Hypoxia-inducible mammalian gene expression analyzed in vivo at a TATA-driven promoter and at an initiator-driven promoter.
We have analyzed protein-DNA interactions in vivo at transcriptional control elements for two hypoxia-inducible genes in mouse hepatoma cells. The promoter for the phosphoglycerate kinase 1 (PGK1) gene contains an initiator element, but no TATA sequence, whereas the promoter for the glucose transporter 1 (Glut1) gene contains a TATA element but no initiator sequence. Our findings reveal hypoxia-inducible, Arnt-dependent occupancy of DNA recognition sites for hypoxia-inducible factor 1 (HIF-1) upstream of both target genes. The conserved recognition motif among the five recognition sites is 5'-CGTG-3'. The PGK1 promoter exhibits constitutive occupancy of a binding site for an unknown protein(s); however, we detect no protein-DNA interaction at the initiator element, in either uninduced or induced cells. The Glut1 promoter also exhibits constitutive protein binding; in addition, the TATA element exhibits partial occupancy in uninduced cells and increased occupancy under hypoxic conditions. We find no evidence for hypoxia-induced changes in chromatin structure of either gene. Time-course analyses of the Glut1 gene reveal a temporal relationship between occupancy of HIF-1 sites and TATA element occupancy. Our findings suggest that the promoters for both hypoxia-responsive genes constitutively maintain an accessible chromatin configuration and that HIF-1 facilitates transcription by recruiting and/or stabilizing a transcription factor(s), such as TFIID, at both promoters.
Okino
S T
ST
Department of Molecular Pharmacology, Stanford University School of Medicine, Stanford, California 94305-5332, USA.
Chichester
C H
CH
Whitlock
J P
JP
D000818:Animals; D051784:Aryl Hydrocarbon Receptor Nuclear Translocator; D001483:Base Sequence; D001665:Binding Sites; D015687:Cell Hypoxia / Q000235:genetics*; D017931:DNA Primers; D004268:DNA-Binding Proteins; D004742:Enhancer Elements, Genetic; D015971:Gene Expression Regulation, Enzymologic; D015972:Gene Expression Regulation, Neoplastic; D051272:Glucose Transporter Type 1; D008114:Liver Neoplasms, Experimental / Q000235:genetics; D051379:Mice; D009004:Monosaccharide Transport Proteins / Q000235:genetics*; D010735:Phosphoglycerate Kinase / Q000235:genetics*; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D018336:Receptors, Aryl Hydrocarbon; D016385:TATA Box; D014157:Transcription Factors / Q000172:deficiency / Q000378:metabolism; D014407:Tumor Cells, Cultured
C497397:Arnt protein, mouse; D017931:DNA Primers; D004268:DNA-Binding Proteins; D051272:Glucose Transporter Type 1; D009004:Monosaccharide Transport Proteins; D018336:Receptors, Aryl Hydrocarbon; C495678:Slc2a1 protein, mouse; D014157:Transcription Factors; D051784:Aryl Hydrocarbon Receptor Nuclear Translocator; D010735:Phosphoglycerate Kinase