (86%), Megalobrama amblycephala (86%), Aspius aspius (84%),

Danio rerio (84%), Gallus gallus (59%), Homo sapiens (57%) and Mus musculus

(55%) (Fig. 1A).

Similarly, we also found that HIF-2α cDNA was 3230 bp in length, which

contained a 5′-UTR (284 bp), an ORF (2346 bp), and a 3′-UTR (600 bp) [GenBank:

HQ432955]. HIF-2α shared amino acid identity with other species orthologous,

Ctenopharyngodon idella (80%), Megalobrama amblycephala (79%), Danio rerio

(77%), Micropogonias undulatus (65%), Ictalurus punctatus (63%), Fundulus

heteroclitus (56%), Gallus gallus (55%), Homo sapiens (54%) and Mus musculus

(52%) (Fig. 1B).

The full-length cDNA of HIF-3α (3374 bp) contained a 5′-UTR (110 bp), an

ORF (1896 bp), and a 3′-UTR (1368 bp) [GenBank: HQ432956]. Chinese sucker

HIF-3α protein shared high amino acid identity with HIF-3α proteins of

Megalobrama amblycephala (84%), Danio rerio (82%), Mustelus canis (68%), Homo

sapiens (45%) and Mus musculus (45%), HIF-4α proteins of Ctenopharyngodon

idella (85%) and Epinephelus coioides (57%) (Fig. 1C).

It was identified that these three proteins contained typical motifs: the basic

helix-loop-helix (bHLH) domain, Per-ARNT-Sim (PAS)-A and -B domains, PAS

associated C-terminal (PAC) domain, oxygen-dependent degradation (ODD) domain,

and N-terminal transactivation (TAD-N) domain (Fig. 1A, 1B and 1C). However,

C-terminal transactivation (TAD-C) domain was not found in HIF-3α protein of

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Chinese sucker (Fig. 1C). Analysis results also showed that a conserved histidine

residue was present in the middle of the PAS-B domain of HIF-1α and terminus of the

bHLH domain of HIF-2α, instead of tyrosine and arginine, respectively, as identified

in other vertebrate species. Valine and alanine also substituted leucine and threonine,

respectively at the upper and middle of PAS-A domain of Chinese sucker HIF-2α, and

a single amino acid was substituted in the middle of the bHLH domain of HIF-3α,

converting aspartic acid to glutamic acid (Fig. 1). In addition, the number of serine

residues at the fish ODD domain (marked by Rahman and Thomas) of HIF-1α

proteins (Rahman and Thomas, 2007) was calculated and varied from 49 to 39 in

Cyprinomorpha (Table 2).

The amino acid sequences of HIF-α from other species based on the closest

homologues by running BLASTP search were taken for phylogenetic analysis.

Multiple protein sequence alignment revealed that Chinese sucker HIF-1α, HIF-2α

and HIF-3α proteins were clustered respectively with the homologues of other

vertebrate species and constructed three distinct clades. The three HIF-α proteins of

Chinese sucker respectively revealed higher identity with the homologues of other

fish, especially Cyprinomorpha than mammals (Fig. 2).

3.2 Tissue-specific expression of HIF-α isoforms

After geNorm analysis, GAPDH was used as the reference gene for the spatial

expression analysis. The qRT-PCR results showed that the HIF-1α, HIF-2α and

HIF-3α mRNA were constitutively expressed in the detected tissues including spleen,

muscle, testis, ovary and liver (Fig. 3). The HIF-1α and HIF-3α mRNA were

significantly higher in liver, testis and ovary than spleen and muscle. HIF-2α mRNA

showed similar expression pattern with HIF-1α and HIF-3α mRNA, whereas was

slightly expressed in the five tissues of Chinese sucker (Fig. 3B).

