Binding of erythropoietin (Epo) receptor (EpoR) on the surface of erythroid progenitor cells stimulates erythropoiesis resulting in molecular events including increases of GATA-1 protein, an erythroid specific transcription factor, and activation of globin genes. We have previously cloned and sequenced the human erythropoietin receptor gene. To examine the transcription control of gene expression, we have identified a 150 bp proximal promoter which contains binding sites for the general transcription factor, SP1, and the erythroid specific transcription factor, GATA-1, but no TATA or CAAT sequences generally associated with cellular promoters. We use gel mobility shift assays with a DNA probe consisting of the erythropoietin receptor proximal promoter to demonstrate the ability of proteins to bind to the GATA-1 and SP1 consensus sequences. These data also indicate that proteins can bind both sites simultaneously. That binding to these sites also occurs within the intact cell was supported by in vivo footprinting of the erythropoietin promoter region within the human erythroid cell line, OCIM1. In vitro footprinting also indicated proteins binding to an AP2 consensus sequence located in the region 5' to the GATA-1 binding site and another region located around -175 bp. Some protection in the region of expected AP2 binding could also be detected in the in vivo footprinting analysis. Transient transfection assays linking deletions of the proximal promoter for the erythropoietin receptor with a luciferase reporter gene in OCIM1 and HeLa cells was used to examine the functional activity of DNA sequences capable of binding nuclear proteins. Reporter gene plasmids containing these deletion mutations indicated that SP1, GATA-1 and AP2 binding sequences modulate hEpoR promoter activity. Surprisingly, residual promoter activity was still present when sequences 5' (upstream) of the SP1 binding site including the GATA-1 sequence were deleted. Co-transfection of the erythropoietin receptor promoter/luciferase reporter gene construct with an eukaryotic expression vector for GATA-1 indicated that GATA-1 is able to transactivate the erythropoietin receptor. Mutation of the GATA-1 site or SP1 site markedly reduced the erythropoietin receptor promoter activity in OCIM1 cells. Together, these data indicate that SP1 is required for activation of the erythropoietin receptor promoter and that GATA-1 likely contributes to the tissue specific expression of the erythropoietin receptor.
