title: Generation of a dominant-negative mutant of endothelial PAS domain protein 1 by deletion of a potent C-terminal transactivation domain.
abstract: Endothelial PAS domain protein 1 (EPAS1) is a basic helix-loop-helix/PAS domain transcription factor that is preferentially expressed in vascular endothelial cells. EPAS1 shares high homology with hypoxia-inducible factor-1alpha (HIF-1alpha) and, like HIF-1alpha, has been shown to bind to the HIF-1-binding site and to activate its downstream genes such as vascular endothelial growth factor (VEGF) and erythropoietin. In this report, we show that EPAS1 increased VEGF gene expression through the HIF-1-binding site. This transactivation was enhanced further by cotransfection of an aryl hydrocarbon receptor nuclear translocator expression plasmid. Deletion analysis of EPAS1 revealed a potent activation domain (amino acids 486-639) essential for EPAS1 to transactivate the VEGF promoter. We confirmed the ability of this domain to activate transcription using a Gal4 fusion protein system. Because a truncated EPAS1 protein lacking the transactivation domain at amino acids 486-639 eliminated induction of the VEGF promoter by wild-type EPAS1, the truncated protein functions as a dominant-negative mutant. Most important, infection of the cells with an adenoviral construct expressing this mutant inhibited the induction of VEGF mRNA under conditions that mimic hypoxia. Our results suggest that EPAS1 is an important regulator of VEGF gene expression. Since VEGF plays a crucial role in angiogenesis, the ability of dominant-negative EPAS1 to inhibit VEGF promoter activity raises the possibility of a novel approach to inhibiting pathological angiogenesis.
authors.affiliation: Cardiovascular Biology Laboratory, Harvard School of Public Health, Children's Hospital, Boston, Massachusetts 02115, USA.
authors.forename: K
authors.initials: K
authors.lastname: Maemura
authors.forename: C M
authors.initials: CM
authors.lastname: Hsieh
authors.forename: M K
authors.initials: MK
authors.lastname: Jain
authors.forename: S
authors.initials: S
authors.lastname: Fukumoto
authors.forename: M D
authors.initials: MD
authors.lastname: Layne
authors.forename: Y
authors.initials: Y
authors.lastname: Liu
authors.forename: S
authors.initials: S
authors.lastname: Kourembanas
authors.forename: S F
authors.initials: SF
authors.lastname: Yet
authors.forename: M A
authors.initials: MA
authors.lastname: Perrella
authors.forename: M E
authors.initials: ME
authors.lastname: Lee
mesh_terms: D000818:Animals; D051792:Basic Helix-Loop-Helix Proteins; D002417:Cattle; D004268:DNA-Binding Proteins / Q000378:metabolism; D003676:Deferoxamine / Q000494:pharmacology; D016228:Endothelial Growth Factors / Q000096:biosynthesis / Q000235:genetics; D004730:Endothelium, Vascular / Q000166:cytology; D005656:Fungal Proteins; D051793:Hypoxia-Inducible Factor 1; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D008222:Lymphokines / Q000096:biosynthesis / Q000235:genetics; D009154:Mutation; D009687:Nuclear Proteins / Q000378:metabolism; D010446:Peptide Fragments / Q000235:genetics; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D018336:Receptors, Aryl Hydrocarbon / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D017384:Sequence Deletion; D015534:Trans-Activators / Q000235:genetics* / Q000378:metabolism; D014157:Transcription Factors; D015533:Transcriptional Activation; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; D000099162:Endothelial PAS Domain-Containing Protein 1
chemical_list: D051792:Basic Helix-Loop-Helix Proteins; D004268:DNA-Binding Proteins; D003676:Deferoxamine; D016228:Endothelial Growth Factors; D005656:Fungal Proteins; D051793:Hypoxia-Inducible Factor 1; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D008222:Lymphokines; D009687:Nuclear Proteins; D010446:Peptide Fragments; D018336:Receptors, Aryl Hydrocarbon; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D015534:Trans-Activators; D014157:Transcription Factors; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; D000099162:Endothelial PAS Domain-Containing Protein 1; C052130:GAL4 protein, S cerevisiae