Transcriptional Regulation of Arylalkylamine‐N‐Acetyltransferase‐2 Gene in the Pineal Gland of the Gilthead Seabream

Dec 23, 2006Journal of neuroendocrinology

Control of a key melatonin-producing gene in the seabream's pineal gland

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Abstract

Pineal aanat2 expression in the gilthead seabream exhibits a rhythmic pattern over a 24-hour cycle.

AANAT activity influences melatonin production in gilthead seabream.
Rhythmic expression of aanat2 mRNA levels was observed in the pineal gland throughout the day.
Constant light conditions reduced the amplitude of aanat2 expression but maintained its rhythmic pattern.
Promoters from seabream and Northern pike enhanced expression in the pineal gland when tested in zebrafish embryos.
A stable seabream aanat2 promoter-luciferase construct showed a circadian rhythm of luciferase activity in zebrafish cells.
The seabream aanat2 promoter is activated by BMAL/CLOCK and OTX5, suggesting regulation by conserved mechanisms.

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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 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 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, 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-specific and rhythmic gene expression). These results suggest that seabream aanat2 is a clock-controlled gene that is regulated by conserved mechanisms.

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Transcriptional Regulation of Arylalkylamine‐N‐Acetyltransferase‐2 Gene in the Pineal Gland of the Gilthead Seabream

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