Abstract
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 beta-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.
Highlights
The synaptic action of norepinephrine is terminated by NaCl-dependent uptake into presynaptic noradrenergic nerve endings, mediated by the norepinephrine transporter (NET)
Isolation and Characterization of human NET (hNET) Genomic Clones—A 0.5-kb rat NET cDNA fragment corresponding to the base pairs 990 –1512 of the coding sequence [21] was radiolabeled by the random primer method and used as the probe to screen a EMBL3 human genomic library derived from placenta chromosomal DNA (CLONTECH, Mountain View, CA)
The experiments presented here have defined critical aspects of the promoter structure and function of hNET gene, including the following: (i) the location of transcription start site(s); (ii) the sequence of the full 5Ј-untranslated leader sequence; (iii) the sequence of approximately 5 kb of the 5Јflanking promoter region, including a search for potential cisregulatory motifs; (iv) most unexpected, an as-yet-unidentified region of the hNET gene that appears by multiple criteria to be the first intron and which resides in the middle of the 5Јuntranslated leader sequence; and (v) that the combination of the upstream 9-kb sequence and the first intron is critical for the high level, noradrenergic-specific transcriptional activity of the hNET gene
Summary
Vol 274, No 10, Issue of March 5, pp. 6507–6518, 1999 Printed in U.S.A. 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*. We demonstrate that the hNET gene contains an as-yet-unidentified intron of 476 base pairs within the 5-untranslated region Both primer extension and 5rapid amplification of cDNA ends analyses identified multiple transcription start sites from mRNAs expressed only in NET-expressing cell lines. Transient transfection assays of these reporter constructs in both NETpositive and NET-negative cell lines show that combination of the 5Ј upstream sequences and the first intron is critical for high level noradrenergic cell type-specific promoter activity of the hNET gene. The paired-like homeodomain transcription factor Phox2a, which is critical for noradrenergic cell type-specific DBH promoter activity [26, 27], appears not to regulate the promoter activity of the hNET gene
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