Multiple Protein Factors Interact with the cis-Regulatory Elements of the Proximal Promoter in a Cell-Specific Manner and Reg\ ulate Transcription of the Dopamine b-Hydroxylase Gene

Abstract

The dopamine ␤-hydroxylase (DBH) gene is expressed selectively in noradrenergic and adrenergic neurons and neuroendocrine cells in the nervous system. A cAMP response element (CRE) residing at Ϫ181 to Ϫ174 bp from the transcription start site of the human DBH gene seems to be essential for DBH transcription. Potential cis-regulatory motifs such as AP1 and YY1 occur proximal to and overlap this CRE, endowing the area with a composite promoter structure. Using the DBHexpressing human neuroblastoma SK-N-BE(2)C and DBHnegative HeLa cell lines as model systems, we report here that this CRE/YY1/AP1 area interacts with multiple nuclear proteins, including CRE-binding protein (CREB) and transcription factor YY1 in a cell-specific manner. In support of the notion that multiple proteins bind to the CRE/YY1/AP1 area, DNase I footprinting analysis has demonstrated that nuclear extracts protect an extended region (from Ϫ186 to Ϫ150 bp) relative to that protected by the purified CREB (from Ϫ186 to Ϫ171 bp).Site-directed mutational analysis has revealed differential roles of potential cis-regulatory motifs in regulation of DBH transcription. Strikingly, the YY1 element positively regulated basal DBH transcription while simultaneously regulating cAMP-mediated induction negatively, which is a novel mechanism of promoter function. Furthermore, three additional DNA-binding sites have been identified by DNase I footprint analysis in the upstream 260 bp promoter region of the human DBH gene, of which two sites are cell-specific. These results support a model whereby multiple proteins bind to the 5Ј-proximal area in a cell-specific manner and coordinately regulate the cell type-specific transcriptional activation of the DBH gene.

Orphan nuclear receptor Nurr1 directly transactivates the promoter activity of the tyrosine hydroxylase gene in a cell‐specific manner

“…A consensus CRE was described in previous studies (Seo et al 1996). The sense and antisense oligonucleotides were annealed, gel-purified, 32 P-labeled with T4 DNA kinase, and used as probes in EMSA.…”

“…EMSA was performed using 40 000 cpm of the labeled probe (approximately 0.05-0.1 ng) and in vitro translated Nurr1 protein in a final volume of 20 lL of 12.5% glycerol, 12.5 mM HEPES (pH 7.9), 4 mM Tris-HCl (pH 7.9), 60 mM KCl, 1 mM EDTA, and 1 mM dithiothreitol with 1 lg of poly(dI-dC) as described previously (Seo et al 1996). Competition binding assays were performed by adding non-radioactive competitor oligonucleotides in a molar excess prior to adding 32 P-labeled oligonucleotides.…”

“…For NL3, pTH2.4CAT was used as a PCR template with a 32 P-labeled 5¢-CTTCC-ATGGTACCCCGAG-3¢, which represents upstream from ) 843 to ) 861 bp of the TH gene and an unlabeled 5¢-ATGGGCCCAGCA-CAACTC-3¢, which represents upstream ) 1003 to ) 985 bp of the TH gene. Approximately 30 000 cpm of the labeled probe was incubated with 27 lL of the in vitro translated Nurr1 protein, and subjected to DNase I digestion in 1 · binding buffer, which contains 20 mM HEPES (pH 7.9), 2 mM MgCl 2 , 50 mM NaCl, 1 mM dithiothreitol, 0.1 mM EDTA, and 10% glycerol, as previously described (Seo et al 1996). The amount of DNase I was adjusted empirically for each reaction to produce an even pattern of partially cleaved DNA fragments.…”

Modeling Transcriptional Control in Gene Networks—Methods, Recent Results, and Future Directions

“…A second issue is that the dynamic possibilities associated with many biochemical architectures observed in genetic regulatory systems have not yet been assessed by modeling. Examples of such architectures are convergence of different signaling pathways through distinct transcription factors onto a particular gene (Howard and Maurer, 1995) heterodimerization of transcription factors in two separate pathways with a common third transcription factor (Hunter et al, 1996), and conditional regulation by a single TF [e.g., the TF YY1 mediating both enhancement of basal transcription and suppression of cAMP-induced transcription of the dopamine β-hydroxylase gene (Seo et al, 1996)]. Such architectures may be expected to provide alternative mechanisms for generating dynamic phenomena such as multistability and oscillations.…”

MAP Kinase–Independent Signaling in Angiotensin II Regulation of Neuromodulation in SHR Neurons

“…23,24 The signaling pathway activates transcription factors relevant to AP-1 binding sites (Fos, Jun, serum response factor), which are proposed to interact with the promotor regions of the NET, TH, and dopamine ␤-hydroxylase (D␤H) genes and to stimulate their transcription. [24][25][26] In view of this recently uncovered signaling mechanism and our previous observations that AT 1 receptor gene expression in SHR neurons is higher compared with that in Wistar-Kyoto rat (WKY) neurons, 13 we decided to compare the AT 1 receptor-mediated signal transduction mechanism between the 2 strains of neurons. We hypothesized that the Ras-Raf-1-MAP kinase pathway would be stimulated by AT 1 receptor activation in parallel with its increased expression of these receptors in the SHR neurons.…”