Molecular mechanisms of the mammalian circadian clock have been studied primarily by genetic perturbation and behavioral analysis. Here, we used bioluminescence imaging to monitor Per2 gene expression in tissues and cells from clock mutant mice. We discovered that Per1 and Cry1 are required for sustained rhythms in peripheral tissues and cells, and in neurons dissociated from the suprachiasmatic nuclei (SCN). Per2 is also required for sustained rhythms, whereas Cry2 and Per3 deficiencies cause only period length defects. However, oscillator network interactions in the SCN can compensate for Per1 or Cry1 deficiency, preserving sustained rhythmicity in mutant SCN slices and behavior. Thus, behavior does not necessarily reflect cell-autonomous clock phenotypes. Our studies reveal previously unappreciated requirements for Per1, Per2, and Cry1 in sustaining cellular circadian rhythmicity and demonstrate that SCN intercellular coupling is essential not only to synchronize component cellular oscillators but also for robustness against genetic perturbations.

Summary Two decades of research identified more than a dozen clock genes and defined a biochemical feedback mechanism of circadian oscillator function. To identify additional clock genes and modifiers, we conducted a genome-wide siRNA screen in a human cellular clock model. Knockdown of nearly a thousand genes reduced rhythm amplitude. Potent effects on period length or increased amplitude were less frequent; we found hundreds of these and confirmed them in secondary screens. Characterization of a subset of these genes demonstrated a dosage-dependent effect on oscillator function. Protein interaction network analysis showed that dozens of gene products directly or indirectly associate with known clock components. Pathway analysis revealed these genes are overrepresented for components of insulin and hedgehog signaling, the cell cycle, and the folate metabolism. Coupled with data showing many of these pathways are clock-regulated, we conclude the clock is interconnected with many aspects of cellular function.

During fasting, mammals maintain glucose homeostasis by stimulating hepatic gluconeogenesis1. Elevations in circulating glucagon (GLU) and epinephrine trigger the cAMP mediated phosphorylation of Creb and dephosphorylation of the Creb coactivator Crtc22. Although the underlying mechanism is unclear, hepatic gluconeogenesis is also regulated by the circadian clock, which coordinates glucose metabolism with changes in the external environment3–6. Here we show that Creb activity during fasting is modulated by Cryptochromes (Cry1 and Cry2), core components of the clock that are rhythmically expressed in the liver. Cry was elevated during the night/day transition, when it reduced fasting gluconeogenic gene expression by blocking GLU-mediated increases in intracellular cAMP concentrations and in the PKA-mediated phosphorylation of Creb. In biochemical reconstitution studies, we found that Cry inhibited accumulation of cAMP in response to G protein coupled receptor (GPCR) activation but not to forskolin, a direct activator of adenyl cyclase. Cry appeared to modulate GPCR activity directly through interaction with Gsα . As hepatic over-expression of Cry lowered blood glucose concentrations and improved insulin sensitivity in insulin resistant db/db mice, our results suggest that compounds which enhance Cry activity may provide therapeutic benefit to individuals with type II diabetes.