A 9.3 kb human erythropoietin (hEPO) encoding genomic clone has been isolated from a leukocyte genomic library and completely sequenced. Based on the sequence analysis, a CAAT box, TATA boxes and other transcriptional regulatory elements in its 5'-flanking region were identified. We also found that 3'-flanking region contains nitrogen-regulatory/oxygen-sensing consensus sequences, tissue-specific regulatory elements, lymphokine responsive element as well as binding sites for AP and SP1. Since the 5'- flanking region contains cytokine-responsive consensus sequences and metal-responsive elements, the effects of cytokines and metal ions on the expression of hEPO gene have been examined in the Hep3B cell line. We have demonstrated that the hEPO gene expression is stimulated by cobalt chloride and the stimulation is further enhanced by the addition of IL6 and IL11 to the culture medium. We also have initiated mapping of the 5'-flanking region in order to identify cellular factors that interact with the IL6 responsive elements and metal responsive element. We will also attempt to determine how such interactions regulate the expression of EPO.
