|关键词||长牡蛎 低氧 低氧诱导因子|
1. HIF家族的鉴定及功能验证。我们通过加权基因共表达网络（weighted correlation network analysis，WGCNA）法分析牡蛎干露表达谱数据。通过对筛选获得的1330个差异基因进行网络构建，最终识别8个基因模块。功能富集分析发现其中一个模块主要富集在调控代谢方面，我们寻找该模块的枢纽基因，发现HIF-α处于较核心位置，推测HIF-α是低氧适应过程中的关键基因。随后，对牡蛎HIF家族进行功能验证。克隆鉴定牡蛎体内存在HIF家族的两个α亚基和一个β亚基，尤其是发现一条新的HIF-α类似物CgHIFα-like，其C-端保守性较差，且具有四个不同的mRNA异构体。qRT-PCR结果显示，HIF家族成员均在鳃组织中表达量最高。低氧下，牡蛎HIF家族的两个α亚基（CgHIF-α和CgHIFα-like）在mRNA和蛋白水平表达量均升高。而CgHIF-β的mRNA和蛋白水平不受低氧影响。另外，结合酵母双杂交，荧光素酶双报告和凝胶阻滞分析结果，证实CgHIF-α和CgHIFα-like蛋白均能够与CgHIF-β形成异源二聚体，并通过结合在低氧反应元件HRE上来转录激活低氧报告基因，表明牡蛎HIF家族成员具有低氧诱导因子功能活性。
2. 通过分析PHD酶的羟基化活性及其对HIF-α的影响，阐明PHD的氧感受机制。克隆鉴定两个牡蛎PHD2（CgPHD2A和CgPHD2B）及一个CgVHL基因，其中CgPHD2B具有三种可变剪切形式，除CgPHD2B-iso1，CgPHD2B-iso2和iso3均编码不完整蛋白。qRT-PCR结果显示，CgPHD2A广泛表达于各发育阶段及成体各组织中，而CgPHD2B三个剪切体具有不同的时间表达模式，其中CgPHD2B-iso1在发育早期表达量最高，且在发育过程中表达量整体呈下降趋势，而CgPHD2B-iso2和iso3表达量逐渐升高，表明具有功能活性的CgPHD2B-iso1可能参与细胞发育过程。功能研究表明，CgPHD2A能够羟基化修饰CgHIF-α上的P404和P504位点，从而抑制CgHIF-α的转录活性和蛋白水平，而且CgPHD2A对C-端的脯氨酸作用更强。通过构建N-端缺失突变体，进一步将CgPHD2A上与底物CgHIF-α结合相关的区域锁定在aa 176-283区域，该区域包含β2β3环结构域；而CgPHD2B蛋白不能够羟基化修饰CgHIF-α，也不会影响CgHIF-α的转录活性，蛋白模型预测表明CgPHD2B与CgPHD2A的主要区别在于β2β3环结构域处多了一小段氨基酸序列，可能会影响其对底物CgHIF-α的结合。
3. 阐明长牡蛎HIF-α对PHD的负反馈调节机制。蛋白亚细胞定位结果表明，CgPHD2A 和CgPHD2B蛋白均定位在细胞质中，低氧能够增加CgPHD2A 和CgPHD2B蛋白表达量，但不影响其亚细胞定位情况。另外，qRT-PCR分析表明短期低氧能增加CgPHD2A的mRNA表达量，通过将CgPHD2A基因启动子区序列构建到荧光素酶报告基因上，结果显示CgHIF-α能调控CgPHD2A的启动子活性。进一步研究表明CgPHD2A基因启动子区的-491到-125区间序列介导CgHIF-α诱导的转录。在该区域中，发现并证实了一个具有功能活性的低氧反应元件HRE；而CgPHD2B只在长期低氧下转录水平略微升高。荧光素酶报告基因实验结果表明CgPHD2B不受CgHIF-α转录调控。
|其他摘要||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-α 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-α and the negative feedback loop formed between HIF-α and PHD. Finally, we analyzed the transcriptional regulation of HIF-α 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-α showed relatively higher connectivity with other genes, suggesting that HIF-α 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 α and one β subunit of HIF, especially a novel HIF-α family member (CgHIFα-like), which encoded a protein with lower conservation in the C-terminal domain. CgHIFα-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-α and CgHIFα-like, whereas it did not change the CgHIF-β 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 CgHIFα-like and CgHIF-α were both capable of heterdimerize with CgHIF-β 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 CgPHD2A and CgPHD2B, and CgPHD2B contains three alternatively spliced transcripts, two of which encoded inactive polypeptides. Gene expression analysis showed that CgPHD2A mRNA was highly detected in all developmental stages and tissues, wheras the three spliced variants of CgPHD2B 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 CgPHD2A hydroxylated CgHIF-α (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 β2β3 loop) in CgPHD2A was responsible for substrate discrimination; CgPHD2B, however, failed to interact with CgHIF-α, which might attribute to the block formed in the β2β3 loop.
3. The negative feedback mechanism of HIF-α on PHD. When transfected into mammalian cells, CgPHD2A and CgPHD2B were located in the cytoplasm and their protein levels were similarly responsive to hypoxia. qRT-PCR analysis showed that the mRNA levels of CgPHD2A and B were differentially regulated under hypoxia, and a functional hypoxia response element (HRE) was identified at position -320 upstream of CgPHD2A that was responsible for this induction, while CgPHD2B was not transcriptionally regulated by CgHIF-α.
4. Transcriptional regulation of HIF-α 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 mRNA 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-α.
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.
|王婷. 长牡蛎低氧信号通路分子作用机制研究[D]. 北京. 中国科学院大学,2017.|