Yak response to high-altitude hypoxic stress by altering mRNA expression and DNA methylation of hypoxia-inducible factors.

Hypoxia-inducible factors (HIFs) are oxygen-dependent transcriptional activators, which play crucial roles in tumor angiogenesis and mammalian development, and regulate the transcription of genes involved in oxygen homeostasis in response to hypoxia. However, information on HIF-1α and HIF-2α in yak (Bos grunniens) is scarce. The complete coding region of yak HIF-2α was cloned, its mRNA expression in several tissues were determined, and the expression levels were compared with those of closely related low-altitude cattle (Bos taurus), and the methylation status of promoter regions were analyzed to better understand the roles of HIF-1α and HIF-2α in domesticated yak. The yak HIF-2α cDNA was cloned and sequenced in the present work reveals the evolutionary conservation through multiple sequence alignment, although 15 bases changed, resulting in 8 amino acid substitutions in the translated proteins in cattle. The tissue-specific expression results showed that HIF-1α is ubiquitously expressed, whereas HIF-2α expression is limited to endothelial tissues (kidney, heart, lung, spleen, and liver) and blood in yak. Both HIF-1α and HIF-2α expressions were higher in yak tissues than in cattle. The HIF-1α expression level is much higher in yak than cattle in these organs, except for the lung (P < 0.05), but the HIF-2α gene is significantly different in the heart, spleen, and kidney (P < 0.05). Furthermore, the methylation levels in the 5' flanking regulatory regions of HIF-1α and HIF-2α in yak kidney were significantly decreased than cattle counterparts (P < 0.05). Identifying these genes and the comparison of different expressions facilitates the understanding of the biological high-altitude hypoxic stress response mechanism and may assist current medical research to understand hypoxia-related diseases.

Xiong X ，Fu M ，Lan D ，Li J ，Zi X ，Zhong J ... -  《-》

Adaptive response of reproduction to high-altitude hypoxic stress by altering mRNA expression of hypoxia-inducible factors in female yaks (Bos grunniens).

Hypoxia-inducible factors (HIFs) are oxygen-dependent transcriptional activators, but there is little information about their role in yak () reproduction. The present study, for the first time, investigated the adaptive mechanism of yak reproduction to high-altitude hypoxic stress by comparing the expression of HIF mRNAs between female yaks at high-altitude and cattle at low-altitude. Hypothalamus, anterior pituitary, oviduct, ovary and uterus tissue samples were collected from five adult female yaks and cattle. mRNA expression was determined by the quantitative real-time polymerase chain reaction. Both and were expressed in all five tissues examined from both species, albeit at different levels. In yaks, the highest mRNA levels of and occurred in the oviduct and anterior pituitary, respectively. Both and mRNA levels were higher in yaks than in cattle ( < 0.01). These data provide evidence that adaptation of reproduction to hypoxic conditions is associated with a greater expression of and in the reproductive axis of female yaks than cattle.

He XD ，Xia Y ，Jige MT ，Wang H ，Zi XD ... -  《-》

cDNA cloning, gene organization and variant specific expression of HIF-1 alpha in high altitude yak (Bos grunniens).

Hypoxia-inducible factor 1 (HIF-1) is a heterodimeric basic-helix-loop-helix-PER-ARNT-SIM (bHLH-PAS) transcription factor consisting of HIF-1alpha and HIF-1beta subunits. HIF-1alpha is the oxygen-regulated subunit of HIF-1, which regulates the transcription of genes involved in oxygen homeostasis in response to hypoxia. Yak (Bos grunniens), a mammal native to high altitude (HA) region ( approximately 3500-5500 m), has successfully adapted over many generations to the chronic hypoxia of HA. In the present work, cDNA encoding HIF-1alpha has been cloned from the blood of yak. Tissue specific expression of the mRNA was analyzed in blood, heart, lung, liver and kidney by RT-PCR with primers from three different regions of cDNA. The HIF-1alpha expression was liver and blood specific. The HIF-1alpha mRNA contains 823 bp long 3'UTR that is AU-rich and contains ten AUUUA pentamers and two overlapping copies of the nonamer UUAUUUAUUUAUU. Three potential microRNAs, hsa-miR-107/mmu-miR-107/rno-miR-107, hsa-miR-18b and hsa-miR-135a/mmu-miR-135a/rno-miR-135a, targeting 3'UTR of yak HIF-1alpha, were identified by using target prediction software. The CDS encodes for 823 residues of amino acids and showed 99%, 95%, 92%, 90% and 90% similarity to domestic cattle, human, plateau pika, mouse and rat HIF-1alpha, respectively. HIF-1alpha cDNA, cloned and sequenced in the present work has revealed the evolutionary conservation through multiple sequence alignment. Liver and blood specific stability of HIF-1alpha mRNA appears miR-107 regulated.

