Positive and negative regulation of the erythropoietin gene

In order to investigate positive and negative regulatory elements of the erythropoietin (Epo) gene, synthetic oligonucleotides were designed to control Epo transcription by means of an antigen strategy. We devised a new method for detecting regulatory elements of genes that have a weak promoter. Synthetic oligonucleotides were incubated with Hep3B cells in the presence or absence of CoCl2 or hypoxia. To exclude the effect of translational regulation, Epo mRNA concentration was determined by competitive polymerase chain reaction. The addition of antisense oligonucleotide for CACCC elements decreased the production of Epo mRNA in a dose-dependent fashion when cells were stimulated by CoCl2 or hypoxia. In contrast, the addition of antisense oligonucleotide for the GATA element caused a dose-dependent stimulation of Epo mRNA production either in the presence or absence of CoCl2 or hypoxia. Triple helix formation was revealed by electrophoresis. CACCC elements were demonstrated to be positive regulatory elements of the Epo gene, whereas the GATA element was a negative regulatory element. Furthermore, by gel mobility shift assays, we demonstrated evidence for the presence of factors in Hep3B cell nuclear extract that specifically bind to CACCC or GATA elements. Based on these observations, we presented the possibility that triple helix formation could serve as a novel means for transcriptional regulation of the gene.