In vivo analysis of DNA-protein interactions on the human erythropoietin enhancer.
The erythropoietin (EPO) gene is one of the best examples of a mammalian gene controlled by oxygen tension. The DNA elements responsible for hypoxia-induced transcription consist of a short region of the proximal promoter and a <50-bp 3' enhancer. The elements act cooperatively to increase the transcriptional initiation rate approximately 100-fold in response to low oxygen tension in Hep3B cells. Two distinct types of transactivating proteins have been demonstrated to bind the response elements in the human EPO enhancer in vitro: one shows hypoxia-inducible DNA binding activity, while the other activity binds DNA under normoxic and hypoxic conditions. We have investigated the DNA-protein interactions on the human EPO enhancer in living tissue culture cells that produce EPO in a regulated fashion (Hep3B) and in cells that do not express EPO under any conditions tested (HeLa). We have identified in vivo DNA-protein interactions on the control elements in the human EPO enhancer by ligation-mediated PCR technology. We show that the putative protein binding sites in the EPO enhancer are occupied in vivo under conditions of normoxia, hypoxia, and cobalt exposure in EPO-producing cells. These sites are not occupied in cells that do not produce EPO. We also provide evidence for a conformational change in the topography of the EPO enhancer in response to hypoxia and cobalt exposure.
Hu
B
B
Center for Excellence in Cancer Research, Treatment and Education, Louisiana State University Medical Center, Shreveport 71130, USA.
Wright
E
E
Campbell
L
L
Blanchard
K L
KL
D001483:Base Sequence; D001665:Binding Sites; D006528:Carcinoma, Hepatocellular; D015687:Cell Hypoxia / Q000235:genetics; D003035:Cobalt / Q000494:pharmacology; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D018983:DNA Footprinting / Q000379:methods; D019175:DNA Methylation; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004742:Enhancer Elements, Genetic / Q000235:genetics*; D004921:Erythropoietin / Q000096:biosynthesis / Q000235:genetics*; D005796:Genes / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D016133:Polymerase Chain Reaction / Q000379:methods; D015534:Trans-Activators / Q000378:metabolism; D014407:Tumor Cells, Cultured
D004268:DNA-Binding Proteins; D015534:Trans-Activators; D004921:Erythropoietin; D003035:Cobalt; D004247:DNA; C018021:cobaltous chloride