Characterization of CgHIFα-Like, a Novel bHLH-PAS Transcription Factor Family Member, and Its Role under Hypoxia Stress in the Pacific Oyster Crassostrea gigas.
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.
Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao, China.|University of Chinese Academy of Sciences, Beijing, China.|Laboratory for Marine Biology and Biotechnology, Qingdao National Laboratory for Marine Science and Technology, Qingdao, China.|National & Local Joint Engineering Laboratory of Ecological Mariculture, Institute of Oceanology, Chinese Academy of Sciences, Qingdao, China.
Ting
T
Wang
Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao, China.|Laboratory for Marine Fisheries and Aquaculture, Qingdao National Laboratory for Marine Science and Technology, Qingdao, Shandong, China.|National & Local Joint Engineering Laboratory of Ecological Mariculture, Institute of Oceanology, Chinese Academy of Sciences, Qingdao, China.
Jie
J
Meng
Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao, China.|Laboratory for Marine Fisheries and Aquaculture, Qingdao National Laboratory for Marine Science and Technology, Qingdao, Shandong, China.|National & Local Joint Engineering Laboratory of Ecological Mariculture, Institute of Oceanology, Chinese Academy of Sciences, Qingdao, China.
Li
L
Li
Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao, China.|Laboratory for Marine Biology and Biotechnology, Qingdao National Laboratory for Marine Science and Technology, Qingdao, China.|National & Local Joint Engineering Laboratory of Ecological Mariculture, Institute of Oceanology, Chinese Academy of Sciences, Qingdao, China.
Guofan
G
Zhang
D000818:Animals; D051792:Basic Helix-Loop-Helix Proteins / Q000235:genetics*; D003001:Cloning, Molecular / Q000379:methods; D049896:Crassostrea / Q000235:genetics*; D018076:DNA, Complementary / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics; D015870:Gene Expression / Q000235:genetics; D017930:Genes, Reporter / Q000235:genetics; D000860:Hypoxia / Q000235:genetics*; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000235:genetics; D020033:Protein Isoforms / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D013312:Stress, Physiological / Q000235:genetics*
D051792:Basic Helix-Loop-Helix Proteins; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D020033:Protein Isoforms; D012333:RNA, Messenger