Journal of Neurochemistry Lippincott—Raven Publishers, Philadelphia © 1996 International Society for Neurochemistry The Roles of CRE, TRE, and TRE-Adjacent S 1 Nuclease Sensitive Element in the Regulation of Tyrosine Hydroxylase Gene Promoter Activity by Angiotensin II Ella L. Kim, Frederico M. Esparza, and Michal K. Stachowiak Laboratory of Molecular Neurobiology, Barrow Neurological Institute, Phoenix, Arizona, U.S.A. Abstract: The c/s elements mediating activation of the tyrosine hydroxylase gene by angiotensin II were exam- ined by transfecting tyrosine hydroxylase promoter—luci- ferase constructs into cultured bovine adrenal medullary cells. Angiotensin li-responsive elements are located within —54/+25-bp and —269/—55-bp promoter regions and were identified, respectively, as cyclic AMP (ORE)- and 1 2-O-tetradecanoylphorbol 1 3-acetate responsive element (TRE)-like sequences. Unlike ORE, TRE also supports basal promoter activity. Mutations of TRE or ORE that reduced angiotensin II stimulation abolished in vitro binding of nuclear proteins to those elements, sug- gesting that proteins forming ORE- 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 pro- teins in vitro. In supercoiled DNA it exhibited Si nuclease sensitivity and was recognized by a DNA cruciform-spe- cific antibody consistent with the extrusion of a cruciform structure that overlaps with the TRE. A mutation that abol- ished 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. Key Words: Tyrosine hydroxylase—Gene pro- moter—Angiotensin Il—DNA structure. J. Neurochem. 67, 26—36 (1996). Angiotensin II (All) is involved in the reflex stimu- lation of catecholaminergic cells of both the PNS and the CNS (reviewed by Peach, 1981; Stachowiak and Goc, 1992; Hong and Stachowiak, 1995). All en- hances depolarization-induced release of catechola- mines (Garcia-Sevila et al., 1979; Foucart et al., 1991) and increases their biosynthesis (Boadle-Biber et al., 1972). Until recently the mechanisms underlying the effects of All were unknown. We have demonstrated that stimulation of All receptors in adrenal medullary (AM) cells increases activity of tyrosine hydroxylase (TH), the first and rate-limiting enzyme in catechola- mine biosynthesis (Stachowiak et al., 1990a). An in- crease in the synthesis and steady-state levels of TH mRNA precedes an increase in enzyme activity, indi- cating transcriptional activation of the TH gene by All (Stachowiak et al., 1 990a,b). Activation of the bovine TH gene by All receptors is mediated by sequences upstream from the transcription start site (Goc and Stachowiak, 1994). The promoter region —2691—194 bp is essential for maximal stimulation by All. It con- tains a 1 2-O-tetradecanoylphorbol 1 3-acetate-respon- sive element (TRE) -like site capable of binding c-Fos and c-Jun proteins, which are induced in AM cells by stimulation of All receptors (Stachowiak et al., 1990 C; Goc et al., 1992). Because the expression of c-Fos and c-Jun from transfected plasmids is sufficient to activate the TH promoter (Goc et al., 1992), we have hypothe- sized that a TRE-like site could serve as the All-re- sponsive element. Additional All-responsive elements may be located upstream or downstream from the TRE site (Goc and Stachowiak, 1994). Although the partic- ipation of TRE and cyclic AMP-responsive element (CRE) in the activation of the TH gene promoter by specific second messenger pathways has been exam- ined (Huang et al., 1991; Kim et al., 1994), their role as well as the role of other promoter sequences in All receptor-mediated regulation of gene expression remains unknown. Ca 2~ mediates All-dependent acti- vation of TH gene expression, and calmodulin and protein kinase C are involved in this process (Stachow- iak et al., 1 990a,b). Stimulation of All receptors also is associated with increased levels of cyclic AMP in AM cells (Boarder et a!., 1988). Thus, activation of the TH gene by All could involve multiple, interacting Received December 19, 1995; revised manuscript received Febru- ary 17, 1996; accepted February 27, 1996. Address correspondence and reprint requests to Dr. M. K. Sta- chowiak at Laboratory of Molecular Neurobiology, Barrow Neuro- logical Institute, 350 West Thomas Road, Phoenix, AZ 85013, U.S.A. Abbreviations used: All, angiotensin II; AM, adrenal medullary; CRE, cyclic AMP-responsive element; DSE, dyad symmetry ele- ment; Luc, luciferase; TH, tyrosine hydroxylase; TRE, I 2-O-tetrade- canoylphorbol 13-acetate responsive element. 26