Distinct and Separable Roles for Endogenous CRY1 and CRY2 within the Circadian Molecular Clockwork of the Suprachiasmatic Nucleus, as Revealed by the Fbxl3^AfhMutation

Apr 26, 2013The Journal of neuroscience : the official journal of the Society for Neuroscience

Different roles of natural CRY1 and CRY2 proteins in the brain's daily clock revealed by the Fbxl3(Afh) mutation

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Abstract

Increased dosage of the Fbxl3(Afh) mutant leads to longer circadian periods in both CRY1- and CRY2-deficient mice.

Circadian rhythms of behavior and SCN bioluminescence lengthened with increased Fbxl3(Afh) dosage in CRY-deficient backgrounds.
CRY1 is more effective than CRY2 at slowing the circadian clock.
CRY1 prolongs the duration of transcriptional suppression in SCN slices, whereas CRY2 does not.
Both CRY proteins act as transcriptional repressors of clock-controlled genes, but CRY1 is the predominant repressor.
Cry1(-/-);Cry2(-/-) mice exhibit arrhythmic behavior despite having short period rhythms (~18 h) in the SCN.
Stabilization of CRY1 and CRY2 is necessary to explain the period lengthening effects associated with Fbxl3(Afh/Afh).

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The circadian clock of the suprachiasmatic nucleus (SCN) drives daily rhythms of behavior. Cryptochromes (CRYs) are powerful transcriptional repressors within the molecular negative feedback loops at the heart of the SCN clockwork, where they periodically suppress their own expression and that of clock-controlled genes. To determine the differential contributions of CRY1 and CRY2 within circadian timing in vivo, we exploited the N-ethyl-N-nitrosourea-induced afterhours mutant Fbxl3(Afh) to stabilize endogenous CRY. Importantly, this was conducted in CRY2- and CRY1-deficient mice to test each CRY in isolation. In both CRY-deficient backgrounds, circadian rhythms of wheel-running and SCN bioluminescence showed increased period length with increased Fbxl3(Afh) dosage. Although both CRY proteins slowed the clock, CRY1 was significantly more potent than CRY2, and in SCN slices, CRY1 but not CRY2 prolonged the interval of transcriptional suppression. Selective CRY-stabilization demonstrated that both CRYs are endogenous transcriptional repressors of clock-controlled genes, but again CRY1 was preeminent. Finally, although Cry1(-/-);Cry2(-/-) mice were behaviorally arrhythmic, their SCN expressed short period (~18 h) rhythms with variable stability. Fbxl3(Afh/Afh) had no effect on these CRY-independent rhythms, confirming its circadian action is mediated exclusively via CRYs. Thus, stabilization of both CRY1 and CRY2 are necessary and sufficient to explain circadian period lengthening by Fbxl3(Afh/Afh). Both CRY proteins dose-dependently lengthen the intrinsic, high-frequency SCN rhythm, and CRY2 also attenuates the more potent period-lengthening effects of CRY1. Incorporation of CRY-mediated transcriptional feedback thus confers stability to intrinsic SCN oscillations, establishing periods between 18 and 29 h, as determined by selective contributions of CRY1 and CRY2.

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Distinct and Separable Roles for Endogenous CRY1 and CRY2 within the Circadian Molecular Clockwork of the Suprachiasmatic Nucleus, as Revealed by the Fbxl3^AfhMutation

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