3.3 Temporal expression of HIF-α isoforms

Temporal expression patterns of three subunits were analyzed during

embryogenesis using qRT-PCR, and EF1α was selected by the geNorm program as

the reference gene. The expression of HIF-1α and HIF-3α showed similar expression

trend with a gradual increase of expression during the development of Chinese sucker,

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and the highest expression was detected at 360 hpf (Fig. 4A). HIF-2α mRNA revealed

similar expression patterns; however, the transcript level was much lower than those

of HIF-1α and HIF-3α (Fig. 4B).

3.4 HIF-α expression after hypoxia and re-oxygenation

ACTB was used as the reference gene for this assay. The HIF-1α and HIF-3α

mRNA level were significantly increased with the gradual decline of oxygen

concentration; however, there was not significant difference among different hypoxia

groups after re-oxygenation (Fig. 5A, 5C). HIF-2α expression also showed a gradual

increase with decline of oxygen concentration after hypoxia, but still remained higher

expression after re-oxygenation, although the gene had an extremely lower expression

level than the other two genes (Fig. 5B).

4. DISCUSSION

The present study was the first report describing cloning and characterization of

three HIF-α cDNAs from an endangered hypoxia-sensitive freshwater teleost,

Chinese sucker. The predicted amino acid sequences of Chinese sucker HIF-1α/2α/3α

were very similar to those of other vertebrates and contained all of the characteristic

motifs of HIF-α proteins, indicating they have similar functions in adaptation to

hypoxia as in other vertebrate species. The three HIF-α genes of Chinese sucker

showed higher similarity of amino acid sequences to that of other fish (59–87% to

HIF-1α, 54–80% to HIF-2α and 68-85% to HIF-3α) than mammalian HIF-α isoforms

(57–59% to HIF-1α, 52–54% to HIF-2α and 45-45% to HIF-3α). The core ODD

domains (Fig. 1) in the Chinese sucker HIF-α proteins had 100% sequence identity to

other vertebrates, suggesting a high degree of evolutionary conservation in the

degradation of all HIF-α proteins (Rahman and Thomas, 2007). Analysis about the

length of HIF-1α sequences showed that fish HIF-1α protein sequences were shorter

than those of air-breathing vertebrates, which it was suggested that air-breathing

vertebrates could have more complex hypoxia regulation mechanism (Terova et al.,

2008). Several unique amino acid sequence substitutions have been suggested to

affect protein conformation and could possibly function to counteract effects of low

temperature on HIF-1α conformation in other vertebrate (Morin and Storey, 2005).

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The phylogenetic tree showed the HIF-3α proteins were clustered with the

HIF-4α proteins, indicating a relatively close evoluntionary relationship between the

two kinds of proteins. The long branch of the Chinese sucker HIF-α proteins in the

divergence of Cyprinomorpha fishes, was suggested that Chinese sucker was evolving

rapidly relative to other Cyprinomorpha fishes (Fig. 2). This could lead to Chinese

sucker quickly responded to environment changes, pollution, eutrophication,

increased water temperature as well as physiological changes of the fish, including

high respiratory rate, anemia, decreased growth and even death. The phylogenetic tree

also revealed both Ctenopharyngodon idella and Megalobrama amblycephala, two

herbivorous fish HIF-αs were clustered in the same small branch, indicating that

Megalobrama amblycephala (suffocation point: 0.5 mg/L) might represent a hypoxia

tolerant fish (Pan et al., 2007).

The enhancement of transcriptional activation is dependent upon the stability and

translocation of specific HIF-α proteins from the cytoplasm to the nucleus, and their

subsequent binding to HREs of target genes (Wenger, 2002). Furthermore, the

stability and DNA-binding activity of rainbow trout HIF-1α (rtHIF-1α) is regulated by

a redox mechanism which is very similar to mammalian HIF-2α (Nikinmaa et al.,

2004). This effect has been attributed to an amino acid difference in the NH2-terminal

portion of the protein: mammalian HIF-2α contains a cysteine at position 25 in the

bHLH domain, which aligns with a serine at position 28 in mammalian HIF-1α

(Nikinmaa and Rees, 2005). On the basis of these results, we calculated the number of

serine residues in the fish ODD domain (Rahman and Thomas, 2007) of HIF-1α

proteins, and presumed that the amount of serine residues increased fish resistance to

hypoxia, with 40 residues representing a threshold value (Table 2). This fish ODD

domain could be considered as a standard to judge hypoxia-tolerant/sensitive fishes.

