Sequence and functional characterization of hypoxia-inducible factors, HIF1α, HIF2αa, and HIF3α, from the estuarine fish, Fundulus heteroclitus.
ABSTRACT: The hypoxia-inducible factor (HIF) family of transcription factors plays central roles in the development, physiology, pathology, and environmental adaptation of animals. Because many aquatic habitats are characterized by episodes of low dissolved oxygen, fish represent ideal models to study the roles of HIF in the response to aquatic hypoxia. The estuarine fish Fundulus heteroclitus is found in habitats prone to hypoxia. It responds to low oxygen via behavioral, physiological, and molecular changes, and one member of the HIF family, HIF2α, has been previously described. Herein, cDNA sequencing, phylogenetic analyses, and genomic approaches were used to determine other members of the HIFα family from F. heteroclitus and their relationships to HIFα subunits from other vertebrates. In vitro and cellular approaches demonstrated that full-length forms of HIF1α, HIF2α, and HIF3α independently formed complexes with the β-subunit, aryl hydrocarbon receptor nuclear translocator, to bind to hypoxia response elements and activate reporter gene expression. Quantitative PCR showed that HIFα mRNA abundance varied among organs of normoxic fish in an isoform-specific fashion. Analysis of the F. heteroclitus genome revealed a locus encoding a second HIF2α-HIF2αb-a predicted protein lacking oxygen sensing and transactivation domains. Finally, sequence analyses demonstrated polymorphism in the coding sequence of each F. heteroclitus HIFα subunit, suggesting that genetic variation in these transcription factors may play a role in the variation in hypoxia responses among individuals or populations.
Project description:Hypoxia-inducible factor-? (HIF-?) and cytochrome P450 1A (CYP1A) are biomarkers of environmental exposure to hypoxia and organic xenobiotic chemicals that act through the aryl hydrocarbon receptor, respectively. Many aquatic environments heavily contaminated with organic chemicals, such as harbors, are also hypoxic. Recently, we and other scientists reported HIF-? genes are upregulated by hypoxia exposure in aquatic organisms, but the molecular mechanisms of hypoxia regulation of CYP1A expression have not been investigated in teleost fishes. As a first step in understanding the molecular mechanisms of hypoxia modulation of CYP1A expression in fish, we characterized CYP1A cDNA from croaker liver. Hypoxia exposure (dissolved oxygen, DO: 1.7 mg/L for 2 to 4 weeks) caused significant decreases in hepatic CYP1A mRNA and protein levels compared to CYP1A levels in fish held in normoxic conditions. In vivo studies showed that the nitric oxide (NO)-donor, S-nitroso-N-acetyl-DL-penicillamine, significantly decreased CYP1A expression in croaker livers, whereas the competitive inhibitor of NO synthase (NOS), N(?)-nitro-L-arginine methyl ester, restored CYP1A mRNA and protein levels in hypoxia-exposed (1.7 mg DO/L for 4 weeks) fish. In vivo hypoxia exposure also markedly increased interleukin-1? (IL-1?, a cytokine), HIF-2? mRNA and endothelial NOS (eNOS) protein levels in croaker livers. Pharmacological treatment with vitamin E, an antioxidant, lowered the IL-1?, HIF-2? mRNA and eNOS protein levels in hypoxia-exposed fish and completely reversed the down-regulation of hepatic CYP1A mRNA and protein levels in response to hypoxia exposure. These results suggest that hypoxia-induced down-regulation of CYP1A is due to alterations of NO and oxidant status, and cellular IL-1? and HIF-? levels. Moreover, the present study provides the first evidence of a role for antioxidants in hepatic eNOS and IL-1? regulation in aquatic vertebrates during hypoxic stress.
