title: Rational design of minimal hypoxia-inducible enhancers.
abstract: The hypoxia-inducible factor (HIF) activates transcription via binding to the highly variable hypoxia-responsive elements (HREs). All hypoxia-inducible constructs described to date utilize multimers of naturally occurring HREs. Here, we describe the rational design of minimal hypoxia-inducible enhancers, conceptually equivalent to using an optimized HIF-binding site (HBS) as the building block. Optimizations of the HBS, spacing between HBSs, the distance from the minimal promoter, and orientation of HBSs allowed us to design constructs with high hypoxic activity. Activation of the 4xopt HBS (36bp) construct by hypoxia or HIF-1alpha and HIF-2alpha was comparable with that of the 4xEPO HRE (208bp) construct. The strong synergism between the properly arranged optimized HBSs was due to stimulation of high affinity HIF binding. Our data prove, for the first time, that it is possible to assemble artificial hypoxia-inducible enhancers from a single type of regulatory element-optimized HBS.
authors.affiliation: Department of Microbiology and Molecular Genetics, Medical Science I, B210, University of California at Irvine, College of Medicine, CA 92697-4025, USA. skaluz@uci.edu
authors.forename: Stefan
authors.initials: S
authors.lastname: Kaluz
authors.forename: Milota
authors.initials: M
authors.lastname: Kaluzová
authors.forename: Eric J
authors.initials: EJ
authors.lastname: Stanbridge
mesh_terms: D000693:Anaerobiosis / Q000235:genetics; D000818:Animals; D001483:Base Sequence; D051792:Basic Helix-Loop-Helix Proteins / Q000378:metabolism*; D001665:Binding Sites; D002460:Cell Line; D004742:Enhancer Elements, Genetic / Q000235:genetics*; D005786:Gene Expression Regulation; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000378:metabolism*; D007527:Isoenzymes / Q000235:genetics; D007770:L-Lactate Dehydrogenase / Q000235:genetics; D000080864:Lactate Dehydrogenase 5; D051379:Mice; D010735:Phosphoglycerate Kinase / Q000235:genetics; D017395:Plasminogen Activator Inhibitor 1 / Q000235:genetics; D051381:Rats; D016385:TATA Box / Q000235:genetics*; D000099162:Endothelial PAS Domain-Containing Protein 1
chemical_list: D051792:Basic Helix-Loop-Helix Proteins; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D007527:Isoenzymes; D007770:L-Lactate Dehydrogenase; D000080864:Lactate Dehydrogenase 5; D010735:Phosphoglycerate Kinase; D017395:Plasminogen Activator Inhibitor 1; D000099162:Endothelial PAS Domain-Containing Protein 1; C528165:Pgk1 protein, mouse