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

Abstract

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 Fbxl3Afh 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 Fbxl3Afh 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 Ϫ/Ϫ ;Cry2 Ϫ/Ϫ mice were behaviorally arrhythmic, their SCN expressed short period (ϳ18 h) rhythms with variable stability. Fbxl3Afh/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 Fbxl3Afh/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.

Regulating the Suprachiasmatic Nucleus (SCN) Circadian Clockwork: Interplay between Cell-Autonomous and Circuit-Level Mechanisms

“…The circadian period is also sensitive to the relative abundance of CRY isoforms, CRY1 and CRY2. Analysis of the compound Cry and Fbxl3 mutants has revealed that, although CRY2 is a negative regulator within the SCN clockwork, it is less potent than CRY1 and indeed antagonizes CRY1 inhibition (Anand et al 2013). Under normal circumstances, circadian period is therefore set by the abundance and cellular localization of CRY1 and CRY2 and these competing ligases.…”

“…In the absence of VPAC2 expression, ERK/MAPK and pCREB signaling in the SCN is responsive to retinal activation at all circadian phases. This loss of a "circadian gate" is likely the result of a 2011b andAn et al 2013. ) proportion of desynchronized SCN neurons being in the responsive phase at any time point ).…”

Clock genes in human alcohol abuse and comorbid conditions

“…CRY2 has a critical role in being the power of balance and tuning the circadian period by opposing the action of CRY1 and thereby denying CRY1 access to DNA targets too early (Anand et al, 2013).…”

CRY1 and CRY2 genetic variants in seasonality: A longitudinal and cross-sectional study

“…Here, the cryptochrome circadian clock 2 (CRY2) and cryptochrome circadian clock 1 (CRY1) proteins are the key repressors in the core of the circadian clock (Dardente et al, 2007;Ozturk et al, 2007;Ye et al, 2011Ye et al, , 2014. However, CRY2 has a key role in balancing the expression of cryptochromes, as it not only acts as a general repressor, but also opposes in specific the actions of CRY1 and inhibits CRY1 from accessing to its targets too early (Anand et al, 2013). See Fig.…”