Abstract
Hypoxia-inducible factor (HIF), a critical member of the basic-helix-loop-helix (bHLH)-containing Per-Arnt-Sim (PAS) protein family, is a master transcription factor involved in maintaining oxygen homeostasis. In the present study, we isolated and characterized a novel bHLH-PAS family member, CgHIFα-like gene, from the Pacific oyster Crassostrea gigas, and determined its importance during hypoxia stress. The 3020-bp CgHIFα-like cDNA encoded a protein of 888 amino acids. The predicted CgHIFα-like amino acid sequence was conserved in the N-terminal bHLH, PAS, and PAC domains (but not in the C-terminal domain) and was most closely related to the HIF family in the bHLH-PAS protein phylogenic tree. Similar to the mammalian HIF-1α, CgHIFα-like could be expressed as four mRNA isoforms containing alternative 5′-untranslated regions and different translation initiation codons. At the mRNA level, these isoforms were expressed in a tissue-specific manner and showed increased transcription to varying degrees under hypoxic conditions. Additionally, the western blot analysis demonstrated that CgHIFα-like was induced by hypoxia. Electrophoretic mobility shift assay indicated that CgHIFα-like could bind to the hypoxia responsive element (HRE), whereas dual-luciferase reporter analysis demonstrated that CgHIFα-like could transactivate the reporter gene containing the HREs. In addition to CgHIFα-like, we identified CgARNT from the C. gigas, analyzed its expression pattern, and confirmed its interaction with CgHIFα-like using a yeast two-hybrid assay. In conclusion, this is the first report on the cloning and characterization of a novel hypoxia transcription factor in mollusks, which could accumulate under hypoxia and regulate hypoxia related gene expression by binding to HRE and dimerizing with CgARNT. As only one member of HIF has been identified in invertebrates to date, our results provide new insights into the unique mechanisms of hypoxia tolerance in mollusks.
Highlights
Oxygen is critical for energy generation by aerobic respiration, its deprivation being lethal for many animals
To functionally analyze the novel C. gigas HIFα-like (CgHIFα-like) protein, we examined whether: (i) CgHIFα-like could be induced by hypoxia treatment both at the mRNA and protein levels, (ii) the CgHIFα-like gene product could interact with CgARNT, and (iii) CgHIFα-like protein was capable of binding to hypoxia response element (HRE) and possessed HRE-dependent transcriptional activity
BHLH-PAS proteins were classified into two groups: Class I included the aryl hydrocarbon receptor (Ahr), neuronal PAS domain protein (NPAS), hypoxia inducible factors (HIF-1α/2α/3α), single-minded proteins (Sim1/2), and trachealess (Trh) protein; Class II consisted of the ubiquitous aryl hydrocarbon receptor nuclear translocator (ARNT) and the circadian rhythm related proteins, brain-muscle-ARNT-like transcription factor (Bmal) and circadian locomotor output cycles protein kaput (Clock; Fig 1)
Summary
Oxygen is critical for energy generation by aerobic respiration, its deprivation being lethal for many animals. Oysters have movement disability; they are, more susceptible to such hypoxia stress [4,5,6] Their survival for up to 47.8 days under desiccated conditions at 4°C indicates that oysters have evolved powerful mechanisms for tolerance to varying degree of hypoxia [2, 7,8,9,10]. Oysters first use physiological mechanisms to stay aerobic, and an increase in ventilation of respiratory surfaces (mainly gills) is a common response to maintain their rate of oxygen consumption. This mechanism remains functional until the hypoxia threshold is reached (around 2mg/L O2), the oxygen tension below which is referred to as “severe hypoxia” [11, 12]. Upon exposure to severe hypoxia, anaerobiosis commences and oysters present biochemical adaptions including (1) extensive consumption of substrates for energy provision during the beginning of severe hypoxia (mainly phosphagens, aspartate and glycogen), (2) optimized fermentation with higher ATP output and the formation of non-acidic or volatile end-products such as succinate, propionate, and acetate [13,14,15], (3) strong depression of metabolic rate [16, 17], and (4) high expression of selective hypoxia-related genes [18, 19]
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