p53 gene cloning and response to hypoxia in the plateau zokor, Myospalax baileyi

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Subterranean rodents are small mammals that spend their entire lives underground in sealed burrows.Severe hypoxia and hypercapnia is common in burrows (Fan & Shi, 1982;Nevo, 1999Nevo, , 2011;;Nevo et al., 2001;Shams et al., 2005).In evolution, subterranean rodents have over the long term developed a series of phenotypic, physiological and genomic strategies to adapt to the harsh environment.Firstly, the eyes of subterranean rodents are degenerated, but they display photosensitivity in their photoperiodic response.These rodents have also developed skeletal muscle that is conducive to digging burrows (Pevet et al., 1984;Nevo, 1999;Fang et al., 2014).Secondly, in these animals the surface area of the pulmonary alveoli and the alveolar capillary volume are enlarged.This change benefits the lungs by increasing the capacity for oxygen uptake (Widmer et al., 1997;Wang et al., 2008a).Thirdly, the increased erythrocyte count and haemoglobin and myoglobin content allows for efficient oxygen transport (Arieli et al., 1986;Wei et al., 2001aWei et al., , b, 2006)).Fourthly, they have reduced the distance for oxygen diffusion to the mitochondria, which ensures efficient oxygen delivery by the structural adaption of tissues (Arieli & Ar, 1981;Edoute et al., 1988).A series of genes have also been shown to be adapted at the molecular level to the underground habitat.This Downloaded from Brill.com09/15/2023 11:47:54AM via free access modification includes a higher expression level for genes like hypoxia-inducible factor 1α (HIF-1α), vascular endothelial growth factor (VEGF), and erythropoietin (EPO) (Shams et al., 2004(Shams et al., , 2005;;Ke & Costa, 2006;Zheng et al., 2011;Wang et al., 2012), and the substitution of amino acids in haemoglobin, haptoglobin and myoglobin (Gurnett et al., 1984;Kleinschmidt et al., 1984Kleinschmidt et al., , 1985;;Ben-Shlomo & Maeda, 1989).In addition, subterranean rodents display exceptional longevity.Heterocephalus glaber and Spalax judaei have maximum lifespans exceeding 30 years (Buffenstein & Jarvis, 2002;Buffenstein, 2008;Kim et al., 2011;Tian et al., 2013) and 21 years (Edrey et al., 2012), respectively.In addition to longevity, reporter assays have shown a striking resistance to cancer.Multi-year observations of large samples did not detect a single incidence of cancer (Buffenstein & Jarvis, 2002;Buffenstein, 2008;Delaney et al., 2013).Previous p53 studies revealed that an arginine (R) was substituted by lysine (K) in residue 172 (Arg174 in human) in Spalax.This substitution is similar to certain mutations found in human tumours; therefore, it implies that Spalax undergoes similar adaptations to hypoxia to those undergone by p53 in human cancers.The mutation reduces the transcription of apoptosis genes, such as Apaf-1, Puma, Pten and Noxa, and enhances the cell cycle arrest and p53 stabilization/homeostasis genes Mdm2, p21 and CycG (Ashur-Fabian et al., 2004;Avivi et al., 2007).Genome and transcriptome analysis of the p53 signal pathway has shown that eight p53 target genes are differentially regulated by hypoxia in Spalax and rat (Fang et al., 2014).
The plateau zokor, Myospalax baileyi, is a specialized subterranean rodent living on the Qinghai-Tibet Plateau.This species inhabits sealed burrows at an altitude of 2800 to 4200 m (Wang et al., 1979;Fan & Shi, 1982).Like other subterranean rodents, the plateau zokor has strategies to adapt to hypoxic and hypercapnic underground environments.In M. baileyi, the p53 expression level was significantly decreased in the liver when mimicking oxygen levels at an altitude of 7000 m (8.0%O 2 ) for 8 h compared to an altitude of 3352 m (Wang et al., 2013;Zhao et al., 2013).Comparison of p53 sequence data and functional studies found that a mutation in codon 104 from serine (S) to asparagine (N) activated the apoptosis-related genes IGFBP3, Apaf-1 and Bax, but had no significant influence on cell cycle arrest genes, including p21 and Mdm2 (Zhao et al., 2013).
To date, the sequence characteristics of p53 and the response to hypoxia in the plateau zokor are not understood well.In this study, we clone the coding DNA sequence (CDS) of p53, analyse and characterize p53 in other rodents, and measure p53's expression differences between high and low altitudes in plateau zokor tissues using quantitative real-time PCR.

