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Regulatory elements of the erythropoietin gene
S Imagawa, MA Goldberg, J Doweiko and HF Bunn
Laboratory of the Howard Hughes Medical Institute, Boston, MA.
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.
Volume 77,
Issue 2,
pp. 278-285,
01/15/1991
Copyright © 1991 by The American Society of Hematology
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