Transcriptional regulation of arylalkylamine-N-acetyltransferase-2 gene in the pineal gland of the gilthead seabream.

BINA ZILBERMAN-PELED; LIOR APPELBAUM; DANIELA VALLONE; NICHOLAS FOULKES; SARIT ANAVA; ANA ANZULOVICH; STEVEN COON; DAVID KLEIN; JACK FALCON; BENNY RON; YOAV GOTHILF

JOURNAL OF NEUROENDOCRINOLOGY.

Blackwell Publishing Ltd

Año: 2007 vol. 19 p. 46 - 53

0953-8194

Pineal serotonin-N-acetyltransferase (arylalkylamine-N-acetyltransferase, AANAT) is considered the key enzyme in the generation of circulating melatonin rhythms; the rate of melatonin production is determined by AANAT activity. In all examined species AANAT activity is regulated at the post-translational levels and, to a variable degree, at the transcriptional level. Here, the transcriptional regulation of pineal aanat (aanat2) of the gilthead seabream (Sparus aurata) was investigated. Real-time PCR quantification of aanat2 mRNA levels in the pineal gland collected throughout the 24-hour cycle revealed a rhythmic expression pattern. In cultured pineal glands the amplitude was reduced, yet the daily 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 (EGFP) is driven by the aanat2 promoters of seabream and Northern pike. In vivo transient expression analyses 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 zebrafish cell line, 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 orthehodenticle homeobox 5 (OTX5). Promoter sequence analyses revealed the presence of the photoreceptor conserved element (PCE) and an extended E-box ? the binding sites for BMAL/CLOCK and OTX5 that were previously associated with pineal-specific and rhythmic gene expression. These results suggest that seabream aanat2 is a clock-controlled gene that is regulated by conserved mechanisms.