A Response Element for the Homeodomain Transcription Factor Ptx3 in the Tyrosine Hydroxylase Gene Promoter Pilar Cazorla, Marten P. Smidt, *Karen L. O’Malley, and J. Peter H. Burbach Rudolf Magnus Institute for Neurosciences, Department of Medical Pharmacology, Medical Faculty, Utrecht University, Utrecht, The Netherlands; and *Washington University School of Medicine, St. Louis, Missouri, U.S.A. Abstract: Tyrosine hydroxylase (TH) is the rate-limiting enzyme in the biosynthesis of catecholamines, which takes place in different types of neuronal systems and nonneuronal tissues. The transcriptional regulation of the TH gene, which is complex and highly variable among different tissues, reflects this heterogeneity. We recently isolated a homeodomain transcription factor, named Ptx3, that is uniquely expressed in the dopaminergic neurons of the substantia nigra pars compacta and ven- tral tegmental area, which together form the mesence- phalic dopaminergic system. This strict localization and its coinciding induction of expression with the TH gene during development suggested a possible role for this transcription factor in the control of the TH gene. We report here the presence of a responsive element for Ptx3 located at position -50 to -45 of the rat TH promoter. Transient transfections using TH promoter constructs and electrophoretic mobility shift assays using Ptx3-con- taining nuclear extracts demonstrated that this region binds Ptx3 protein and confers a transcriptional effect on the TH gene. Depending on the cell type, the effect of Ptx3 was an eight- to 12-fold enhancement of TH pro- moter activity in Neuro2A neuroblastoma cells, or a 60 – 80% repression in nonneuronal human embryonic kidney 293 cells. Despite the close association of the Ptx3- binding site and the major cyclic AMP-response element in the TH gene, no interplay was found between Ptx3 and cyclic AMP-modulating agents. In combination with the orphan nuclear receptor Nurr1, which is required for the induction of the TH gene in mesencephalic dopaminergic neurons, the TH promoter activity to Ptx3 was enhanced in Neuro2A cells. Nurr1 alone displayed only very weak activity on the TH promoter in this cell type. The results demonstrate that the homeodomain protein Ptx3 has the potential to act on the promoter of the TH gene in a markedly cell type-dependent fashion. This suggests that Ptx3 contributes to the regulation of TH expression in mesencephalic dopaminergic neurons. Key Words: Ty- rosine hydroxylase —Ptx3—Mesencephalic dopaminer- gic neurons—Tissue specificity—Bicoid-type binding element. J. Neurochem. 74, 1829 –1837 (2000). Tyrosine hydroxylase (TH; EC 1.14.16.2) is the first and rate-limiting enzyme in the synthesis pathway of the catecholamines. Due to this role, TH expression is crit- ically modulated by multiple regulatory mechanisms (for review, see Kumer and Vrana, 1996). In the brain, TH is expressed in several cell groups, including the dopami- nergic neurons of the substantia nigra, ventral tegmental area, hypothalamus, and olfactory bulb, the noradrener- gic neurons of the locus coeruleus and lateral tegmental system, and the adrenergic neurons of the brainstem. In the periphery, TH is expressed in sympathetic ganglia and adrenal chromaffin cells. Given this wide distribu- tion, it is clear that different transcriptional regulatory mechanisms may account for regulated expression of the TH gene in different cell populations. Some positive and negative cis-regulatory elements have been identified in the control of cell type-specific expression within the TH gene (Yang et al., 1998a), but their sequence and posi- tion are not always conserved across species (Gandelman et al., 1990). The 5'-flanking region of the TH gene has been studied in great detail by using in vitro and in vivo techniques. There are several binding sites for ubiq- uitous transcription factors in the proximal TH pro- moter. These include binding sites for activating pro- tein-1 (AP1) (Angel et al., 1987), SP1 (Jones et al., 1986), and activating protein-2 (AP2) (Imagawa et al., 1987), two cyclic AMP-response elements (CRE) (Comb et al., 1986; Best et al., 1995), the Oct/HEPT motif (Dawson et al., 1994), and an E box/dyad ele- ment (Yoon and Chikaraishi, 1992). However, their relative contribution to the transcriptional activity var- ies among cell types (Wong et al., 1994) and may be differentially used in different developmental stages Received October 11, 1999; revised manuscript received December 27, 1999; accepted January 2, 2000. Address correspondence and reprint requests to Dr. J. P. H. Burbach at Rudolf Magnus Institute for Neurosciences, Department of Medical Pharmacology, Medical Faculty, Utrecht University, Universiteitsweg 100, 3584 CG Utrecht, The Netherlands. E-mail: J.P.H.Burbach@med.uu.nl Abbreviations used: AP1, activating protein-1; AP2, activating pro- tein-2; BBE, bicoid-type binding element; CRE, cyclic AMP-response element; DTT, dithiothreitol; E, embryonic day; EMSA, electro- phoretic mobility shift assay; HEK 293, human embryonic kidney 293; mesDA, mesencephalic dopaminergic; TH, tyrosine hydroxylase. 1829 Journal of Neurochemistry Lippincott Williams & Wilkins, Inc., Philadelphia © 2000 International Society for Neurochemistry