Project description:Copper (Cu) regulates hypoxia-inducible factor-1 (HIF-1) transcription activity by affecting the selectivity of HIF-1α targeting to the promoters of the affected genes. Here, we made an effort to provide a comprehensive understanding of Cu regulation of the selectivity of HIF-1α targeting across genome. We used tetraethylenepentamine (TEPA), a Cu selective chelator, to reduce Cu content in the cells. In hypoxia, we conducted chromatin immunoprecipitation combined with massively parallel DNA sequencing (ChIP-seq) to globally map the binding sites of HIF-1α, Pol Ⅱ (RNA polymeraseⅡ) and histone H3K27ac. We also performed RNA-sequencing (RNA-seq) in EA.hy926 cells under hypoxia (1% O2) with or without Cu depression to determine the profile of mRNA expression. Our analyses identified 3197 HIF-1α binding sites under hypoxia. Cu depression by TEPA reduced 1820 binding sites from the 3197, but induced additional 274 new binding sites. We analyzed these binding sites in the promoter and putative enhancer regions, coupled with their mRNA expression profiles, and found 281 Cu-dependent and 10 Cu-independent HIF-1α target genes. We found that the core bases “GGAA” and “TTCC” constituted the critical motifs for the binding sites of Cu-dependent genes. This study thus revealed that Cu, by affecting the binding of HIF-1α to the critical motifs in the promoter and putative enhancer regions of HIF-1 regulated genes, leads to the selectivity of HIF-1 regulated expression of Cu-dependent genes.
Project description:Copper (Cu) regulates hypoxia-inducible factor-1 (HIF-1) transcription activity by affecting the selectivity of HIF-1α targeting to the promoters of the affected genes. Here, we made an effort to provide a comprehensive understanding of Cu regulation of the selectivity of HIF-1α targeting across genome. We used tetraethylenepentamine (TEPA), a Cu selective chelator, to reduce Cu content in the cells. In hypoxia, we conducted chromatin immunoprecipitation combined with massively parallel DNA sequencing (ChIP-seq) to globally map the binding sites of HIF-1α, Pol Ⅱ (RNA polymeraseⅡ) and histone H3K27ac. We also performed RNA-sequencing (RNA-seq) in EA.hy926 cells under hypoxia (1% O2) with or without Cu depression to determine the profile of mRNA expression. Our analyses identified 3197 HIF-1α binding sites under hypoxia. Cu depression by TEPA reduced 1820 binding sites from the 3197, but induced additional 274 new binding sites. We analyzed these binding sites in the promoter and putative enhancer regions, coupled with their mRNA expression profiles, and found 281 Cu-dependent and 10 Cu-independent HIF-1α target genes. We found that the core bases “GGAA” and “TTCC” constituted the critical motifs for the binding sites of Cu-dependent genes. This study thus revealed that Cu, by affecting the binding of HIF-1α to the critical motifs in the promoter and putative enhancer regions of HIF-1 regulated genes, leads to the selectivity of HIF-1 regulated expression of Cu-dependent genes.
Project description:HIF-1 regulated VEGF expression were upregulated in reponse to ischemic injury. However, the VEGF mRNA level were increased in the early stage but decreased in the late stage of myocardial infarction. We found that activities of HIF-1 binding to the HRE sites were possiblely responsible for the differential expression of VEGF. The HIF-1 binding activities were regulated by copper. After ischemic injury, copper concentrations were decreased in the infarct myocardium in the late stage of myocardial infarction, which might reduce VEGF expression through insufficient HIF-1 binding to HRE sites.
Project description:Reduced or absent cytotrophoblast invasion of the maternal uterine spiral arteries is a common clinical finding in studies of pregnancies complicated by preeclampsia, suggesting that the mechanisms behind invasion of these cells is perturbed. The placenta initially develops in a low oxygen environment of 1-2% oxygen until after the 10th week of pregnancy. During this time oxygen concentration exerts a major influence over trophoblast activity and, in vitro, hypoxia inducible factors are proposed to be one of many key regulators of first trimester trophoblast behaviour. We used a global gene expression microarray approach to identify signalling pathways involved in invasion of the first trimester trophoblast cell line HTR8/SVneo under hypoxic conditions where HIF-1 was active. Additionally, first trimester placental samples from different gestational age groups were labelled with anti HIF-1 and HIF-2 to evaluate whether HIFs are differentially expressed and localised across the period of development characterised by hypoxia (6-8 weeks) and maternal blood perfusion (10-12 weeks). Eighty-eight genes were differentially expressed between cells cultured in 1% oxygen (where HIF-1 was localised to the nucleus) and 5% oxygen (where HIF-1 was cytoplasmic). 65% of the genes were predicted to contain HIF-1α:ARNT transcription factor binding sites. Increased nuclear localisation of HIF-1α was seen in extravillous cytotrophoblasts in early first trimester compared with late, while cellular expression of HIF-2α in the villous stroma was higher in late first trimester. While HIFs and their downstream targets are clearly induced in trophoblasts during early placental development, and in vitro hypoxic conditions, the mechanism and pathways by which invasion is increased under hypoxic conditions is not clear from the gene expression profile. Further insight beyond the transcription level is required to fully understand this complex phenomenon.
Project description:Hypoxia Inducible Factor (HIF) prolyl hydroxylase domain (PHD) enzymes catalyse the posttranslational hydroxylation of conserved prolyl residues in the alpha-subunit of the HIF transcription factor. These modifications, which promote the degradation of HIF-alpha subunits by the pVHL E3 ligase complex and impart oxygen-dependent regulation of the HIF transcriptional response, have been extensively characterised at the molecular, cellular and organismal level. Since the discovery of the PHDs, a range of less well-characterised non-HIF substrates have been reported with the potential to confer oxygen-sensitivity to a diverse range of cellular processes. We sought to systematically compare all of the reported non-HIF substrates for their ability to support PHD-catalysed hydroxylation. We performed a comprehensive series of in vitro hydroxylation assays reacting synthetic peptides and full-length protein substrates generated by in vitro transcription and translation with purified recombinant enzyme preparations. Prolyl hydroxylation was assayed directly by mass spectrometry and radiochemical assay for hydroxyproline. Both methods enabled quantitative appraisal of enzyme-catalysed hydroxylation, with liquid chromatography mass spectrometry methods employing NMR-quantified peptide standards to calibrate retention time signatures and determine ionisation efficiencies for each target peptide. Using these approaches we assayed hydroxylation on 23 different proteins. Surprisingly, we did not detect measurable hydroxylation on any of the reported non-HIF substrates using either method. In contrast, control assays with HIF1-alpha substrate supported high stoichiometry (typically >90%) hydroxylation. Our findings suggest that PHD substrates are more restricted than has been reported.
Project description:Chronic hypoxia induces pulmonary vascular remodeling and pulmonary hypertension (PH). While it is established that transcription factors, hypoxia-inducible factors (HIF-1α/HIF-2α) activate gene programs that drive hypoxia-induced PH, the mechanism of HIF-1/2 activation is less clear. Here, we report that carboxylterminus of Hsp70-interacting protein (CHIP or Stub1) modulates HIF-1α and HIF-2α transcription rather than reducing their stability. Knocking-down Stub1 reduced hypoxic activation of HIF-1α mRNA, protein, and activity while enhancing hypoxic induction of HIF-2α mRNA, protein, and target genes in pulmonary vascular cells. Mechanistically, CBP/p300-mediated acetylation of lysine (K287) inactivates the ubiquitin ligase activity of Stub1 and triggers its translocation from the cytoplasm into the nucleus. There, it recognizes the HIF promoter and hypoxia response elements (HREs) in target genes. Expression of Stub1-K287Q mutant (mimicking acetylation) enhanced hypoxia-induced HIF-1α expression, while acetyl-deficient Stub1-K287R mutant had the opposite effect on HIF-α but enhanced hypoxia-induced HIF-2α transcriptional activity. Endothelial-Stub1 transgenic mice tolerated chronic hypoxia better, had less pulmonary vascular remodeling, reduced pulmonary vascular resistance, and greater cardioprotection. Thus, Stub1 nuclear translocation enhances hypoxic induction of HIF-1α activity while suppressing deleterious effects of HIF-2α. These observations indicate that nuclear-Stub1 synergizes with HIF-1α to promote transcriptional responses and antagonizes HIF2α-driven PH in chronic hypoxia.