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

Abstract

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.

Molecular characterization of circumventricular organs and third ventricle ependyma in the rat: potential markers for periventricular tumors

“…Many were shown to be overexpressed in the rat PG in a recent microarray study 35 . Three genes, Tph , Aanat and Asmt , which encode enzymes involved in melatonin synthesis, have been previously reported to be expressed in the rat PG 42,43 . Other genes found to be overexpressed in our study are implicated in light reception ( Sag , Opn4 and Pg10.2 ) and transduction ( Aipl1 , Prph2 , Pdc , Rhok , Rxrg , Guca1b and Cngb1 ) consistent with the common origin in vertebrates of pinealocytes and photoreceptors 35 .…”

Seeing the light to change colour: An evolutionary perspective on the role of melanopsin in neuroendocrine circuits regulating light‐mediated skin pigmentation

“…The melanopsin-controlled primary physiological response may be regulated in a circadian manner. Light changes the levels/activity of most of the genes involved in the regulation of circadian biological clocks, including specific transcription factors (CLOCK and Baml1; Chong, Bernard, & Klein, 2000;Zilberman-Peled et al, 2007) and the period circadian homologs protein 1 and 2 (Per1 and Per2), in a cyclic manner to phase the biological clocks to environmental light levels (Klein, 2006;Simonneaux & Ribelayga, 2003). These proteins are expressed in isolated X. laevis melanophores, and melanopsin-sensitive night, while opn4m mRNA shows little circadian entrainment (Moraes, de Lima et al, 2014).…”

“…In mammals, seasonal skin pigmentation correlates with αMSH levels that peak during the spring, inducing change in the pelage when short photoperiods (low melatonin) begin [Colour figure can be viewed at wileyonlinelibrary.com] mediated by the same specific transcription factors and proteins described in melanophores (e.g., clock, bmal1, per1 and per2). Melatonin synthesis by the pineal gland is cyclical, with a key enzyme in the biosynthetic pathway, arylalkylamine N-acetyltransferase (AANAT; Figure 4), regulated at the transcriptional and post-translational level by transcription factors (CLOCK and Baml1; Chong et al, 2000;Zilberman-Peled et al, 2007) and proteins (Per1 and Per2) whose level/activity is high at night (Klein, 2006;Simonneaux & Ribelayga, 2003), and low during the day (Figure 4). In mammals and teleosts, pineal gland per1 and per2 increase at night to stimulate melatonin synthesis, which in LEI organisms causes melanosome aggregation.…”

A survey of molecular details in the human pineal gland in the light of phylogeny, structure, function and chronobiological diseases

“…The pineal gland of Zebrafish is just one of many tissues that endow a circadian clock [231]. Notably, there is increasing evidence that the Zebrafish Aanat gene is endogenously clock‐controlled in the pineal gland [232–234].…”