Project description:Hypoxia-inducible factors (HIF)-1α and HIF2α are major transcription factors required for adaptive responses to hypoxia. HIFs form a complex with aryl hydrocarbon receptor nuclear translocator (ARNT) to bind to the regulatory regions of target genes. The acetylation of histones by histone acetyltransferases (HATs) is one of the epigenetic marks associated with active chromatin. Indeed, HIFs recruit p300 HAT to hypoxia response elements (HREs) within gene regulatory regions. Here, we report an unusual HIF-mediated transcriptional activation in ovarian clear cell carcinoma (CCC). While characterizing coagulation factor VII (FVII) gene induction during hypoxic conditions, we observed that the interaction of HIF2α with Sp1, but not with ARNT, could induce transcription of FVII in a HRE-independent manner. Unexpectedly, this gene activation is associated with histone deacetylation. We found that a class II HDAC, HDAC4, is recruited with HIF2α to the FVII promoter as a co-activator, while p300 HAT negatively regulated this process. Furthermore, this mechanism can be synergistically enhanced via a deacetylation-dependent pathway when cells are simultaneously exposed to hypoxic and serum-free conditions. These results suggest the presence of a stress-responsive transcription mediated by the HIF2α/Sp1/HDAC4 network and explain how CCC shed their procoagulant activity under hypoxia.
Project description:MicroRNAs (miRNAs) are involved in a wide variety of cellular processes and post-transcriptionally regulate several mechanism and diseases. However, contribution of miRNAs functioning during hypoxia and DNA methylation together is less understood. The current study was aimed to find a shared miRNAs signature upstream to hypoxia (via HIF gene family members) and methylation (via DNMT gene family members). This was followed by the global validation of the hypoxia related miRNA signature using miRNA microarray meta-analysis of the hypoxia induced human samples. We further concluded the study by looking into thrombosis related terms and pathways enriched during protein-protein interaction (PPI) network analysis of these two sets of gene family. Network prioritization of these shared miRNAs reveals miR-129, miR-19band miR-23b as top regulatory miRNAs. A comprehensive meta-analysis of microarray datasets of hypoxia samples revealed 29 differentially expressed miRNAs. GSEA of the interacting genes in the DNMT-HIF PPI network indicated thrombosis associated pathways including "Hemostasis", "TPO signaling pathway" and "angiogenesis". Interestingly, the study has generated a novel database of candidate miRNA signatures shared between hypoxia and methylation, and their relation to thrombotic pathways, which might aid in the development of potential therapeutic biomarkers.
Project description:HIF (Hypoxia-inducible factor) gene family members function as master regulators of cellular and systemic oxygen homeostasis during changes in oxygen availability. Qinghai-Tibet Plateau is a natural laboratory for for long-term hypoxia and cold adaptation. In this context, T. scleroptera that is restricted to >3500 m high-altitude freshwater rivers was selected as the model to compare with a representative species from the plain, P. dabryanus. We cloned different HIF-? and carried out a phylogenetic analysis from invertebrates to vertebrates for identifying HIF-? genes and analyzing their evolutionary history. Intriguingly, the HIF-? has undergone gene duplications might be due to whole-genome duplication (WGD) events during evolution. PAML analysis indicated that HIF-1?A was subjected to positive selection acted on specific sites in Triplophysa lineages. To investigate the relationship between hypoxia adaptation and the regulation of HIF-? stability by pVHL in plateau and plain fish, a series of experiments were carried out. Comparison the luciferase transcriptional activity and protein levels of HIF-?s and the differing interactions of HIF-?s with pVHL, show clear differences between plateau and plain fish. T. scleroptera pVHL could enhance HIF-? transcriptional activity under hypoxia, and functional validation through pVHL protein mutagenesis showed that these mutations increased the stability of HIF-? and its hetero dimerization affinity to ARNT. Our research shows that missense mutations of pVHL induced evolutionary molecular adaptation in Triplophysa fishes living in high altitude hypoxic environments.
