In order to characterize the specificity of expression of the neurotransmitter biosynthetic gene dopamine beta-hydroxylase (DBH), the identification of proteins that interact with the DB1 enhancer was initiated. A homeobox-containing cDNA was isolated from a PC12 expression cDNA library screened with the DB1 enhancer. The homeodomain is a member of the paired-like class, and is encoded by several nonidentical cDNAs. The cDNAs contain the same sequence in the homeodomain and 3' coding and noncoding sequences, but diverge in sequence 5' to the homeodomain. This family of homeobox-containing cDNAs is named Arix. Arix mRNA transcripts are found only in noradrenergic, DBH-positive tissues, and in cell lines derived from those tissue. The DB1 enhancer contains two binding sites for the Arix homeodomain, and both sites contribute to basal activity of the DBH promoter. When introduced into tissue culture, Arix regulates the transcriptional activity from the DBH promoter, and also from the promoter of the tyrosine hydroxylase gene, encoding the initial enzyme of the catecholamine biosynthetic pathway. The pattern of expression of the Arix transcripts, the presence of the homeodomain, and the transcriptional regulatory properties suggest that this family of proteins may be involved in the specificity of expression of the catecholamine biosynthetic genes.
Expression of the gene encoding the neurotransmitter biosynthetic enzyme dopamine β‐hydroxylase (DBH) is regulated in a tissue‐specific pattern, and transcription is influenced by environmental stimuli. Using the promoter proximal region of the rat DBH gene and nuclear extracts from SHSY‐5Y neuroblastoma cells, a DNA‐protein complex was identified that is competitive with oligonucleotides containing the recognition site of transcription factor AP‐2. DNase footprint analysis identified an AP‐2 binding site between −136 and −115 of the DBH promoter. Mutation of that AP‐2 site results in a sevenfold reduction of basal reporter gene expression, but second messenger‐stimulated activity is retained. Cotransfection of an AP‐2 expression vector and a DBH promoter‐reporter construct into cultured cells results in a sixfold stimulation of reporter gene expression, demonstrating the ability of AP‐2 to trans‐activate the DBH promoter. These results identify a new regulatory element on the rat DBH gene and suggest that the AP‐2 site plays a role in maintaining basal levels of DBH transcription.
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