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

Abstract

Hypoxia-inducible factor 1 (HIF-1) is a heterodimeric basic-helix-loop-helix-PER-ARNT-SIM (bHLH-PAS) transcription factor consisting of HIF-1α and HIF-1β subunits. HIF-1α 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 (∼ 3500–5500 m), has successfully adapted over many generations to the chronic hypoxia of HA. In the present work, cDNA encoding HIF-1α 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-1α expression was liver and blood specific. The HIF-1α 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-1α, 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-1α, respectively. HIF-1α cDNA, cloned and sequenced in the present work has revealed the evolutionary conservation through multiple sequence alignment. Liver and blood specific stability of HIF-1α mRNA appears miR-107 regulated.

Introduction

Organisms vary widely in their tolerance to high altitude (HA) hypoxia and certain vertebrate species have evolved capabilities to survive under limited supplies of oxygen (Hochachka and Lutz, 2001). One such vertebrate species, Himalayan yak (Bos grunniens) has successfully adapted over many generations to the chronic hypoxia of HA (∼ 3500–5500 m) despite belonging to the genus Bos; contrary to this, the closely related domestic bovine members, when exposed to similar conditions, are known for their high hypoxic pulmonary vascular response (HPVR) and the development of severe pulmonary hypertension (brisket disease) (Cruz et al., 1980, Weir et al., 1974, Will et al., 1975). Domestic cattle (Bos taurus) transported from sea level have a higher (40–50%) incidence of brisket disease, whereas, the incidence is as low as 2% in cattle native to HA. Yak and few mammalian species indigenous to high altitude have adapted to hypobaric hypoxia by elimination of the hypoxic vasoconstrictive drive (Anand et al., 1986). This finding is, although, strongly suggestive of genetic selection to resist the disease (Anand et al., 1986, Grover, 1965), however, the factor(s) responsible for the extraordinary ability of the yak to survive with endurance at HA are yet to be established.

In acute HA exposure, current evidence from diverse studies of both human and animal models indicates that responses to hypoxia are initiated by several oxygen sensing and signal transduction pathways (Rhodes, 2005). Adaptive responses to hypoxia at molecular level involve transcriptional activation of target genes by Hypoxia-Inducible Factor-1 (HIF-1). HIF-1 functions as a global regulator of O₂ homeostasis facilitating both O₂ delivery and adaptation to O₂ deprivation (Semenza, 1999). HIF-1, a basic-helix-loop-helix-PER-ARNT-SIM (bHLH-PAS) transcription factor, consists of HIF-1α and HIF-1β subunits (Wang et al., 1995). Decreased oxygen concentration increases HIF-1α stability through post-translational modifications (Huang et al., 1998), which induces several target genes involved in erythropoiesis, glycolysis, glucose transport, vasodilatation and angiogenesis (Carmeliet et al., 1998, Iyer et al., 1998, Palmer et al., 1998, Pugh and Ratcliffe, 2003, Ratcliffe, 2002, Ryan et al., 1998). HIF-1α thus functions as a key regulator of O₂ homeostasis, and as a consequence plays an essential role in cellular responses to hypoxia. Abnormal expression of HIF-1α is related to various hypoxia associated diseases (Covello and Simon, 2004).

Yak, because of their adaptation to HA environment along with high endurance for work, ought to be a model animal for understanding the molecular basis of adaptation. As HIF-1α is an essential mediator of adaptive response to hypoxia, we decided to clone and sequence HIF-1α cDNA from yak. The aim of the present study was to characterize yak HIF-1α cDNA, find sequence variations compared to other mammals and study variant specific expression of the HIF-1α mRNA in blood, heart, lung, liver and kidney. The CDS was found to be conserved through evolution and the mRNA variant expression was liver and blood specific out of the five tissues analyzed. Additionally microRNAs (miRNAs), a class of non-coding RNA genes that post-transcriptionally regulate gene expression and play vital role in tissue specification or cell lineage decisions (Bartel, 2004, Lagos-Quintana et al., 2002), were exhaustively searched through literature and software tools to help explain how the mRNA was so specifically expressed in the tissues.