Project description:Many aerobic organisms have developed molecular mechanism to tolerate hypoxia, but the specifics of these mechanisms remain poorly understood. It is important to develop genetic methods that confer increased hypoxia tolerance to intensively farmed aquatic species, as these are maintained in environments with limited available oxygen. As an asparaginyl hydroxylase of hypoxia-inducible factors (HIFs), factor inhibiting HIF (FIH) inhibits transcriptional activation of hypoxia-inducible genes by blocking the association of HIFs with the transcriptional coactivators CREB-binding protein (CBP) and p300. Therefore, here we sought to test whether fih is involved in regulating hypoxia tolerance in the commonly used zebrafish model. Overexpressing the zebrafish fih gene in epithelioma papulosum cyprini (EPC) cells and embryos, we found that fih inhibits the transcriptional activation of zebrafish HIF-α proteins. Using CRISPR/Cas9 to obtain fih-null zebrafish mutants, we noted that the fih deletion makes zebrafish more tolerant of hypoxic conditions than their WT siblings, but does not result in oxygen consumption rates that significantly differ from those of WT fish. Of note, we identified fewer apoptotic cells in adult fih-null zebrafish brains and in fih-null embryos, possibly explaining why the fih-null mutant had greater hypoxia tolerance than the WT. Moreover, the fih deletion up-regulated several hypoxia-inducible genes in fih-null zebrafish exposed to hypoxia. The findings of our study suggest that fih plays a role in hypoxia tolerance by affecting the rate of cellular apoptosis in zebrafish.
Project description:Cable bacteria belonging to the family Desulfobulbaceae couple sulfide oxidation and oxygen reduction by long-distance electron transfer over centimeter distances in marine and freshwater sediments. In such habitats, aquatic plants can release oxygen into the rhizosphere. Hence, the rhizosphere constitutes an ideal habitat for cable bacteria, which have been reported on seagrass roots recently. Here, we employ experimental approaches to investigate activity, abundance, and spatial orientation of cable bacteria next to the roots of the freshwater plant Littorella uniflora. Fluorescence in situ hybridization (FISH), in combination with oxygen-sensitive planar optodes, demonstrated that cable bacteria densities are enriched at the oxic-anoxic transition zone next to roots compared to the bulk sediment in the same depth. Scanning electron microscopy showed cable bacteria along root hairs. Electric potential measurements showed a lateral electric field over centimeters from the roots, indicating cable bacteria activity. In addition, FISH revealed that cable bacteria were present in the rhizosphere of Oryza sativa (rice), Lobelia cardinalis and Salicornia europaea. Hence, the interaction of cable bacteria with aquatic plants of different growth forms and habitats indicates that the plant root-cable bacteria interaction might be a common property of aquatic plant rhizospheres.
Project description:Pheochromocytomas and paragangliomas (PPGLs) with activated pseudohypoxic pathways are associated with an immature catecholamine phenotype and carry a higher risk for metastasis. For improved understanding of the underlying mechanisms we investigated the impact of hypoxia and pseudohypoxia on catecholamine biosynthesis in pheochromocytoma cells naturally lacking Hif2α (MPC and MTT) or expressing both Hif1α and Hif2α (PC12). Cultivation under extrinsic hypoxia or in spheroid culture (intrinsic hypoxia) increased cellular dopamine and norepinephrine contents in all cell lines. To distinguish further between Hif1α- and Hif2α-driven effects we expressed Hif2α in MTT and MPC-mCherry cells (naturally lacking Hif2α). Presence of Hif2α resulted in similarly increased cellular dopamine and norepinephrine under hypoxia as in the control cells. Furthermore, hypoxia resulted in enhanced phosphorylation of tyrosine hydroxylase (TH). A specific knockdown of Hif1α in PC12 diminished these effects. Pseudohypoxic conditions, simulated by expression of Hif2α under normoxia resulted in increased TH phosphorylation, further stimulated by extrinsic hypoxia. Correlations with PPGL tissue data led us to conclude that catecholamine biosynthesis under hypoxia is mainly mediated through increased phosphorylation of TH, regulated as a short-term response (24-48 h) by HIF1α. Continuous activation of hypoxia-related genes under pseudohypoxia leads to a HIF2α-mediated phosphorylation of TH (permanent status).
