Use of a marked erythropoietin gene for investigation of its cis-acting elements

To examine the function of conserved noncoding regions in the erythropoietin (Epo) gene, we have prepared clones and pools of Hep3B cells stably transfected with a marked 4.1-kilobase Epo gene and deletions thereof. The marked transcripts had single base substitutions at three sites in the coding portion of Exam 5, enabling them to be distinguished from endogenous Epo mRNA by ribonuclease protection and competitive polymerase chain reaction. The basal expression and hypoxic induction of the marked Epo gene that had no deletions were indistinguishable from that of the endogenous Epo gene. Likewise, deletion of conserved intervening sequence 1 had minimal effect on hypoxic induction. In contrast, a 3'-deletion that included the conserved 3'-enhancer element resulted in a substantial, but not complete, suppression of hypoxic induction while a 3'-deletion downstream of the enhancer resulted in enhancement. A 188-base pair deletion of a conserved 3'- untranslated region in Exon 5 had minimal effect on hypoxic induction. However, the truncated Epo mRNA had a markedly prolonged half-life (15 h) in comparison to the endogenous Epo mRNA (2.0 h) or the marked full-length Epo mRNA (2.1 h). Further deletions in the 3'-UTR showed that a relatively small region of approximately 50 bases is responsible for the relatively rapid turnover of Epo mRNA. These experiments provide formation on cis-acting elements of the Epo gene that cannot be obtained from conventional reporter gene transfection experiments.