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Journal of Neuroscience, Vol 10, 520-530, Copyright © 1990 by Society for Neuroscience
Identification of a functional glucocorticoid response element in the phenylethanolamine N-methyltransferase promoter using fusion genes introduced into chromaffin cells in primary culture
ME Ross, MJ Evinger, SE Hyman, JM Carroll, L Mucke, M Comb, DJ Reis, TH Joh and HM Goodman
Department of Molecular Biology, Massachusetts General Hospital, Boston 02114.
The rat gene encoding phenylethanolamine N-methyltransferase (PNMT) was
cloned and a consensus sequence for a glucocorticoid response element (GRE)
was found at -513 bp, 5' to the transcriptional start site. In order to
define the function of this element, fusion genes containing the PNMT
promoter and a chloramphenicol acetyltransferase (CAT) reporter gene were
constructed. These constructs did not express after transfection into any
of 7 continuous cell lines, none of which endogenously produce PNMT. A
system for transfecting chromaffin cells in primary culture was therefore
devised using constructs containing 200 bp of the proenkephalin (ENK)
promoter, whose expression characteristics are well known. pENK beta GAL-1,
containing the ENK promoter with a lac Z reporter, was introduced into
these cells and beta-galactosidase activity was visualized in situ.
Approximately 90% of cells transfected were chromaffin; transfection
efficiency was 5%. High levels of CAT activity were measured in chromaffin
cells transfected with pENKAT12, possessing a CAT reporter. In contrast to
tumor cell lines, pENKAT12 induction in these cells by forskolin and
phorbol esters did not require a phosphodiesterase inhibitor. In this
chromaffin system, both basal and regulated expression of the PNMT fusion
genes were detected. Dexamethasone (dex) induced expression of pPNMT3000
and pPNMT900, containing the putative GRE and 3000 bp or 863 bp of PNMT
promoter sequence, 4- to 10-fold. Expression of pPNMT300 and pPNMT100,
which lack the GRE and contain 273 bp or 99 bp of PNMT promoter sequence,
was unaffected by dex. Addition of the PNMT region spanning -490 to -863 bp
conferred full dex responsiveness to a thymidine kinase promoter. Deletion
of the putative GRE sequence by site-directed mutagenesis abolished the dex
response. These data identify the sequence at -513 bp in the rat PNMT gene
as a functional, positively acting GRE. Primary cultures of bovine
chromaffin cells provide a biologically relevant expression system for
transcriptional studies of catecholamine genes and their related
neuropeptides.
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