Project description:Aquatic mammals, such as cetaceans experience various depths, with accordingly diverse oxygenation, thus, cetaceans have developed adaptations for hypoxia, but mechanisms underlying this tolerance to low oxygen are unclear. Here we analyzed VHL and HIF-2?, in the hypoxia signaling pathway. Variations in VHL are greater than HIF-2? between cetaceans and terrestrial mammals, and beluga whale VHL (BW-VHL) promotes HIF-2? degradation under hypoxia. BW-VHL catalyzes BW-HIF-2? to form K48-linked poly-ubiquitin chains mainly at the lysine 429 of BW-HIF-2? (K429) and induces BW-HIF-2? for proteasomal degradation. W100 within BW-VHL is a key site for BW-VHL functionally and BW-VHL enhances transcriptional activity of BW-HIF-2? under hypoxia. Our data therefore reveal that BW-VHL has a unique function that may contribute to hypoxic adaptation.
Project description:Alternative RNA splicing analysis in Hep3B cell cultured under 21% (N1,3,5) or 1.2% (H2,4,6) oxygen Hypoxia is a common characteristic of many solid tumors. The hypoxic microenvironment stabilizes hypoxia-inducible transcription factor 1α (HIF1α) and 2α (HIF2α) to activate gene transcription, which promotes tumor cell survival. 95% of human genes are alternatively spliced, producing RNA isoforms that code functionally distinct proteins. Thus, effective hypoxia response requires increased HIF target gene transcription as well as proper RNA splicing of these HIF target genes. However, it is unclear if and how hypoxia regulates RNA splicing of HIF target genes. This study determined the effects of hypoxia on alternative splicing (AS) of HIF and non-HIF target genes in Hep3B cells and characterized the role of HIF in regulating AS of HIF induced genes. The results indicated that hypoxia generally promotes exon inclusion for hypoxia-induced, but reduces exon inclusion for hypoxia reduced genes. Mechanistically, HIF activity, but not hypoxia per se is found to be necessary and sufficient to increase exon inclusion of several HIF target genes including pyruvate dehydrogenase kinase 1 (PDK1). PDK1 splicing reporters confirmed that transcriptional activation by HIF is sufficient to increase exon inclusion of PDK1 splicing reporter. In contrast, transcriptional activation of the PDK1 minigene by other transcription factor in the absence of endogenous HIF target gene activation fails to alter PDK1 RNA splicing, demonstrating a novel role of HIF target gene(s) in regulating RNA splicing of HIF target genes. Implications:This study demonstrates a novel function of HIF in regulating RNA splicing of HIF target genes. We analyzed total RNA from Hep3B cells cultured under 21% (N1,3,5) or 1.2% (H2,4,6) oxygen using the Affymetrix Human Exon 1.0 ST platform. Array data was processed by Altanalyze software version 2.0.7. Techinical replicates were performed for Nx and Hx treated Hep3B cells
Project description:High altitude pulmonary edema (HAPE) susceptibility is associated with EGLN1 polymorphisms, we hypothesized that HAPE-susceptible (HAPE-S, had HAPE episode in past) subjects may exhibit abnormal HIF1α levels in normoxic conditions. We measured HIF1α levels in HAPE-S and HAPE resistant (HAPE-R, no HAPE episode) individuals with similar pulmonary functions. Hemodynamic responses were also measured before and after normobaric hypoxia (Fi02 = 0.12 for 30 min duration at sea level) in both groups. . HIF1α was higher in HAPE-S (320.3 ± 267.5 vs 58.75 ± 33.88 pg/ml, P < 0.05) than HAPE-R, at baseline, despite no significant difference in baseline oxygen saturations (97.7 ± 1.7% and 98.8 ± 0.7). As expected, HAPE-S showed an exaggerated increase in pulmonary artery pressure (27.9 ± 6 vs 19.3 ± 3.7 mm Hg, P < 0.05) and a fall in peripheral oxygen saturation (66.9 ± 11.7 vs 78.7 ± 3.8%, P < 0.05), when exposed to hypoxia. HIF1α levels at baseline could accurately classify members of the two groups (AUC = 0.87). In a subset of the groups where hemoglobin fractions were additionally measured to understand the cause of elevated hypoxic response at baseline, two of four HAPE-S subjects showed reduced HbA. In conclusion, HIF 1 α levels during normoxia may represent an important marker for determination of HAPE susceptibility.