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J Biol Chem, Vol. 274, Issue 10, 6507-6518, March 5, 1999
From the Department of Neurology and Department of Anatomy and
Neurobiology, University of Tennessee College of Medicine,
Memphis, Tennessee 38163 and The synaptic action of
norepinephrine is terminated by NaCl-dependent uptake into
presynaptic noradrenergic nerve endings, mediated by the norepinephrine
transporter (NET). NET is expressed only in neuronal tissues that
synthesize and secrete norepinephrine and in most cases is co-expressed
with the norepinephrine-synthetic enzyme dopamine
A Previously Undescribed Intron and Extensive 5' Upstream
Sequence, but Not Phox2a-mediated Transactivation, Are Necessary
for High Level Cell Type-specific Expression of the Human
Norepinephrine Transporter Gene
, and Department of Psychiatry,
Veterans Affairs Connecticut Health Care System and Yale University
School of Medicine, West Haven, Connecticut 06516
-hydroxylase
(DBH). To understand the molecular mechanisms regulating human NET
(hNET) gene expression, we isolated and characterized an hNET genomic
clone encompassing approximately 9.5 kilobase pairs of the 5' upstream
promoter region. Here we demonstrate that the hNET gene contains an
as-yet-unidentified intron of 476 base pairs within the 5'-untranslated
region. Furthermore, both primer extension and 5'-rapid amplification
of cDNA ends analyses identified multiple transcription start sites
from mRNAs expressed only in NET-expressing cell lines. The start
sites clustered in two subdomains, each preceded by a TATA-like
sequence motif. As expected for mature mRNAs, transcripts from most
of these sites each contained an additional G residue at the 5'
position. Together, the data strongly support the authenticity of these
sites as the transcriptional start sites of hNET. We assembled
hNET-chloramphenicol acetyltransferase reporter constructs containing
different lengths of hNET 5' sequence in the presence or the absence of
the first intron. Transient transfection assays indicated that the
combination of the 5' upstream sequence and the first intron supported
the highest level of noradrenergic cell-specific transcription.
Forced expression of the paired-like homeodomain transcription factor Phox2a did not affect hNET promoter activity in NET-negative cell lines, in marked contrast to its effect on a DBH-chloramphenicol acetyltransferase reporter construct. Together with our previous studies suggesting a critical role of Phox2a for noradrenergic-specific expression of the DBH gene, these data support a model in which distinct, or partially distinct, molecular mechanisms regulate cell-specific expression of the NET and DBH genes.
Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.
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