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P. M. Luvone, F. G. Ali, R. Biscoglia, J. Weller, D. C. Klein, R. Haque; Circadian Regulation of Melatonin Synthesis in Chick Photoreceptor Cells: Roles of CLOCK and NPAS2 in the Circadian Oscillator and the Regulation of Aanat Expression. Invest. Ophthalmol. Vis. Sci. 2009;50(13):3236.
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Circadian rhythms are generated by self-sustained biological oscillators consisting of transcription-translation feedback loops. Critical to the function of these circadian oscillators are BMAL1:CLOCK heterodimers and BMAL1:NPAS2 heterodimers, which bind to circadian E-box enhancer elements in promoters of other circadian clock genes and clock-controlled genes. Previous studies using transfected COS cells have shown that these heterodimers control expression of the clock-controlled gene Aanat (J.Biol. Chem 275: 32991, 2000). Aanat encodes arylalkylamine N-acetyltransferase, the enzyme that regulates the circadian rhythm in melatonin synthesis The present study investigated whether NPAS2 and/or CLOCK regulates Aanat expression in intact chicken photoreceptors.
Photoreceptors were isolated from frozen sections of retinas of 2 week old chicks by laser capture microdissection. Photoreceptor cell cultures prepared from embryonic day 6 neural retina were incubated under a 14h:10h light-dark cycle of illumination for 8 days and then transferred to constant (24/day) darkness (DD). Gene-specific Npas2 and Clock miRNA were transfected on 4th day of culture. Cells were harvested every 6h in DD beginning at zeitgeber time 2. Transcripts of Bmal1, Npas2, Clock, Per2, Aanat, and Hprt were quantified by the qRT-PCR. Chromatin immunoprecipitation (ChIP) using antibodies to NPAS2 and CLOCK was performed to determine if they bound to the Aanat promoter.
Bmal1, Clock and Npas2 transcripts were expressed in photoreceptors in vivo and in vitro. Npas2, Bmal1, Per2, and Aanat transcripts were rhythmically expressed in cultures of vector transfected cells (control), whereas Clock transcript levels were constitutive. Clock miRNA significantly inhibited the expression of CLOCK protein but had no effect on Bmal1 transcript level. However, Clock knockdown significantly dampened the circadian expression of Npas2, Per2, and Aanat transcripts. Npas2 miRNA significantly reduced Npas2 expression at the transcript and protein levels. Npas2 knockdown had no effect on the expression of any of the clock genes examined, although it dampened the circadian rhythm of Aanat mRNA. NPAS2 and CLOCK were found to be bound to the Aanat E-box by ChIP analysis.
The clock-generated rhythm of Aanat expression is regulated by binding of either NPAS2 or CLOCK, presumably as BMAL1 heterodimers, to the E-Box in the Aanat promoter. CLOCK plays a more significant role than NPAS2 in sustaining the core circadian oscillator.
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