Human erythropoietin gene expression in liver and kidney is inducible by anemia or hypoxia. DNase I-hypersensitive sites were identified 3' to the human erythropoietin gene in liver nuclei. A 256-base-pair region of 3' flanking sequence was shown by DNase I protection and electrophoretic mobility-shift assays to bind four or more different nuclear factors, at least two of which are induced by anemia in both liver and kidney, and the region functioned as a hypoxia-inducible enhancer in transient expression assays. These results provide insight into the molecular basis for the regulation of gene expression by a fundamental physiologic stimulus, hypoxia.In mammals, erythropoietin (EPO) is the primary humoral regulator of red blood cell production and thus of blood oxygen-carrying capacity. EPO RNA levels increase several hundredfold in rodent liver and kidney in response to anemia or hypoxia (1, 2). EPO gene expression is believed to be induced by hypoxia or anemia via a single mechanism (reviewed in ref.3). The signal sensed by EPO-producing cells is probably a decrease in local tissue oxygen tension, whether due to decreased blood oxygen-carrying capacity (anemia) or decreased ambient oxygen concentration (hypoxia). EPO gene expression in Hep3B human hepatoma cells can be induced in 1% 02(4, 5), demonstrating that the same cell type can sense hypoxia and respond by increasing its steady-state level of EPO RNA. Nuclear extracts prepared from Hep3B cells cultured in 1% 02 support a higher level of EPO gene transcription in vitro than extracts from cells cultured in 20% 02 (6). EPO gene expression in Hep3B cells (4, 5) and in vivo (2, 7) can also be stimulated by CoC12 administration.By introducing DNA containing the human EPO gene into the mouse genome via pronuclear microinjection, we have identified cis-acting DNA sequences that regulate tissuespecific, inducible human EPO gene expression. Transgenes of 4 kilobases (kb) (tgEP04) and 10 kb (tgEP0O10) containing the human EPO gene, a 3' flanking region of 0.7 kb, and 5' flanking regions of 0.4 kb and 6 kb, respectively, are inducibly expressed in adult liver but not in kidney (8, 9). In the liver of anemic tgEPO10 mice, human EPO RNA is synthesized specifically by perivenous hepatocytes and the amount of EPO RNA per cell increases as anemia is made more severe (10). When tgEP04 or tgEPO10 mice are made anemic, human EPO RNA increases by several orders of magnitude in liver compared with the uninduced state, indicating the presence of sequences mediating inducible liver expression in close proximity to the human EPO gene (8, 9). MATERIALS AND METHODSDNase I-Sensitivity Studies. Nuclei were isolated and then digested with DNase I at 0, 1, 2, or 5 ,ug/ml for 2 min at 25°C, and DNA was isolated as described (11).Nuclear Extracts. Liver (41 g) and kidney (12.6 g) were isolated from 21 untreated mice, kidney (7.6 g) was isolated from 15 anemic mice treated with phenyihydrazine (mean hematocrit, 24%), and liver (9.7 g) was isolated from 5 phenylhydrazine-treated m...