Serine and cysteine are polar amino acids containing hydroxyl and thiol groups,

respectively. We suppose that due to the existence of large number of hydroxyl groups,

it is difficult for proline hydroxylase enzymes to access the target sites, resulting in

accumulation of HIF-1α.

In this study, expression of three HIF-α mRNAs of Chinese sucker was analyzed

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at different developmental stages. The results showed that the mRNA levels of three

subunits were the lowest at 48 hpf for the pharyngula period, when the fish start to

form organs, and reached highest expression at 360 hpf (Fig. 4), which the fish is

during the post-larval period and the larval begin to food intake (Kimmel et al, 1995;

Wang et al, 2004), therefore, it is suggested that larval fish with first food intake was

sensitive to hypoxia condition, and fishmen should reinforce management to improve

larval fish survival.

HIF-1α, HIF-2α and HIF-3α transcriptions were detected in all the Chinese

sucker tissues examined, spleen, muscle, testis, ovary and liver (Fig. 3). The HIF-αs

mRNA levels were expressed more highly in liver than in other four tissues, which

was consistent with the previous study in Megalobrama amblycephala (Shen et al.,

2010). However, in the present study, the total mRNA expression levels of Chinese

sucker HIF-1α and HIF-3α were higher than HIF-2α. The distinctive expression

pattern of the Chinese sucker HIF-1α/2α/3α mRNA in tissues implied their possible

involvements in different physiological functions.

Previous studies showed that HIF-1α mRNA levels increased significantly under

hypoxia in grass carp (Law et al., 2006) while in other studies, HIF-1α mRNA

remained stable under hypoxic conditions (Soitamo et al., 2001). In Megalobrama

amblycephala, mRNA levels of HIF-2α (but not HIF-1α) were markedly up-regulated

under hypoxia stress (Shen et al., 2010). However, Chinese sucker HIF-2α mRNA

level increased in re-oxygenation treatment groups (Fig. 5), suggesting that HIF-2α

mRNA had different expression pattern during adaption to hypoxia from HIF-1α and

HIF-3α mRNA, though the physiological implications of the differential regulation of

HIF-1α/3α and HIF-2α were unclear. HIF-2α mRNA levels increased under hypoxia

as well as after re-oxygenation, since HIF-2α is altered depending on the

HIF-expressing conditions or genes under hypoxia (Koizume et al., 2008).

Hypoxia was the most critical factor for proper fish health. In the present study,

HIF-2α mRNA had low expression in all experimental conditions, suggesting Chinese

sucker HIF-2α mRNA had similar expression mechanism as in mammals, because

under baseline conditions, HIF-2α mRNA was not detectable but had marked hypoxia

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induction appearance in rats (Wiesener et al., 2003). All the above results suggested

that various responsive mechanisms on HIF-αs might be evolved in different fish

species to accommodate themselves to scare-oxygen environments (Shen et al., 2010).

In conclusion, we identified and characterized three distinct HIF-α isoforms

(HIF-1α, HIF-2α and HIF-3α) in the hypoxic-sensitive Chinese sucker. All isoforms

were expressed and regulated under hypoxic conditions and different developmental

stages. These results suggested that the three isoforms played important roles in

development and hypoxia regulation and could also be helpful when performing

artificial reproduction of this endangered species.

ACKNOWLEDGEMENTS

The research was supported by National Natural Science Foundation of China (3

0901099), International Foundation of Science (A4525-1) and Natural Science Found

ation of Hubei Province of China (2008CBB102).

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Fig.1. Multiple alignment of the deduced amino acid sequences of Chinese sucker (A,

HIF-1α; B, HIF-2α; C, HIF-3α). Domains of typical characterization of HIF-αs were

marked on the alignment. The two conserved proline residues within the ODD

domain were indicated by open arrow, and the asparagine residue (Asn-803 in human)

in TAD-C which controlled HIF-1 binding to CBP/p300 was indicated by arrowheads.

The closed arrow indicated the serine and cysteine residues within bHLH domain and

the asterisks indicated the cord ODD domain. The fish ODD domain in Fig.1.A was

marked according to Rahman and Thomas (2007).

Fig.2. Phylogenetic tree of Chinese sucker HIF-1α, HIF-2α and HIF-3α proteins. The

bootstrap support (ClustalX software) for each branch (1000 replications) was shown.

Asterisks represented the Chinese sucker HIF-α proteins.

Fig.3. mRNA expression patterns of HIF-α isoforms in different tissues of Chinese

sucker (A, HIF-1α and 3α relative mRNA expression; B, HIF-2α relative mRNA

expression). Reference gene: GAPDH. Y-axes represents the mean ± SE (N = 5-6).

Different letters above bars represented significant difference in the expression levels

of HIF-1α, HIF-2α, and HIF-3α at different tissues of Chinese sucker (p<0.05), and

same letters above bars indicated no significant difference.

Fig.4. mRNA expression patterns of HIF-α isoforms in Chinese sucker embryos of

different developmental stages (A, HIF-1α and 3α relative mRNA expression; B,

HIF-2α relative mRNA expression). Reference gene: EF1α. Y-axes represents the

mean ± SE (N = 5-6) and all fishes were exposed under normoxic conditions. The

X-axis represented the developmental stage, hpf, hour post-fertilization. Different

letters above bars represented significant difference in the expression levels of HIF-1α,

HIF-2α, and HIF-3α at different development stages of Chinese sucker (p<0.05), and

same letters above bars indicated no significant difference.

Fig.5. Effects of short-term (24 hours) hypoxia exposure on relative HIF-α mRNA

expression in Chinese sucker (A, B, C: relative mRNA expression of HIF-1α, HIF-2α,

and HIF-3α, respectively). Y-axes represents the mean ± SE (N = 5-6). CTL, control;

DO, dissolved oxygen; R-1, R-2 and R-3, re-oxygenation groups (with 7.2 mg/L of

DO and 24.6±1°C of water temperature in the recirculation water system) with 3.2,

2.8 and 2.2 mg/L DO, respectively. Different letters above bars represented

significant difference in the expression levels of HIF-1α, HIF-2α and HIF-3α in

hypoxia of different concentration and re-oxygenation treatment groups (p<0.05), and

same letters above bars indicated no significant difference.

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Fig. 1

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Megalobrama amblycephala HIF-3αCtenopharyngodon idella HIF-4α

Danio rerio HIF-3αMyxocyprinus asiaticus HIF-3α�

Mustelus canis HIF-3α

Epinephelus coioides HIF-4αHomo sapiens HIF-3α

Mus musculus HIF-3αCtenopharyngodon idella HIF-2α

Megalobrama amblycephala HIF-2α

Danio rerio HIF-2αMyxocyprinus asiaticus HIF-2α�

Ictalurus punctatus HIF-2αMicropogonias undulatus HIF-2α

Fundulus heteroclitus HIF-2α

Homo sapiens HIF-2αMus musculus HIF-2α

Gallus gallus HIF-2αHemiscyllium ocellatum HIF-2α

Homo sapiens HIF-1α

Mus musculus HIF-1αGallus gallus HIF-1α

Acipenser gueldenstaedtii HIF-1αSander lucioperca HIF-1αGymnocephalus cernuus HIF-1α

Perca fluviatilis HIF-1αGasterosteus aculeatus HIF-1α

Micropogonias undulatus HIF-1αThymallus thymallus HIF-1α

Salmo salar HIF-1α

Esox lucius HIF-1αIctalurus punctatus HIF-1α

Myxocyprinus asiaticus HIF-1α�Cyprinus carpio HIF-1α

Gymnocypris przewalskii HIF-1αCarassius carassius HIF-1α

Danio rerio HIF-1αPseudorasbora parva HIF-1αHypophthalmichthys molitrix HIF-1α

Megalobrama amblycephala HIF-1αCtenopharyngodon idella HIF-1α

Aspius aspius HIF-1α0.5

Fig.2

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A B Fig.3

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A B Fig.4

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A

B

C Fig.5

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Table 1 Primers used for PCR and mRNA expression.

