The Molecular Mechanism Of Hypoxia Signal Pathway In The Pacific Oyster(Crassostrea Gigas)

Oxygen deprivation is lethal for most animals.Thus,oxygen homeostasis must be strictly regulated.Marine mollusks,such as the Pacific oyster Crassostrea gigas,suffered from oxygen limitation in intertidal zones.During low tide,the oysters are daily exposed to desiccation,potentially leading to hypoxia.Hypoxia can also occur in the seawaters,especially due to the increasing water eutrophication and global warming.As the main ecological stressor in marine environments,hypoxia has become a crucial factor limiting the mariculture.However,oysters can survive up for 47.8 days at 4°C in desiccation,which indicated that oysters must have developed powerful mechanisms for hypoxia tolerance.In mammals,PHD-HIF pathway is the only oxygen sensing system observed at the molecular level.In this study,we constructed a gene co-expression network to screen hypoxia response genes in the Pacific oyster and found that HIF-? plays a key role in hypoxia adaptation process.Subsequently,the functional study of the oyster HIF family was carried out to clarify its transcriptional regulation mechanism.At the same time,we analyzed the interaction between HIF and its hydroxylase PHD,verifying the hydroxylase activity of PHD,its effect on HIF-? and the negative feedback loop formed between HIF-? and PHD.Finally,we analyzed the transcriptional regulation of HIF-? on the key glycolytic genes,to elucidate oyster energy metabolism mechanism under hypoxia stress.The main results are summarized as follows:1.Transcriptional regulation mechanism of HIF family members.In this study,WGCNA analysis was performed to analyze RNA-seq data from the Pacific oyster exposed to dessication,and eight modules were identified.Functional enrichment analysis revealed a gene module related to the regulation of metabolism.In addition,we found the intramodule hub genes,and HIF-? showed relatively higher connectivity with other genes,suggesting that HIF-? may play a key role in the process of hypoxia adaptation.Then,we characterized the oyster HIF family members and determined their importance under hypoxia.The Pacific oyster contained two ? and one ? subunit of HIF,especially a novel HIF-? family member(CgHIF?-like),which encoded a protein with lower conservation in the C-terminal domain.CgHIF?-like was expressed as four mRNA isoforms with alternative 5?-untranslated regions.qRT-PCR analysis showed that all the oyster HIFs mRNAs were highest in gills.Hypoxia treatment increased both the mRNA and protein levels of CgHIF-? and CgHIF?-like,whereas it did not change the CgHIF-? levels.Besides,we observed the transcriptional mechanism of oyster HIFs.Yeast two-hybrid assay,electrophoretic mobility shift assay and dual-luciferase reporter assay collectively indicated that CgHIF?-like and CgHIF-? were both capable of heterdimerize with CgHIF-? and transactive reporter gene in a hypoxia response element-dependent manner.2.Functional study of the oxygen sensor PHD.We characterized two PHD homologs from the Pacific oyster,namely CgPHD2 A and CgPHD2 B,and CgPHD2 B contains three alternatively spliced transcripts,two of which encoded inactive polypeptides.Gene expression analysis showed that CgPHD2 A mRNA was highly detected in all developmental stages and tissues,wheras the three spliced variants of CgPHD2 B showed variant temporal expression patterns.Among which,CgPHD2B-iso1 transcript was highest at the early developmental stage,and its mRNA level generally decreased during development;CgPHD2B-iso2 and-iso3 mRNAs remained at low levels until the morula stage and increased gradually thereafter.Functional study showed that CgPHD2 A hydroxylated CgHIF-?(P404 and P504)to regulate its protein level and transcriptional activity,with a differential preference for the two critical prolines.And we verified that the amino acid region 176-283(containing the ?2?3 loop)in CgPHD2 A was responsible for substrate discrimination;CgPHD2B,however,failed to interact with CgHIF-?,which might attribute to the block formed in the ?2?3 loop.3.The negative feedback mechanism of HIF-? on PHD.When transfected into mammalian cells,CgPHD2 A and CgPHD2 B were located in the cytoplasm and their protein levels were similarly responsive to hypoxia.qRT-PCR analysis showed that the mRNA levels of CgPHD2 A and B were differentially regulated under hypoxia,and a functional hypoxia response element(HRE)was identified at position-320 upstream of CgPHD2 A that was responsible for this induction,while CgPHD2 B was not transcriptionally regulated by CgHIF-?.4.Transcriptional regulation of HIF-? on the key glycolytic genes.At the beginning of hypoxia,the oxygen consumption rate of oysters increased,indicating that oysters stayed aerobic respiration at this time.Subsequently,the oxygen consumption decreased continuously,and anaerobic end products such as succinate,acetate and propionate were successively produced,suggesting the onset of anaerobiosis.In addition,RNA-seq analysis showed that most of the glycolytic genes m RNA levels decreased under low oxygen.PEPCK,however,is an exception,the transcript of which was highly induced by hypoxia,and we further confirmed that the PEPCK promoter activity was regulated by CgHIF-?.In conclusion,this is the first report on the whole understanding of the PHD-HIF oxygen sensing and transduction pathway in mollusks.It indicated that the intertidal shellfish may have a more complex hypoxia regulation mechanism to adapt to the low oxygen environment,providing theoretical foundation for the assessment of creatures hypoxia tolerance degree and warning of low oxygen area.