European Journal of Neuroscience, Vol. 10, pp. 508–521, 1998 © European Neuroscience Association CRE and TRE sequences of the rat tyrosine hydroxylase promoter are required for TH basal expression in adult mice but not in the embryo Caryn Trocme ´, 1,2 Chamsy Sarkis, 1,2 Jean-Michel Hermel, 2 Rene ´ Duchateau, 2 Stephen Harrison, 3,5 Michel Simonneau, 4,6 Raya Al-Shawi 3,7 and Jacques Mallet 2 1 Both authors contributed equally to this work. 2 Laboratoire de Ge ´ne ´tique Mole ´culaire de la Neurotransmission et des Processus Neurode ´ge ´ne ´ratifs, CNRS-UMR C9923, Ho ˆpital de la Pitie ´ Salpe ˆ trie `re, Paris, France 3 Center for Genome Research, AFRC, University of Edinburgh, Edinburgh, UK 4 Laboratoire de Neurobiologie Cellulaire et Mole ´culaire, CNRS, Gif-sur-Yvette, France 5 Present address: National Institute of Medical Research, the Ridgeway, Mill Hill, London, UK 6 Present address: Laboratoire de Pharmacoge ´ne ´tique et Abords The ´rapeutiques des Maladies He ´re ´ditaires, INSERM U458, Ho ˆpital Robert Debre ´, Paris, France 7 Present address: Department of Anatomy and Developmental Biology, Royal Free Hospital School of Medicine, Rowland Hill Street, London, UK Keywords: cAMP-responsive element, embryonic development, gene regulation, tissue-specific expression, TPA-responsive element, transgenic mice, tyrosine hydroxylase Abstract Tyrosine hydroxylase (TH), the rate-limiting enzyme in the biosynthesis of catecholamine neurotransmitters, is expressed in a restricted number of areas, and subject to numerous regulations during development and in adulthood. Two transcription factor binding sites present in the proximal region of the TH gene, the TPA- responsive element (TRE) and the c-AMP responsive element (CRE), have been shown to play important roles in TH gene regulation in vitro. In order to elucidate in vivo the role of these two sites, we produced transgenic mice bearing a 5.3-kb fragment from the 5' flanking sequence of the TH gene with mutations in either the CRE- or TRE-sites. Using the intact 5.3-kb fragment fused to two different reporter genes (HSV1-tk and lacZ), we show that this promoter fragment is able to specifically direct expression in catecholaminergic tissues both in adult mice and embryos. Interestingly, the CRE- and TRE-mutated transgenes were not expressed in adult mice, contrary to the situation in embryos where they were specifically expressed in catecholaminergic regions. These results demonstrate that the CRE and TRE play an essential role in basal TH expression in adult tissues in vivo. Moreover, they suggest that distinct transcription factors are involved in TH regulation in developing and adult tissues. In support of this, gel mobility shift experiments revealed a complex present only in embryonic tissues. Taken together, these data highlight the diversity of the mechanisms underlying the establishment and maintenance of the catecholaminergic phenotype. Introduction Tyrosine hydroxylase (TH, EC 1.14.16.2) is the first and rate-limiting enzyme in the biosynthesis of catecholamines, which are involved in a wide variety of important physiological functions. Therefore, the regulation of TH protein level and activity represents the central means for controlling the synthesis of these neurotransmitters. Nearly every form of documented regulation has been shown to modulate TH expression (Kumer & Vrana, 1996), as illustrated for example by the multiple studies concerning the trans-synaptic induction of TH in the adaptive response to diverse environmental stimuli (Faucon Biguet et al., 1986; Zigmond et al., 1989; Icard-Liepkalns et al., 1992). TH is expressed in a restricted number of brain nuclei, in sympathetic Correspondence: J. Mallet, as above. Received 8 July 1997, revised 1 September 1997, accepted 16 September 1997 ganglia, and in adrenal medulla (Ho ¨kfelt et al., 1984). During develop- ment, TH is also transiently expressed in neurones which do not maintain a catecholaminergic phenotype in the adult, as well as in some non-neural cells (Cochard et al., 1978; Teitelman et al., 1981; Specht et al., 1981b; Jaeger & Joh, 1983; Berger et al., 1985; Jonakait et al., 1985; Nagatsu et al., 1990; Hess & Wilson, 1991). Thus, it is very likely that the TH gene requires multiple regulatory elements to allow for the plasticity and diversity of its expression. Numerous studies aimed at delineating cis-acting DNA elements that direct basal, tissue-specific and induced TH expression (for a review, see Kumer & Vrana, 1996). The first 212 bp of the rat TH