Primers name

Primer sequence（5'-3'） Expected product/bp

HIF-1α-UAP-F CATGGGCCTCACACAGTTTG

HIF-1α-UAP-R ATTGACCTCGCAGTCGTAGC 1918

HIF-1α- GSP3'-O GGATTACTGTTTCCAAGTGGAC

HIF-1α- GSP3'-I CTAGAGATGCTGGCTCCATACA

HIF-1α- GSP5'-O CGGCTAAGGAAAGTCTTGCTGT

HIF-1α- GSP5'-I GGTCACAGATGAGCACAAGGT

HIF-1α- qRT-PCR-F TGGACTGGGTGCTTTGCTT

HIF-1α- qRT-PCR-R ATGATGGCGGTCTTGAACTG 232

HIF-2α-UAP-F AGTAARGARACVGAGGTGTT

HIF-2α-UAP-R ACTTSAGGTTSACRGTGCG 473

HIF-2α- GSP3'-O AGTAARGARACVGAGGTGTT

HIF-2α- GSP3'-I ACGCACCGTCAACCTCAAG

HIF-2α- GSP5'-O GTCCATCAGTCTGTCTACCTC

HIF-2α- GSP5'-I GGTCACAGATGAGCACAAGGT

HIF-2α- qRT-PCR-F GCTCAATCCCATCTGCCAAG

HIF-2α- qRT-PCR-R CTGACGCCCTCCCATAGAAG 280

HIF-3α-UAP-F CAARTCHGCYACVTGGAAGGT

HIF-3α-UAP-R GCTGRAARTCRTCATCCAT 969

HIF-3α- GSP3'-O GTATCCCAGAATGCCCTCAG

HIF-3α- GSP3'-I TTCTTCGCCCTCAAACCTG

HIF-3α- GSP5'-O CTGTGTGAAAGTCAGGTCCA

HIF-3α- GSP5'-I TATGGCGTGTGAGGAATGTG

HIF-3α- qRT-PCR-F CACAGTGTGACGGACGTGT

HIF-3α- qRT-PCR-R AAGCCTCCATTCTTAGCCA 184

EF1α-F CTTCTCAGGCTGACTGTGC

EF1α-R CCGCTAGCATTACCCTCC 362

β-actin-F ACCCACACCGTGCCCATCTA

β-actin-R CGGACAATTTCTCTTTCGGCTG 204

GAPDH-F YGCYGGCATCTCCCTCAA

GAPDH-R TCAGCAACACGRTGGCTGTAG 86

Abbreviations: UAP, universal amplified primers; GSP, gene-specific primer; qRT-PCR, quantitative reverse transcription-polymerase chain reaction; F, forward; R, reverse; S, C or G; R, A or G; V, not T; H, not G; Y, C or T. For other abbreviations, see text.

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Table 2 The Number of serine counted at the open box region in Fig. 1A of HIF-1α. Underline means that this species information was coming from references.

Species Status of

taxonomy Number of serine

Hypoxia-sensitive

Suffocation point (mg/L)

Reference

Cyprinus carpio Cyprinomorpha 49 No 0.30-0.34 Qi et al., 2007; Yoshikawa et

al, 1995

Pseudorasbora parva Cyprinomorpha 49 No

Carassius carassius Cyprinomorpha 49 No 0.11-0.13 Qi et al., 2007; Fu et al, 2011

Ctenopharyngodon idella Cyprinomorpha 47 No 0.30-0.51 Qi et al., 2007; Law et al,

2006

Hypophthalmichthys molitrix Cyprinomorpha 47 No 0.30-0.40 Chung et al, 1980;