Animals and sample collection
Plateau zokors were live-trapped in the Zongjiagou region in Huangyuan country, Qinghai Province, China.They were divided into two groups: (1) high altitude (3300 m group), which were zokors collected from the Zongjiagou region in Huangyuan country at an altitude of 3300 m with an oxygen content of 193.4 g/m 3 ; (2) low altitude (2260 m), which were zokors collected from Zongjiagou in the Huangyuan region and raised in Xining City, Qinghai Province, China at an altitude of 2260 m for eight days with an oxygen content of 226.7 g/m 3 .Sprague-Dawley (SD) rats were bought from Lanzhou, Gansu Province, China.Rats were divided into two groups: (1) high altitude (3300 m Group), raised in Zongjiagou region in Huangyuan country for eight days; (2) low altitude (2260 m), raised in Xining City at an altitude of 2260 m for eight days.The sample size was 8 for each of the above groups.All animals were anaesthetized with sodium pentobarbital (5%) and sacrificed using cervical dislocation immediately before dissection.Liver, lung, stomach, intestine and skeletal muscles were rapidly removed and quickly frozen in liquid nitrogen.All procedures involved in the handling and care of animals were in accordance with the China Practice for the Care and Use of Laboratory Animals and approved by the China Zoological Society (permit number: GB 14923-2010).

Selection pressure analysis
Sequence alignments were obtained using ClustalX1.81(Jeanmougin et al., 1998), and format conversion was performed with MEGA 7.0 software (Kumar et al., 2016).The maximum-likelihood method was used to estimate the positive sites using branch-site models in codeml from the PAML package based on the mammalian species tree.The significance of the likelihood ratio test (LRT) statistic was determined using a χ 2 distribution, and the positively selected sites were identified using Bayes Empirical Bayes (BEB) analysis (Zhang et al., 2005;Yang, 2007).

Convergent evolution analysis
Ten mammalian species were used to infer the convergent sites.We constructed ancestral p53 amino acid sequences using the Ancestors programme in the MEGA 7.0 software (Tamura et al., 2011).Ancestral inferences appeared reliable because mean posterior probabilities for the entire protein exceeded 99% for all nodes.We attempted to identify convergent changes by comparing ancestral and extant p53 protein sequences.Next, we calculated the probability that the observed convergent sites exceeded the expectation due to random chance using the Jones-Taylor-Thornton (JTT) and Poisson models (Zhang & Kumar, 1997).

RNA extraction and quantification of p53 mRNA using qRT-PCR
Total RNA was isolated from the liver, lung, stomach, intestine and skeletal muscle using TRIzol reagent (Invitrogen Corp.).The RNA concentration and purity were assessed using UV spectrophotometry (1.8 < A260/A280 < 2.0).RNA integrity was checked using electrophoresis.A reverse transcription reaction was performed starting with 3.8 μg of total RNA and the First Strand cDNA Synthesis kit (Tiangen).

Statistical analysis
Statistical analyses were performed using SAS 8.2 software.The expression levels of p53 between high and low altitudes were compared using Student's t-test.A value of P < 0.05 was considered to be statistically significant.

Comparison and phylogenetic tree of Myospalax baileyi p53 sequences
The p53 CDS was cloned and sequenced from M. baileyi.The complete coding sequence was 1179 bp, and the deduced protein consisted of 392 amino acid residues.Alignment by ClustalW2 found that the M. baileyi p53 protein is identical to 98.98%, 95.41%, 87.02%, 82.99% with E. cansus, S. judaei, H. glaber and R. norvegicus p53 proteins, respectively.The phylogenetic tree demonstrates that plateau zokor had the highest homology with E. cansus, followed by S. judaei (fig.1).
Multiple sequence alignment analysis showed that the mutation of codon 309 from glycine (G) to aspartic acid (D) in M. baileyi is unique to the plateau zokor compared to E. cansus, S. judaei and H. glaber p53.

Positive selection sites analysis
To detect the positively selected sites of p53 in plateau zokor, we treated the plateau zokor branch as the foreground branch.Using the LRT based on the branch-site model for gene p53, we observed that there are no positive selection sites in plateau zokor p53 (table 1).

Convergent evolution analysis
To detect the sites that are convergent in subterranean rodents in response to hypoxic environments, we attempted to identify convergent changes by comparing ancestral and extant p53 protein sequences.We observed that two sites experienced convergent evolution in subterranean rodents.The 78th position of p53 was proline (P) on the ancestral branch.However, it was replaced by serine (S) on the subterranean rodent branch of M. baileyi and S. judaei.This change was caused by a C to T transition at the first position of the codon at site 78.In addition, the 84th position was alanine (A) on the ancestral branch, and it was replaced by proline (P) in the subterranean rodent branch (fig.3).According to a statistical test, the 78th and 84th positions were likely convergent sites, rather than chance substitution (P < 0.01).
The sequence alignment showed that the amino acid sequences of the plateau zokor HIF-1α ODD domain were 92.61% identical to those in rat.Compared with plateau zokor, rat had 12 mutation sites within the ODD domain: Asp440Asn, Asp441Glu, Thr444Asn, Asn465Ser, Pro480Ser, Asn481Ser, Ala482Pro, Glu486Gly, Thr552Ala, Ser586Asn, Asn589Ser, Ala599Val.The motif 397 to 436 of plateau zokor is 100% identical with that of rat, and the other motif (507 to 579) of plateau zokor is 98.63% identical with that of rat.Compared with plateau zokor, rat had one mutation sites within the motif 507 to 579: Thr552Ala (fig.5).

Discussion
The plateau zokor is a specialized rat on the Qinghai-Tibet Plateau.This species was reported to have adaptations in a series of genes involved in hypoxia response, including p53 (Fan & Shi, 1982;Zheng et al., 2011;Wang et al., 2012;Zhao et al.,   2013).According to our phylogenetic gene tree, the plateau zokor is placed with rodentia and shares many homologies with E. cansus.These two species are grouped on a small branch with S. judaei as sister group.In an analysis of reporter assays, E. cansus and S. judaei were grouped on a small branch subtended by plateau zokor (Zhao et al., 2013).These species all grouped into a large branch with R. norvegicus.Heterocephalus glaber belongs to the family of subterranean rodents; however, this species was far removed from other subterranean rodents.Studies of the phylogenetic relationship based on the genome and transcriptome analysis found that the plateau zokor diverged from the rat approximately 52 million years ago (Shao et al., 2015).A molecular phylogenetic tree based on the p53 and cytochrome b protein sequences reported previously showed that plateau zokor and E. cansus were grouped on a branch with S. judaei as sister group (Zhao et al., 2013).Eospalax cansus is a typical subterranean rodent, living on the Loess Plateau in China (Li, 1989).Severe hypoxia and hypercapnia are common characteristics of burrows (Fan & Shi, 1982;Nevo, 1999).During long-term evolution, M. baileyi and E. cansus developed a series of strategies to evolve adaptively and convergently in the hypoxic environment.They have a higher oxygen utilization rate in their tissues to adapt to hypoxic conditions, with a high oxygen pressure and saturation in arterial blood, and a low oxygen pressure and saturation in venous blood; the number of red blood cells, and the hemoglobin and myoglobin concentrations are also significantly higher.In addition, they have substitutions of amino acids in hemoglobin and myoglobin to increase the affinity to oxygen, and have enhanced their capacity to adapt to hypoxic environments (Wei et al., 2006a, b;Yang et al., 2006;Wang et al., 2008b).Thus, our results are consistent with previously published reports and imply that the molecular evolution of the p53 gene in plateau zokor is similar to that in E. cansus, and this also indicates that subterranean rodents have evolved adaptively and convergently to hypoxia.Acting as a tumour suppressor gene, p53 has mutations that are also observed in approximately 50% of human cancers (Hernandez et al., 2003;Klein, 2004).Several mutations were reported at the mutation 'hotspots' at residues R175, G245, R248, R249, R273, and R282.The mutation occurs in more than 90% of human cancers (Bullock & Fersht, 2001;Freed-Pastor & Prives, 2012).Compared to the amino acid sequence of p53 protein in E. cansus, S. judaei and H. glaber, we found that in plateau zokor codon 309 is mutated from glycine (G) to aspartate (D); this mutation is unique for plateau zokor.We predict that the Gly309Asp mutation may play a significant role in the adaptation of the plateau zokor to its extreme environment, but this warrants further investigation.We also observed that the subterranean rodents (M.baileyi, E. cansus, S. judaei, H. glaber) had seven mutation sites in common compared to human p53.There were two mutation sites in common with the human hotspots: Gln246Arg (248 in human) and His271Arg (273 in human).These two mutations are reported to be the most frequently altered residues in p53 protein.In p53, these mutations cause a loss of wild-type p53 tumour suppressor activity and promote tumourigenesis, resulting in cell cycle G1 arrest in cells (Cho et al., 1994;Hollstein et al., 1994;Vousden & Lu, 2002;Menendez et al., 2006).Using site-directed mutagenesis to study the function of p53, the Arg174Lys amino acid substitution in Spalax was found to reduce transcription of the apoptosis genes and enhance cell cycle arrest and p53 stabilization/homeostasis genes (Ashur-Fabian et al., 2004;Avivi et al., 2007).The Ser104Asp amino acid substitution in plateau zokor activated the apoptosis genes and had no significant influence on cell cycle arrest genes, including p21 and Mdm2 (Zhao et al., 2013).The mutations in different subterranean rodents were species-specific and could be attributed to their specific microenvironment.Therefore, we propose that the variations do not promote tumourigenesis in subterranean rodents, but enhance their adaptability to the hypoxic environment.
Subterranean rodents live in dark, hypoxic and hypercapnic underground environments (Fan & Shi, 1982;Nevo, 1999Nevo, , 2011;;Nevo et al., 2001;Shams et al., 2005), and p53 plays an important role in hypoxic environments.Genome and transcriptome data have shown that p53 is not positively selected in subterranean rodents (Kim et al., 2011;Fang et al., 2014), and previous studies have shown that plateau zokor p53 does not have positively selected sites (Zhao et al., 2013).In this study, the LRT based on branch-site model A for gene p53 indicated there are no positive selection sites in plateau zokor.In addition, we observed that p53 amino acid sequences have two convergent sites in M. baileyi and S. judaei.We propose that the two substitution sites might enhance subterranean rodent adaptation to hypoxic environments.
Studies have demonstrated that the expression level of p53 is related to the degree of hypoxia.Under normal conditions, wild-type p53 is expressed at low levels in most cells and has a short half-life.In contrast, its level increases and stabilizes in response to hypoxia (Giaccia & Kastan, 1998;Koumenis et al., 2001).However, later study showed that hypoxic induction of p53 requires the concomitant induction of HIF-1α, demonstrating a direct interaction between p53 and HIF-1α (Hansson et al., 2002;Fels & Koumenis, 2005;Sánchez-Puig et al., 2005).The oxygen-dependent degradation (ODD) domain lies within amino acids 401 to 603 in human HIF-1α (Huang et al., 1998).In the ODD domain, the two proline residues (P402 and P564), play a pivotal role in regulating HIF-1 activity and expression by the ubiquitin-proteasome pathway, and act as switches for the oxygen-dependent regulation of HIF-1α (Huang et al., 1998;Masson et al., 2001).Studies have identified two p53-binding sequence motifs in human HIF-1α; one motif (397 to 436) is adjacent to the ODD domain, the other motif (507 to 579) is coincident with the ODD domain of HIF-1α (Hansson et al., 2002).Under normoxic conditions, the expression levels of p53 and HIF-1α are low.When the oxygen concentration decreases, HIF-1α stabilizes, and the p53 level remains low or lower than before.Under severe hypoxia or anoxic conditions, p53 is accumulated and stabilizes and binds to the ODD domain of HIF-1α (Fels & Koumenis, 2005;Sánchez-Puig et al., 2005).When comparing oxygen levels at a mimicked altitude of 7000 m (8.0%O 2 ) for 8 h with an altitude of 3352 m, the p53 expression level was significantly decreased in plateau zokor liver and significantly increased in rat.However, there was no significant difference between 10.8% O 2 and 16.0% O 2 (Wang et al., 2013;Zhao et al., 2013).In this study, we found that the relative expression levels of p53 in the tissues of liver, lung, stomach, intestine and skeletal muscle of the plateau zokor are increased significantly from 2260 m to 3300 m, but there was no significant difference under the same conditions in rat.Thus, the expression level of p53 showed different patterns among plateau zokor and rat in various tissues.This could be due to different regulatory mechanisms in the two species that are adaptive for the hypoxic environment.Doing a multiple sequence alignment of the two p53binding sequence motifs in HIF-1α, our results show that plateau zokor and rat are highly homologous to human, with the two proline residues (P402 and P564) not mutated in plateau zokor or rat.Compared with plateau zokor, rat had one mutation site within the motif 507 to 579: a polar amino acid (Thr, T) was substituted by a non-polar amino acid (Ala, A) in residue 552 in rat.The results imply that the substitution in the motif 507 to 579 decreases the affinity of HIF-1α to p53-binding sites in rat, and the p53-binding sites in HIF-1α of plateau zokor are stronger than that of rat.

