The Roles of CRE, TRE, and TRE-Adjacent S1 Nuclease Sensitive Element in the Regulation of Tyrosine Hydroxylase Gene Promoter Activity by Angiotensin II

Abstract: The cis elements mediating activation of the tyrosine hydroxylase gene by angiotensin II were examined by transfecting tyrosine hydroxylase promoter-luciferase constructs into cultured bovine adrenal medullary cells. Angiotensin II-responsive elements are located within −54/+25-bp and −269/−55-bp promoter regions and were identified, respectively, as cyclic AMP (CRE)- and 12- O -tetradecanoylphorbol 13-acetate responsive element (TRE)-like sequences. Unlike CRE, TRE also supports basal promoter activity. Mutations of TRE or CRE that reduced angiotensin II stimulation abolished in vitro binding of nuclear proteins to those elements, suggesting that proteins forming CRE- and TRE-inducible complexes may mediate angiotensin II stimulation. The TRE is adjacent to a dyad symmetry element. Those two sites form a common regulatory unit in which the dyad symmetry element acts as a repressor of the TRE site. Isolated dyad symmetry element did not bind nuclear proteins in vitro. In supercoiled DNA it exhibited S1 nuclease sensitivity and was recognized by a DNA cruciform-specific antibody consistent with the extrusion of a cruciform structure that overlaps with the TRE. A mutation that abolished formation of the cruciform correlated with a loss of repressor activity. We propose a novel model of tyrosine hydroxylase gene regulation in which functions of the TRE are modulated via structural transition in the adjacent DNA.     

Sequences Distal to the AP1/E Box Motif Are Involved in the Cell Type-Specific Expression of the Rat Tyrosine Hydroxylase Gene

Abstract: In order to define cell type-specific elements associated with the catecholamine biosynthetic enzyme, tyrosine hydroxylase (TH), transient transfections of promoter deletion constructs were used to test relative reporter-gene activities in TH-expressing and-nonexpressing cell lines. Such assays demonstrated that a region between-503 and-578 contributed to rat TH promoter activity in the pheochromocytoma cell line PC12. Deletion of these sequences resulted in a 66% loss in cell type-specific activity. Mutations within the E box/dyad symmetry element (CAGGTGCCTGTGACAGTG) did not affect the basal and cell type-specific pattern of expression exhibited by the rat TH promoter. Promoter fusion constructs between the rat TH promoter (-741 and-197) and the human TH promoter (-197 and +1) exhibited reporter-gene activities equivalent to that of wild-type-741 rat TH constructs, further demonstrating that sequence elements upstream of the rat E box/dyad symmetry are important for cell type-specific expression. Gel-shift experiments indicated that a PC12 nuclear factor could bind to a 39-bp sequence within this region in a cell type-specific manner. The size of this factor was 52 kDa as determined by UV cross-linking experiments.