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Genome-wide analysis of HIF-2? chromatin binding sites under normoxia in human bronchial epithelial cells (BEAS-2B) suggests its diverse functions.

ABSTRACT: Constitutive functional HIF-2? was recently identified in cancer and stem cell lines under normoxia. In this study, BEAS-2B, a bronchial epithelial cell line, was shown to constitutively express active HIF-2? under normoxia and exhibit markers of pluripotency including Oct-4, Nanog, and sphere formation. Oct-4 expression was reduced after knockdown of HIF-2? under normoxia. Global enrichment analysis of HIF-2? demonstrated the diverse functions of HIF-2? under normoxia. Bioinformatics analysis of the enriched loci revealed an enhancer role of HIF-2? binding sites, involvement of HIF-2? interacting proteins, and enriched de novo motifs which suggest the diverse role of HIF-2? in pseudohypoxia. The low ratio of the discovered loci overlapping with those revealed in cancer cell lines 786-O (16.1%) and MCF-7 (15.9%) under hypoxia indicated a prevailing non-canonical mechanism. Hypoxia had positive, marginal or adverse effects on the enrichment of the selected loci in ChIP-PCR assays. Deletion of the N-terminal activation domain (N-TAD) of HIF-2? disrupted the reporting activity of two of the loci annotated to ELN and ANKRD31. Hypoxia incurring abundance variation of HIF-2? may misrepresent the N-TAD functions as canonical hypoxia inducible features via C-TAD activation. Elucidation of the pseudohypoxia functions of constitutive HIF-2? is useful for resolving its role in malignancy and pluripotency.

SUBMITTER: Lee MC

PROVIDER: S-EPMC4931692 | biostudies-literature | 2016 Jul

REPOSITORIES: biostudies-literature

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Genome-wide analysis of HIF-2α chromatin binding sites under normoxia in human bronchial epithelial cells (BEAS-2B) suggests its diverse functions.

Lee Meng-Chang MC Huang Hsin-Ju HJ Chang Tzu-Hao TH Huang Hsieh-Chou HC Hsieh Shen-Yuan SY Chen Yi-Siou YS Chou Wei-Yuan WY Chiang Chiao-Hsi CH Lai Ching-Huang CH Shiau Chia-Yang CY

Scientific reports 20160704

Constitutive functional HIF-2α was recently identified in cancer and stem cell lines under normoxia. In this study, BEAS-2B, a bronchial epithelial cell line, was shown to constitutively express active HIF-2α under normoxia and exhibit markers of pluripotency including Oct-4, Nanog, and sphere formation. Oct-4 expression was reduced after knockdown of HIF-2α under normoxia. Global enrichment analysis of HIF-2α demonstrated the diverse functions of HIF-2α under normoxia. Bioinformatics analysis o ...[more]

PMID: 27373565

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Project description:BACKGROUND:Emissions from diesel vehicles and biomass burning are the principal sources of primary ultrafine particles (UFP). The exposure to UFP has been associated to cardiovascular and pulmonary diseases, including lung cancer. Although many aspects of the toxicology of ambient particulate matter (PM) have been unraveled, the molecular mechanisms activated in human cells by the exposure to UFP are still poorly understood. Here, we present an RNA-seq time-course experiment (five time point after single dose exposure) used to investigate the differential and temporal changes induced in the gene expression of human bronchial epithelial cells (BEAS-2B) by the exposure to UFP generated from diesel and biomass combustion. A combination of different bioinformatics tools (EdgeR, next-maSigPro and reactome FI app-Cytoscape and prioritization strategies) facilitated the analyses the temporal transcriptional pattern, functional gene set enrichment and gene networks related to cellular response to UFP particles. RESULTS:The bioinformatics analysis of transcriptional data reveals that the two different UFP induce, since the earliest time points, different transcriptional dynamics resulting in the activation of specific genes. The functional enrichment of differentially expressed genes indicates that the exposure to diesel UFP induces the activation of genes involved in TNF? signaling via NF-kB and inflammatory response, and hypoxia. Conversely, the exposure to ultrafine particles from biomass determines less distinct modifications of the gene expression profiles. Diesel UFP exposure induces the secretion of biomarkers associated to inflammation (CCXL2, EPGN, GREM1, IL1A, IL1B, IL6, IL24, EREG, VEGF) and transcription factors (as NFE2L2, MAFF, HES1, FOSL1, TGIF1) relevant for cardiovascular and lung disease. By means of network reconstruction, four genes (STAT3, HIF1a, NFKB1, KRAS) have emerged as major regulators of transcriptional response of bronchial epithelial cells exposed to diesel exhaust. CONCLUSIONS:Overall, this work highlights modifications of the transcriptional landscape in human bronchial cells exposed to UFP and sheds new lights on possible mechanisms by means of which UFP acts as a carcinogen and harmful factor for human health.

Project description:BackgroundMitochondria is prone to oxidative damage by endogenous and exogenous sources of free radicals, including particulate matter (PM). Given the role of mitochondria in inflammatory disorders, such as asthma and chronic obstructive pulmonary disease, we hypothesized that supplementation of vitamin D may play a protective role in PM-induced mitochondrial oxidative damages of human bronchial epithelial BEAS-2B cells.MethodsBEAS-2B cells were pretreated with 1,25(OH)2D3, an active form of vitamin D, for 1 h prior to 24-hour exposure to PM (SRM-1648a). Oxidative stress was measured by flow cytometry. Mitochondrial functions including mitochondrial membrane potential, ATP levels, and mitochondrial DNA copy number were analyzed. Additionally, mitochondrial ultrastructure was examined using transmission electron microscopy. Intracellular and mitochondrial calcium concentration changes were assessed using flow cytometry based on the expression of Fluo-4 AM and Rhod-2 AM, respectively. Pro-inflammatory cytokines, including IL-6 and MCP-1, were quantified using ELISA. The expression levels of antioxidants, including SOD1, SOD2, CAT, GSH, and NADPH, were determined.ResultsOur findings first showed that 24-hour exposure to PM led to the overproduction of reactive oxygen species (ROS) derived from mitochondria. PM-induced mitochondrial oxidation resulted in intracellular calcium accumulation, particularly within mitochondria, and alterations in mitochondrial morphology and functions. These changes included loss of mitochondrial membrane integrity, disarrayed cristae, mitochondrial membrane depolarization, reduced ATP production, and increased mitochondrial DNA copy number. Consequently, PM-induced mitochondrial damage triggered the release of certain inflammatory cytokines, such as IL-6 and MCP-1. Similar to the actions of mitochondrial ROS inhibitor MitoTEMPO, 1,25(OH)2D3 conferred protective effects on mtDNA alterations, mitochondrial damages, calcium dyshomeostasis, thereby decreasing the release of certain inflammatory cytokines. We found that greater cellular level of 1,25(OH)2D3 upregulated the expression of enzymatic (SOD1, SOD2, and CAT) and non-enzymatic (GSH and NADPH) antioxidants to modulate cellular redox homeostasis.ConclusionOur study provides new evidence that 1,25(OH)2D3 acts as an antioxidant, enhancing BEAS-2B antioxidant responses to regulate mitochondrial ROS homeostasis and mitochondrial function, thereby enhancing epithelial defense against air pollution exposure.

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Genome-wide analysis of HIF-2? chromatin binding sites under normoxia in human bronchial epithelial cells (BEAS-2B) suggests its diverse functions.

Publications

Genome-wide analysis of HIF-2α chromatin binding sites under normoxia in human bronchial epithelial cells (BEAS-2B) suggests its diverse functions.

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