Identification and Characterization of a Novel Phorbol Ester-responsive DNA Sequence in the 5′-Flanking Region of the Human Dopamine β-Hydroxylase Gene

Hiroshi Ishiguro,Kouji Yamada,Naohiro Ichino,Toshiharu Nagatsu

doi:10.1074/jbc.273.34.21941

Hiroshi Ishiguro, Kouji Yamada + Show 2 more

Open Access

https://doi.org/10.1074/jbc.273.34.21941

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Abstract

The phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), enhances transcription of many eukaryotic genes, including that for dopamine beta-hydroxylase (DBH). In the present study, we report identification and characterization of a novel sequence motif residing in the 5'-flanking region of the human DBH gene, which mediates transcriptional induction by TPA. Deletional analyses indicated the promoter region between -223 and -187 base pairs to be critical. Whereas this region does not contain any putative regulatory motifs with significant sequence homology to the AP-1 motif, extensive deletional and site-directed mutational analyses indicated that a sequence between -210 and -199 base pairs, 5'-ATCCGCCTGTCT-3', may represent a novel TPA-response element (TRE). In addition, alteration of the YY1-binding site decreased TPA-mediated induction of the DBH promoter activity, suggesting that contiguous cis-regulatory element(s) cooperate with this novel sequence motif. Furthermore, insertional mutation analyses between the YY1-binding site and the cyclic AMP-responsive element indicated that the stereospecificity of these motifs is important for intact transcriptional induction by TPA. Taken together, these data suggest that transcriptional up-regulation of the human DBH gene in response to TPA requires coordination of a novel TRE (human DBH TRE, hDTRE), cyclic AMP-responsive element, and the YY1-binding site.

Highlights

Dopamine ␤-hydroxylase (DBH, dopamine ␤-monooxygenase: EC 1.14.17.1)1 catalyzes the conversion of dopamine to norepinephrine in the biosynthesis pathway of catecholamines
Analyses indicated that a negative regulatory region (Ϫ282 bp to Ϫ232 bp) that controls the tissue-specific expression of the rat DBH gene exists upstream of the DB-1 enhancer and that the activator protein 2 (Ϫ129 bp to Ϫ120 bp) element plays a role in maintaining basal levels of rat DBH transcription [17, 18]
To identify the TPA-responsive DNA region of the human DBH promoter, we examined a series of deletional constructs containing different lengths of the upstream DBH region fused to the reporter luciferase gene in transient transfection assay

Summary

TRE of Human DBH Gene

The zinc finger-type nuclear protein YY1 is widely expressed in the nuclear protein fraction and regulates the transcriptional level of many viral (19 –25) as well as cellular (26 – 41) genes. YY1, TFIIB, and RNA polymerase II function to transcribe the DNA directly without a TATA-binding protein [45]. YY1 interacts with TATA-binding protein, CREB-binding protein, TFIIB, and TAFII55 [46], implying that these basal transcriptional factors may interact with one other in offering the human DBH promoter. We identified a TPA-responsive element (TRE) in the promoter region of the human DBH gene (hDTRE) and a hDTRE-binding protein. The response to TPA stimulation was determined and characterized using combinations of the DNA element with hDTRE-binding protein, YY1, and CREB (or CREB-binding protein). The mechanism of the TPA response in the hDTRE was found to differ from that of AP-1 (TGA(C/G)TCA) in many other genes

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION