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Npas4 is activated by melatonin, and drives the clock gene Cry1 in the ovine pars tuberalis

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Original languageEnglish
Pages (from-to)979-989
Number of pages11
JournalMolecular Endocrinology
Issue number6
Publication statusPublished - 18 Apr 2013


Seasonal mammals integrate changes in the duration of nocturnal melatonin secretion to drive annual physiological cycles. Melatonin receptors within the proximal pituitary region, the pars tuberalis (PT), are essential in regulating seasonal neuroendocrine responses. In the ovine PT, melatonin is known to influence acute changes in transcriptional dynamics coupled to the onset (dusk) and offset (dawn) of melatonin secretion, leading to a potential interval-timing mechanism capable of decoding changes in day-length (photoperiod). Melatonin offset at dawn is linked to cAMP accumulation, which directly induces transcription of the clock gene Per1. The rise of melatonin at dusk induces a separate and distinct cohort, including the clock-regulated genes Cry1 and Nampt, but little is known of the up-stream mechanisms involved. Here, we used next generation sequencing of the ovine PT transcriptome at melatonin onset, and identified Npas4 as a rapidly induced bHLH-PAS domain transcription factor. In vivo we show nuclear localisation of NPAS4 protein in presumptive melatonin target cells of the PT (αGSU-expressing cells), while in situ hybridisation studies identified acute and transient expression in the PT of Npas4 in response to melatonin. In vitro, NPAS4 forms functional dimers with bHLH-PAS domain co-factors ARNT, ARNT2 and ARNTL, transactivating both Cry1 and Nampt ovine promoter reporters. Using a combination of 5` deletions and site-directed mutagenesis we show NPAS4:ARNT transactivation to be co-dependent upon two conserved central midline elements (CMEs) within the Cry1 promoter. Our data thus reveal NPAS4 as a candidate immediate early-response gene in the ovine PT, driving molecular responses to melatonin.

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