Characterization of the Core Mammalian Clock Component, NPAS2, as a REV-ERBα/RORα Target Gene

Abstract

The mammalian clock is regulated at the cellular level by a transcriptional/translational feedback loop. BMAL1/CLOCK (or NPAS2) heterodimers activate the expression of the PERIOD (PER) and CRYPTOCHROME (CRY) genes acting as transcription factors directed to the PER and CRY promoters via E-box elements. PER and CRY proteins form heterodimers and suppress the activity of the BMAL1/CLOCK (or NPAS2) completing the feedback loop. The circadian expression of BMAL1 is influenced by retinoic acid receptor-related orphan receptor ␣ (ROR␣) and REV-ERB␣, two nuclear receptors that target a ROR-response element in the promoter of the BMAL1 gene. Given that BMAL1 functions as an obligate heterodimer with either CLOCK or NPAS2, it is unclear how the expression of the partner is coordinated with BMAL1 expression. Here, we demonstrate that NPAS2 is also a ROR␣ and REV-ERB␣ target gene. Using a ChIP/microarray screen, we identified both ROR␣ and REV-ERB␣ occupancy of the NPAS2 promoter. We identified two functional ROREs within the NPAS2 promoter and also demonstrate that both ROR␣ and REV-ERB␣ regulate the expression of NPAS2 mRNA. These data suggest a mechanism by which ROR␣ and REV-ERB␣ coordinately regulate the expression of the positive arm of the circadian rhythm feedback loop.Circadian rhythms are the natural ϳ24-h cycles that are conserved across a wide variety of organisms, including Arabidopsis, Drosophila, and mammals. In mammals, these rhythms are entrained by light signals and are controlled by the "master clock" in the suprachiasmatic nucleus (SCN) 2 in the brain. In the periphery, semi-autonomous clocks can be entrained to signals from the SCN and signals from other cues, including nutrient status (1). The mammalian circadian clock is controlled by a transcriptional/translational feedback loop involving several key proteins. The expression of these key proteins includes brain and muscle ARNT (aryl hydrocarbon receptor nuclear translocator)-like 1 (BMAL1), circadian locomotor output kaput (CLOCK), PERIOD (PER), and CRYPTOCROME (CRY). BMAL1 and CLOCK are members of the basic helix-loop-helix-PAS family of transcription factors and form a heterodimer that targets E-box DNA elements in the promoters of the PER and CRY genes and activates their transcription. The CRY and PER proteins heterodimerize, and as the levels of this protein complex accumulate it effectively inhibits the activity of BMAL1/CLOCK limiting the expression of the PER and CRY genes. This feedback loop results in the oscillations in expression of BMAL1/CLOCK and CRY/PER that follow a circadian pattern (2, 3).Two classes of nuclear receptors play a critical role in modulation of the mammalian circadian clock. The retinoic acid receptor-related orphan receptors (RORs) and REV-ERBs modulate expression of the BMAL1 gene through two conserved ROR-response elements (ROREs) located within the BMAL1 promoter. Expression of BMAL1 is modulated by competition between RORs and REV-ERBs for binding the BMAL1 promoter and either activation (ROR) or repression (RE...

Oxidative Stress and Inflammation Modulate Rev-erbα Signaling in the Neonatal Lung and Affect Circadian Rhythmicity

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Retinoic Acid-related Orphan Receptor α Regulates Diurnal Rhythm and Fasting Induction of Sterol 12α-Hydroxylase in Bile Acid Synthesis

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