Danio rerio Cyprinomorpha 47 No Rytkönen et al, 2007

Megalobrama amblycephala Cyprinomorpha 47 ? 0.50 (27 ℃) Oyang et al, 2001

Gymnocypris przewalskii Cyprinomorpha 44 No

Ictalurus punctatus Cyprinomorpha 40 No 0.53 (28 ℃) Chen et al, 2001

Myxocyprinus asiaticus Cyprinomorpha 39 Yes 1.10 (30 ℃) Pan et al., 2007

Acipenser gueldenstaedtii Ganoidomorpha 35 Yes

Esox lucius Clupeomorpha 31 No 1.11 (29 ℃) Qiao et al, 2005; Cameron,

1973

Perca fluviatilis Percomorpha 30 Yes 1.76 (29 ℃) Fan et al, 2009;

Sander lucioperca Percomorpha 29 Yes 1.77 (29 ℃) Saulamo and Thoresson,

2005;

Micropogonias undulatus Percomorpha 29 No Rahman and Thomas, 2007

Mus musculus Rodentia 40

Gallus gallus Gallomorphae 38

Homo sapiens Primates 37

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Fig.1. Multiple alignment of the deduced amino acid sequences of Chinese sucker (A,

HIF-1α; B, HIF-2α; C, HIF-3α). Domains of typical characterization of HIF-αs were

marked on the alignment. The two conserved proline residues within the ODD

domain were indicated by open arrow, and the asparagine residue (Asn-803 in human)

in TAD-C which controlled HIF-1 binding to CBP/p300 was indicated by arrowheads.

The closed arrow indicated the serine and cysteine residues within bHLH domain and

the asterisks indicated the cord ODD domain. The fish ODD domain in Fig.1.A was

marked according to Rahman and Thomas (2007).

Fig.2. Phylogenetic tree of Chinese sucker HIF-1α, HIF-2α and HIF-3α proteins. The

bootstrap support (ClustalX software) for each branch (1000 replications) was shown.

Asterisks represented the Chinese sucker HIF-α proteins.

Fig.3. mRNA expression patterns of HIF-α isoforms in different tissues of Chinese

sucker (A, HIF-1α and 3α relative mRNA expression; B, HIF-2α relative mRNA

expression). Reference gene: GAPDH. Y-axes represents the mean ± SE (N = 5-6).

Different letters above bars represented significant difference in the expression levels

of HIF-1α, HIF-2α, and HIF-3α at different tissues of Chinese sucker (p<0.05), and

same letters above bars indicated no significant difference.

Fig.4. mRNA expression patterns of HIF-α isoforms in Chinese sucker embryos of

different developmental stages (A, HIF-1α and 3α relative mRNA expression; B,

HIF-2α relative mRNA expression). Reference gene: EF1α. Y-axes represents the

mean ± SE (N = 5-6) and all fishes were exposed under normoxic conditions. The

X-axis represented the developmental stage, hpf, hour post-fertilization. Different

letters above bars represented significant difference in the expression levels of HIF-1α,

HIF-2α, and HIF-3α at different development stages of Chinese sucker (p<0.05), and

same letters above bars indicated no significant difference.

Fig.5. Effects of short-term (24 hours) hypoxia exposure on relative HIF-α mRNA

expression in Chinese sucker (A, B, C: relative mRNA expression of HIF-1α, HIF-2α,

and HIF-3α, respectively). Y-axes represents the mean ± SE (N = 5-6). CTL, control;

DO, dissolved oxygen; R-1, R-2 and R-3, re-oxygenation groups (with 7.2 mg/L of

DO and 24.6±1°C of water temperature in the recirculation water system) with 3.2,

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2.8 and 2.2 mg/L DO, respectively. Different letters above bars represented

significant difference in the expression levels of HIF-1α, HIF-2α and HIF-3α in

hypoxia of different concentration and re-oxygenation treatment groups (p<0.05), and

same letters above bars indicated no significant difference.

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