GATA-3 regulates the transcriptional activity of tyrosine hydroxylase by interacting with CREB Seok Jong Hong, Youngbuhm Huh, Han Chae, Sunghoi Hong, Thomas Lardaro and Kwang-Soo Kim Molecular Neurobiology Laboratory, McLean Hospital, Harvard Medical School, Belmont, Massachusetts, USA Abstract The zinc finger transcription factor GATA-3 is a master regulator of type 2 T-helper cell development. Interestingly, in GATA-3–/– mice, noradrenaline (NA) deficiency is a proximal cause of embryonic lethality. However, neither the role of GATA-3 nor its target gene(s) in the nervous system were known. Here, we report that forced expression of GATA-3 resulted in an increased number of tyrosine hydroxylase (TH) expressing neurons in primary neural crest stem cell (NCSC) culture. We also found that GATA-3 transactivates the promoter function of TH via specific upstream sequences, a domain of the TH pro- moter residing at )61 to )39 bp. Surprisingly, this domain does not contain GATA-3 binding sites but possesses a binding motif, a cAMP response element (CRE), for the transcription factor, CREB. In addition, we found that site-directed mutation of this CRE almost completely abolished transactivation of the TH promoter by GATA-3. Furthermore, protein–protein interaction assays showed that GATA-3 is able to physically interact with CREB in vitro as well as in vivo. Based on these results, we propose that GATA-3 may regulate TH gene transcription via a novel and distinct protein–protein interaction, and directly con- tributes to NA phenotype specification. Keywords: neural crest stem cells, protein–protein inter- action, sympathetic neuron, transcription factors, tyrosine hydroxylase. J. Neurochem. (2006) 98, 773–781. Vertebrate nervous system development is regulated by a complex regulatory network of extracellular signals and nuclear transcription factors controlling the process of cell fate specification of vast neural subtypes (Marquardt and Pfaff 2001; Goridis and Rohrer 2002). Among the various phenotypes of a particular neuron, neurotransmitter identity is an important feature because it determines the nature of the chemical neurotransmission a given neuron will mediate, and influences its specific connectivity with target cells. As indicated by the group nomenclature of noradrenaline (NA)-producing cells (i.e. group A1–A7) in the CNS, the NA cell type is one of the earliest neurotransmitter systems to be defined by neuroscientists (Cooper et al. 1996). In the CNS, NA is mainly produced in the locus coeruleus (A4 and A6 cell groups) by sympathetic ganglia (SG) in the peripheral nervous system (PNS). The regulatory cascade controlling the NA neurotransmitter phenotype has been extensively studied, leading to the identification and functional characterization of critical signaling molecules and transcription factors. For example, bone morphogenic proteins (BMPs) are essential for sympathetic nervous system (SNS) development and induce the expression of the proneural gene, Mash1 (or Cash1) (Brunet and Pattyn 2002; Goridis and Rohrer 2002; Howard 2005). Thus, Mash1 is the first transcription factor shown to be essential for NA neuron development (Guillemot et al. 1993). Downstream of Mash1 lies the homeodomain transcription factor, Phox2a, which is a critical regulator of NA cell lineage development (Morin et al. 1997; Guo et al. 1999; Lo et al. 1999; Stanke et al. 1999). The closely related transcription factor, Phox2b, is also induced by BMPs independently of Mash1 and is another essential regulator of NA neuron development (Pattyn et al. 1999). Received December 13, 2005; revised manuscript received March 14, 2006; accepted March 15, 2006. Address correspondence and reprint requests to Kwang-Soo Kim, Molecular Neurobiology Laboratory, McLean Hospital, Harvard Medical School, 115 Mill Street, Belmont, MA 02478, USA. E-mail: kskim@mclean.harvard.edu Abbreviations used: aa, amino acid; bZip, basic region-leucine zipper; CEE, chick embryo extract; CRE, cAMP response element; CREB, cAMP response element binding protein; d.b.h., dopamine b-hydroxy- lase; DMEM, Dulbecco’s modified Eagle’s medium; EMSA, electroph- oretic mobility shift assay; GFP, green fluorescent protein; MOI, multiplicity of infection; NA, noradrenaline; NCSC, neural crest stem cells; PNS, peripheral nervous system; RSV, Rous Sarcoma Virus; SG, sympathetic ganglia; SNS, sypathetic nervous system; TH, tyrosine hydroxylase; Th2, type 2 T helper cells. Journal of Neurochemistry , 2006, 98, 773–781 doi:10.1111/j.1471-4159.2006.03924.x Ó 2006 The Authors Journal Compilation Ó 2006 International Society for Neurochemistry, J. Neurochem. (2006) 98, 773–781 773