title: Regulatory elements of the erythropoietin gene.
abstract: Because the human hepatoma cell line Hep3B produces erythropoietin (Epo) in a regulated fashion, it can be used to investigate the cis-acting regulatory elements of the Epo gene. Comparison of primate and mouse sequences shows strong homology not only in the coding sequence but also within the 5' flanking region, the first intron, and the 3' flanking region. These portions of the Epo gene were inserted 5' and 3' to a reporter gene, human growth hormone (GH). 5A is a 1,192-base pair (bp) HindIII-Xbal fragment that extends from 378 bp 5' to the cap site through the first intron. To obviate the problem of false initiation of translation from the Epo ATG start codon, this site was changed to TAG by site-directed mutagenesis. 3A is a 255-bp Accl-BglII fragment that extends 67 bp upstream from the Epo termination codon and covers most of the 3' noncoding region of homology. The plasmid DNAs were transfected by electroporation into Hep3B cells with RSVCAT as an internal standard to correct for transfection efficiency. One aliquot of cells was exposed to 50 mumol/L CoCl2 or to 1% O2. At the end of the incubations, GH and Epo were measured in the cell media and the cell pellet was assayed for CAT. Production of GH was stimulated 1.7-fold by cobalt or hypoxia. Furthermore, addition of 3A to the GH gene, irrespective of orientation, stimulated GH production 2.6-fold with CoCl2 and 2.3-fold with hypoxia. Stable cell lines were produced by cotransfection of the above constructions, along with the selectable marker pSV-Neo. In two clones, exposure to hypoxia resulted in much more marked (16-fold) induction of GH. Stimulus of both GH and Epo production by hypoxia was partially abrogated by carbon monoxide. These results demonstrate the presence of promoter and enhancer elements within the human Epo gene that are appropriately responsive to hypoxia and cobalt.
authors.affiliation: Laboratory of the Howard Hughes Medical Institute, Boston, MA.
authors.forename: S
authors.initials: S
authors.lastname: Imagawa
authors.forename: M A
authors.initials: MA
authors.lastname: Goldberg
authors.forename: J
authors.initials: J
authors.lastname: Doweiko
authors.forename: H F
authors.initials: HF
authors.lastname: Bunn
mesh_terms: D000818:Animals; D002460:Cell Line; D002522:Chlorocebus aethiops; D003035:Cobalt / Q000494:pharmacology; D004921:Erythropoietin / Q000235:genetics*; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D000860:Hypoxia / Q000503:physiopathology; D066298:In Vitro Techniques; D012045:Regulatory Sequences, Nucleic Acid; D014158:Transcription, Genetic; D014162:Transfection
chemical_list: D004921:Erythropoietin; D003035:Cobalt