ORIGINAL ARTICLE Transcriptional Regulation of Arylalkylamine-N-Acetyltransferase-2 Gene in the Pineal Gland of the Gilthead Seabream B. Zilberman-Peled,* 1 L. Appelbaum,*àà 1 D. Vallone,à N. S. Foulkes,à S. Anava,* A. Anzulovich,§ S. L. Coon,– D. C. Klein,– J. Falco ´n,** B. Ron  and Y. Gothilf  *Department of Zoology and  Department of Neurobiochemisty, George S. Wise Faculty of Life Sciences, Tel Aviv University, Israel. àMax-Planck-Institut fur Entwicklungsbiologie, Tubingen, Germany. §Department of Biochemistry and Biological Sciences, Faculty of Chemistry, Biochemistry and Pharmacy, San Luis University, San Luis, Argentina. –National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland, USA. **Laboratoire Arago, CNRS and University Pierre and Marie Curie, Banyuls sur Mer, France.   Israel Oceanographic and Limmnological Research, Eilat, Israel. Endogenous circadian oscillators that drive daily rhythms of physio- logical and behavioural processes exist in all organisms (1). The core molecular mechanism of the circadian oscillator involves intracellu- lar autoregulatory transcriptional ⁄ translational feedback loops, which include positive and negative transcription factors. Typically, the positive proteins bind to a core DNA element, E-box, to activate transcription, whereas negative elements suppress this activity (2–4). This mechanism drives the rhythmic expression of clock-controlled genes (4, 5) and ultimately leads to diverse behavioural and physio- logical rhythms. Rhythmic production and secretion of melatonin from the pineal gland, peaking at night, constitutes an important component of the 1 B.Z.P. and L.A. contributed equally to this study. Journal of Neuroendocrinology Correspondence to: Dr Yoav Gothilf, Department of Neurobiochemistry, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel (e-mail: yoavg@tauex.tau.ac.il). ààCurrent address: Centre for Narcolepsy, Stanford University, Palo Alto, CA 94304 USA. Pineal serotonin-N-acetyltransferase (arylalkylamine-N-acetyltransferase; AANAT) is considered the key enzyme in the generation of circulating melatonin rhythms; the rate of melatonin pro- duction is determined by AANAT activity. In all the examined species, AANAT activity is regulated at the post-translational level and, to a variable degree, also at the transcriptional level. Here, the transcriptional regulation of pineal aanat (aanat2) of the gilthead seabream (Sparus aurata) was investigated. Real-time polymerase chain reaction quantification of aanat2 mRNA levels in the pineal gland collected throughout the 24-h cycle revealed a rhythmic expression pattern. In cultured pineal glands, the amplitude was reduced, but the daily rhythmic expression pattern was maintained under constant illumination, indicating a circadian clock-controlled regulation of seabream aanat2. DNA constructs were prepared in which green fluorescent protein was dri- ven by the aanat2 promoters of seabream and Northern pike. In vivo transient expression analy- ses in zebrafish embryos indicated that these promoters contain the necessary elements to drive enhanced expression in the pineal gland. In the light-entrainable clock-containing PAC-2 zebra- fish cell line, a stably transfected seabream aanat2 promoter-luciferase DNA construct exhibited a clock-controlled circadian rhythm of luciferase activity, characteristic for an E-box-driven expression. In NIH-3T3 cells, the seabream aanat2 promoter was activated by a synergistic action of BMAL ⁄ CLOCK and orthodenticle homeobox 5 (OTX5). Promoter sequence analyses revealed the presence of the photoreceptor conserved element and an extended E-box (i.e. the binding sites for BMAL ⁄ CLOCK and OTX5 that have been previously associated with pineal-speci- fic and rhythmic gene expression). These results suggest that seabream aanat2 is a clock-con- trolled gene that is regulated by conserved mechanisms. Key words: melatonin, AANAT, OTX5, BMAL, CLOCK. doi: 10.1111/j.1365-2826.2006.01501.x Journal of Neuroendocrinology 19, 46–53 ª 2006 The Authors. Journal Compilation ª 2006 Blackwell Publishing Ltd