Neuron-specific expression of the human dopamine beta-hydroxylase gene requires both the cAMP-response element and a silencer region.

H Ishiguro,K.T Kim,T.H Joh,K.S Kim

doi:10.1016/s0021-9258(17)46802-5

H Ishiguro, K.T Kim + Show 2 more

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https://doi.org/10.1016/s0021-9258(17)46802-5

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Abstract

Dopamine beta-hydroxylase (DBH), the enzyme catalyzing the conversion of dopamine to norepinephrine, is specifically expressed in adrenergic and noradrenergic neurons in the central nervous system. DNase I hypersensitive sites were found in the 5'-flanking region of the DBH gene in noradrenergic human neuroblastoma SK-N-BE(2)C cells, but not in DBH-nonexpressing HeLa cells. We report here that the 4.3-kilobase upstream sequence of the human DBH gene confers cell type-specific expression as assessed by transient expression assay. Furthermore, deletional and mutational analyses revealed two genetic regulatory elements required for the regulation of cell type specificity. First, deletion of the cAMP-response element (CRE) abolished > 95% of the transcriptional activity by the DBH upstream promoter, thus implicating the CRE as an essential positive genetic element. Second, deletion of a region between -490 and -263 base pairs resulted in 10-fold increase of reporter gene activity only in HeLa cells, indicating that this region contains a cell-specific silencer. A 13-base pair fragment residing within that region shows 77% sequence identity with the neuron-specific silencer motif recently identified in two neuronal genes, i.e. SCG10 and type II sodium channel genes. We propose that the interplay between the CRE and this neuron-specific silencer region plays an important role in the tissue-specific expression of the DBH gene in noradrenergic cells.

Highlights

Neuron-specific Expressionof the Human Dopamine @-Hydroxylase Gene Requires Both the CAMP-Response Element and a Silencer Region*
We report here the results of experiments to elucidate molecular mechanisms regulating expression of the DBH gene in ments required for the regulatioofncell type specific- the norepinephrine-synthesizinghuman neuroblastoma SK
Deletion of the CAMP-response element N-BE(2)C cell line (Ciccarone et al, 1989), as well as those (CRE)abolished ~ 9 5 %of the transcriptional activity contributing to suppression of DBH expression in the nonby the DBH upstream promoter, implicating the neuronal HeLa cell line

Summary

RESULTS

None of the fusion constructs containing the promoter sequences deleted between -4.3 kb and -486 bp showed CAT activity higher than that directed by pBLCAT3 (Fig. 3C), indicating that the 486-bp upstream cell lines which differed with respect to DBHgene expression, sequence contains sufficient information to suppress expresboth 4.3CAT and 2.6CAT exhibited cell-type specificity in sion in HeLa cells. To further characterize the negative region, we deleted conjunction with the observation that all threeDNase I HSSs the sequence between -490 and -262 bp inthe 604CAT identified in the active DBH gene were localized within 2.6 plasmid (Fig. 5 A ) and compared the promoter activity of the kb upstream of the transcriptional initiation site suggested resulting construct in both cell lines (Fig. 5B).

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Findings

DISCUSSION