Abstract
The rat aromatic l-amino acid decarboxylase (AADC) gene contains alternative promoters which direct expression of neuronal and nonneuronal mRNAs that differ only in their 5'-untranslated regions (UTRs). We have analyzed the expression of the nonneuronal promoter of the rat AADC gene in the kidney epithelial cell line LLC-PK1 and in cells which do not express the nonneuronal form of AADC by transient transfection. These studies revealed that the first 1.1 kilobases of the nonneuronal promoter, including the nonneuronal-specific 5'-UTR (Exon 1), contains sufficient information to direct tissue-specific expression. Serial deletions of this promoter localized the cis-active element to a region between -52 and -28 base pairs upstream of the nonneuronal transcription start site. An A/T-rich sequence, within this region which we have termed KL-1, was found to bind a kidney and liver-specific factor by DNase footprint analysis and was capable of directing tissue-specific expression from a heterologous promoter. Moreover, when the KL-1 sequence was mutated in the context of the entire promoter sequence, all transcriptional activity was abolished. DNA sequence comparison revealed that the KL-1 fragment is highly homologous to the binding site for hepatocyte nuclear factor-1 (HNF-1). Mobility shift studies utilizing an antibody to HNF-1 demonstrated binding of HNF-1 to the KL-1 fragment and cotransfection of HNF-1 cDNA into cells which do not express the nonneuronal form of AADC resulted in activation of transfected AADC nonneuronal promoter constructs. These results strongly suggest that the transcription factor which regulates the tissue-specific expression of the nonneuronal form of AADC mRNA is HNF-1.
Highlights
Aromatic L-amino acid decarboxylase (AADC, EC 4.1.1.28)1 catalyzes the decarboxylation of L-dopa to dopamine and 5-hydroxytryptophan to serotonin, as well as the decarboxylation of the aromatic amino acids tyrosine, tryptophan, and phenylalanine to the corresponding amines [1, 2]
While the coding region is identical in both forms, liver and kidney AADC mRNA contains a short 5Ј-untranslated region (UTR) which is entirely unrelated to the 5Ј-untranslated regions (UTRs) found in AADC mRNA expressed in tissues of neuronal origin
We demonstrate that the KL-1 element is a binding site for hepatocyte nuclear factor-1 (HNF-1) and suggest that HNF-1 is the transcription factor primarily responsible for the tissue-specific expression of the nonneuronal promoter of the AADC gene
Summary
Aromatic L-amino acid decarboxylase (AADC, EC 4.1.1.28) catalyzes the decarboxylation of L-dopa to dopamine and 5-hydroxytryptophan to serotonin, as well as the decarboxylation of the aromatic amino acids tyrosine, tryptophan, and phenylalanine to the corresponding amines [1, 2]. We have previously shown that, within the rat AADC gene, dual promoters direct the expression of these tissue-specific forms of AADC mRNA resulting in the alternative use of two untranslated exons [9]. We have previously shown that a region of the neuronal AADC promoter, containing 2.4 kilobases (kb) upstream of the transcription start site and including the untranslated exon 2, was functional in all cell lines tested, including those which do not express AADC endogenously [14]. These studies identified several cis-active elements within the neuronal promoter which controlled the activity of this promoter, but which appeared to be binding sites for ubiquitously expressed transcription factors. Because of the noradrenergic innervation of the kidney, it has been difficult to demonstrate that dopamine is produced endogenously in the kidney
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