Abstract
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.
Highlights
Perturbations in the environment present a continual challenge to cellular systems responsible for maintaining homeostasis
The designation PAS stands for the three proteins first discovered to contain the domains: Per, a Drosophila protein that helps regulate circadian rhythmicity; Arnt, a protein that heterodimerizes with the mammalian aromatic hydrocarbon receptor (AhR) in mediating transcriptional responses to xenobiotics like dioxin; and Sim, a protein implicated in Drosophila central nervous system development [7]
Hypoxia stabilizes the bHLH/PAS protein known as hypoxia-inducible factor-1␣ (HIF-1␣) thereby causing it to accumulate within the cell; HIF-1␣ heterodimerizes with Arnt to form a DNA-binding transcription factor known as hypoxiainducible factor-1 (HIF-1)
Summary
Perturbations in the environment present a continual challenge to cellular systems responsible for maintaining homeostasis. Our findings (Fig. 2, A and B) reveal that, in uninduced wild-type cells, the DNA encompassing the potential HIF-1 binding sites upstream of the PGK1 and Glut1 genes exhibits a DNase I digestion pattern similar to that of naked DNA.
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