Appelbaum L, Vallone D, Anzulovich A, Ziv L, Tom M, Foulkes N S, Gothilf Y
Department of Zoology, George S Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Isreal.
J Mol Endocrinol. 2006 Apr;36(2):337-47. doi: 10.1677/jme.1.01893.
Daily rhythms of melatonin production are controlled by changes in the activity of arylalkylamine-N-acetyltransferase (AANAT). Zebrafish possess two aanats, aanat1 and aanat2; the former is expressed only in the retina and the latter is expressed in both the retina and the pineal gland. Here, their differential expression and regulation were studied using transcript quantification and transient and stable in vivo and in vitro transfection assays. In the pineal gland, the aanat2 promoter exhibited circadian clock-controlled activity, as indicated by circadian rhythms of Enhanced green fluorescent protein (EGFP) mRNA in AANAT2:EGFP transgenic fish. In vivo transient expression analyses of the aanat2 promoter indicated that E-box and photoreceptor conserved elements (PCE) are required for expression in the pineal gland. In the retina, the expression of both genes was characterized by a robust circadian rhythm of their transcript levels. In constant darkness, the rhythmic expression of retinal aanat2 persisted while the aanat1 rhythm disappeared; indicating that the former is controlled by a circadian clock and the latter is also light driven. In the light-entrainable clock-containing PAC-2 zebrafish cell line, both stably transfected aanat1 and aanat2 promoters exhibited a clock-controlled circadian rhythm, characteristic for an E-box-driven expression. Transient co-transfection experiments in NIH-3T3 cells revealed that the two, E-box- and PCE-containing, promoters are driven by the synergistic action of BMAL/CLOCK and orthehodenticle homeobox 5. This study has revealed a shared mechanism for the regulation of two related genes, yet describes their differential phases and photic responses which may be driven by other gene-specific regulatory mechanisms and tissue-specific transcription factor profiles.
褪黑素生成的日常节律受芳基烷基胺-N-乙酰基转移酶（AANAT）活性变化的控制。斑马鱼有两种AANAT，即aanat1和aanat2；前者仅在视网膜中表达，后者在视网膜和松果体中均有表达。在此，利用转录本定量以及体内和体外的瞬时与稳定转染试验，研究了它们的差异表达和调控。在松果体中，aanat2启动子表现出昼夜节律时钟控制的活性，这在AANAT2:EGFP转基因鱼中增强绿色荧光蛋白（EGFP）mRNA的昼夜节律中得到体现。aanat2启动子的体内瞬时表达分析表明，E-box和光感受器保守元件（PCE）是在松果体中表达所必需的。在视网膜中，这两个基因的表达都具有转录水平强劲的昼夜节律特征。在持续黑暗中，视网膜aanat2的节律性表达持续存在，而aanat1的节律消失；这表明前者受昼夜节律时钟控制，后者也受光驱动。在含有可被光调节的时钟的PAC-2斑马鱼细胞系中，稳定转染的aanat1和aanat2启动子均表现出时钟控制的昼夜节律，这是E-box驱动表达的特征。在NIH-3T3细胞中的瞬时共转染实验表明，这两个含有E-box和PCE的启动子受BMAL/CLOCK和orthodenticle同源盒5的协同作用驱动。这项研究揭示了两个相关基因调控的共同机制，但也描述了它们不同的相位和光反应，这可能由其他基因特异性调控机制和组织特异性转录因子谱驱动。