Figure 1 .
Figure 1.Phylogenetic tree based on the amino acid sequence of p53 from Myospalax baileyi together with 36 other species.The p53 sequences of the other species are from GenBank.Neighbour-joining method with 1000 bootstrap replicates was used.Bootstrap values for the internal nodes are given.

Figure 2 .
Figure 2. Amino acid sequence alignment of p53 protein with Homo sapiens, Myospalax baileyi, Eospalax cansus, Spalax judaei, Heterocephalus glaber and Rattus norvegicus.Amino acids with more than 90% identity are shown in red, 50%-90% identity in blue and the others in black.Arrows indicate that Myospalax baileyi has a mutation at residue 309 in the black box.

Figure 3 .
Figure 3. Evolution of convergent sites in p53 sequences.Amino acids and codons of sites 78 and 84 are shown.Amino acids in Myospalax baileyi and Spalax judaei are highlighted in red, and in the other species in blue.

Figure 5 .
Figure 5. Amino acid sequence alignment of HIF-1α ODD domain in Homo sapiens, Myospalax baileyi and Rattus norvegicus.Amino acids with more than 90% identity are highlighted in red, 50-90% identity in blue and the others in black.The arrow indicates residue 552.

Table 1 .
Likelihood values, parameter estimates, and sites under positive selection for p53 in plateau zokor.