Abstract
Light is one of the strongest environmental time cues for entraining endogenous circadian rhythms. Emerging evidence indicates that CREB-regulated transcription co-activator 1 (CRTC1) is a key player in this pathway, stimulating light-induced Period1 (Per1) transcription in mammalian clocks. Here, we demonstrate a light-independent role of Drosophila CRTC in sustaining circadian behaviors. Genomic deletion of the crtc locus causes long but poor locomotor rhythms in constant darkness. Overexpression or RNA interference-mediated depletion of CRTC in circadian pacemaker neurons similarly impairs the free-running behavioral rhythms, implying that Drosophila clocks are sensitive to the dosage of CRTC. The crtc null mutation delays the overall phase of circadian gene expression yet it remarkably dampens light-independent oscillations of TIMELESS (TIM) proteins in the clock neurons. In fact, CRTC overexpression enhances CLOCK/CYCLE (CLK/CYC)-activated transcription from tim but not per promoter in clock-less S2 cells whereas CRTC depletion suppresses it. Consistently, TIM overexpression partially but significantly rescues the behavioral rhythms in crtc mutants. Taken together, our data suggest that CRTC is a novel co-activator for the CLK/CYC-activated tim transcription to coordinate molecular rhythms with circadian behaviors over a 24-hour time-scale. We thus propose that CRTC-dependent clock mechanisms have co-evolved with selective clock genes among different species.
Highlights
CRTC induces Sik[1] transcription, and elevated SIK1 feeds back to phosphorylate CRTC proteins, blocking their nuclear entry[22,23,24]
Chromosomal deletions covering the crtc locus did not complement these circadian defects in trans-heterozygotes with crtc[] allele (Fig. 1b–d, Supplementary Table 1), further supporting that CRTC is necessary for sustaining robust circadian behaviors in Drosophila
We found that crtc mutants displayed a ~4-hour delay in the increasing phase of circadian mRNA expression in light: dark (LD) cycles while crtc effects on the declining phase were relatively weak (Fig. 3a, top)
Summary
CREB-dependent transcription has long been implicated in different aspects of circadian gene expression. Fasting-activated CREB-CRTC2 stimulates Bmal[1] expression[61], whereas CLOCK-BMAL1–induced CRY rhythmically gates CREB activity in this process by modulating G protein-coupled receptor activity and inhibiting cAMP-induced CREB phosphorylation[62] This molecular feedback circuit mutually links mammalian clocks and energy metabolism in terms of CREB-dependent transcription. Intracellular signaling relays in the SCN converge on the dephosphorylation and nuclear translocation of CRTC1 to activate CRE-dependent transcription[21,24] Under these circumstances, a circadian role of light-sensitive TIM might have degenerated, while per took over a role in the light-entrainment pathway by retaining CREB-CRTC1–dependent transcriptional regulation from the primitive TTFL.
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