Dolt KS ，Mishra MK ，Karar J ，Baig MA ，Ahmed Z ，Pasha MA ... -  《GENE》

HIF-1alpha and -2alpha genes in a hypoxia-sensitive teleost species Megalobrama amblycephala: cDNA cloning, expression and different responses to hypoxia.

Transcriptional responses to hypoxia are primarily mediated by hypoxia-inducible factor (HIF) alpha subunits, i.e. HIF-1alpha, -2alpha and -3alpha. In fish, the molecular constructions, expression characteristics and hypoxic regulation of HIF-alpha subunits are still not well known. In this study, we identified the HIF-1alpha and HIF-2alpha full-length cDNAs in a hypoxia-sensitive fish species Wuchang bream, Megalobrama amblycephala. The whole length of HIF-1alpha cDNA was 3,815bp, consisting of an open reading frame (ORF) encoding 774 amino acid (aa) residues. The HIF-2alpha cDNA totaled 3,121-bp including an 835-aa ORF. The Wuchang bream HIF-1alpha and HIF-2alpha subunits were structurally similar in the DNA-binding and dimerization domains, but differed in the transactivation domain. In adult fish, both HIF-1alpha and HIF-2alpha mRNAs were detected in different tissues under normoxic conditions. HIF-1alpha mRNA was highly expressed in the liver, gill and testis, whereas HIF-2alpha mRNA was abundantly expressed in most of the Wuchang bream tissues. Both HIF-1alpha and HIF-2alpha mRNAs were detected in all stages of embryogenesis and expressed in a ubiquitous pattern. In contrast to HIF-1alpha, the mRNA levels of HIF-2alpha fluctuated in different stages, with higher expression in the zygote, 8-, 28-, 48- and 52-hr post fertilization (hpf) embryos. During hypoxic treatment, the mRNA levels of HIF-2alpha were significantly (p<0.01) up-regulated to 910% in the liver and 320% in the kidney, whereas no significant changes of HIF-1alpha mRNA were observed in the corresponding tissues. These results suggest that the Wuchang bream HIF-1alpha and -2alpha would be involved in different physiological functions under normoxia and hypoxia situations.

Shen RJ ，Jiang XY ，Pu JW ，Zou SM ... -  《-》

Molecular cloning, characterization and expression of two hypoxia-inducible factor alpha subunits, HIF-1alpha and HIF-2alpha, in a hypoxia-tolerant marine teleost, Atlantic croaker (Micropogonias undulatus).

Alteration of gene expression is a crucial component of adaptation by animals to hypoxic conditions and is mediated by specific transcription factors, hypoxia-inducible factors (HIFs), which are composed of alpha and beta subunits. In this study, we report the cloning and characterization of two HIF-alpha subunits, HIF-1alpha and HIF-2alpha, and their expression in various tissues of a hypoxia-tolerant marine teleost, Atlantic croaker (Micropogonias undulatus). The full-length croaker HIF-1alpha (2805 bp) and HIF-2alpha (3205 bp) cDNAs contain open reading frames encoding proteins with 720 and 847 amino acids, respectively, which are highly homologous to the HIF-1alpha and HIF-2alpha proteins of other non-mammalian species. Croaker HIF-1 alpha shares only 43% sequence identity with the croaker HIF-2alpha subunit. However, the basic helix-loop-helix/Per-ARNT-Sim regions appear to be relatively well conserved between the two proteins, with identities of 75-83%. The core oxygen-dependent degradation domain regions in croaker HIFs are well conserved, suggesting a similar mechanism of HIF degradation to that in other vertebrate species. Northern blot analysis showed that croaker HIF-1alpha and HIF-2alpha mRNAs (transcript sizes 3.0-3.8 kb) are highly expressed in the brain, heart, liver, and gonads under hypoxic conditions, whereas muscle tissues show lower levels of expression. Short-term (1.7 mg/L dissolved oxygen, DO for 3 days to 1 week) and long-term (1.7, 2.7 and 3.7 mg/L DO for 3 weeks) hypoxia exposure caused significant increases in HIF-1alpha and HIF-2alpha mRNA expression in croaker ovaries compared to mRNA levels in fish held in normoxic conditions (DO: 6.5 mg/L). However, HIF transcript levels in hypoxia-exposed fish had returned to control values 24 h after the DO in the tanks was restored to normoxic levels. The results suggest that the upregulation of both HIF-1alpha and HIF-2alpha subunits at the transcriptional level is an important component of adaptation of croaker to chronic hypoxia and HIF-alphas are potentially useful molecular indicators of

Rahman MS ，Thomas P 《GENE》