Zilberman-Peled B, Appelbaum L, Vallone D, Foulkes N S, Anava S, Anzulovich A, Coon S L, Klein D C, Falcón J, Ron B, Gothilf Y
Department of Zoology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel.
J Neuroendocrinol. 2007 Jan;19(1):46-53. doi: 10.1111/j.1365-2826.2006.01501.x.
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.
松果体5-羟色胺-N-乙酰基转移酶（芳基烷基胺-N-乙酰基转移酶；AANAT）被认为是循环褪黑素节律产生中的关键酶；褪黑素的产生速率由AANAT活性决定。在所有被检测的物种中，AANAT活性在翻译后水平受到调控，并且在不同程度上也在转录水平受到调控。在此，研究了金头鲷（Sparus aurata）松果体aanat（aanat2）的转录调控。通过实时聚合酶链反应对在24小时周期内收集的松果体中aanat2 mRNA水平进行定量分析，揭示了一种节律性表达模式。在培养的松果体中，振幅降低，但在持续光照下仍保持每日节律性表达模式，表明金头鲷aanat2受生物钟控制。制备了由金头鲷和白斑狗鱼的aanat2启动子驱动绿色荧光蛋白的DNA构建体。在斑马鱼胚胎中的体内瞬时表达分析表明，这些启动子包含驱动松果体中增强表达的必要元件。在含有可被光诱导的生物钟的PAC-2斑马鱼细胞系中，稳定转染的金头鲷aanat2启动子-荧光素酶DNA构建体表现出荧光素酶活性的生物钟控制的昼夜节律，这是E-box驱动表达的特征。在NIH-3T3细胞中，金头鲷aanat2启动子通过BMAL/CLOCK和正齿同源盒5（OTX5）的协同作用被激活。启动子序列分析揭示了光感受器保守元件和一个扩展的E-box的存在（即先前与松果体特异性和节律性基因表达相关的BMAL/CLOCK和OTX5的结合位点）。这些结果表明，金头鲷aanat2是一个受保守机制调控的生物钟控制基因。
