NPAS2 Compensates for Loss of CLOCK in Peripheral Circadian Oscillators

Abstract

Heterodimers of CLOCK and BMAL1 are the major transcriptional activators of the mammalian circadian clock. Because the paralog NPAS2 can substitute for CLOCK in the suprachiasmatic nucleus (SCN), the master circadian pacemaker, CLOCK-deficient mice maintain circadian rhythms in behavior and in tissues in vivo. However, when isolated from the SCN, CLOCK-deficient peripheral tissues are reportedly arrhythmic, suggesting a fundamental difference in circadian clock function between SCN and peripheral tissues. Surprisingly, however, using luminometry and single-cell bioluminescence imaging of PER2 expression, we now find that CLOCK-deficient dispersed SCN neurons and peripheral cells exhibit similarly stable, autonomous circadian rhythms in vitro. In CLOCK-deficient fibroblasts, knockdown of Npas2 leads to arrhythmicity, suggesting that NPAS2 can compensate for loss of CLOCK in peripheral cells as well as in SCN. Our data overturn the notion of an SCN-specific role for NPAS2 in the molecular circadian clock, and instead indicate that, at the cellular level, the core loops of SCN neuron and peripheral cell circadian clocks are fundamentally similar.

Author Summary

In mammals, circadian clocks are based on a core transcriptional–translational feedback loop. BMAL1 and CLOCK activate the transcription of Per1-3 and Cry1/2. PER and CRY proteins inhibit BMAL1/CLOCK, and thus their own transcription. In Clock ^-/- mice, NPAS2 can substitute for CLOCK in the suprachiasmatic nucleus (SCN), the major circadian pacemaker. However, peripheral tissues of Clock ^-/- mice were reported to lack circadian rhythms. Since then, the protein CLOCK has been deemed essential for circadian rhythms in peripheral tissues. However, here we show that Clock ^-/- peripheral cells and tissues exhibit stable, autonomous circadian rhythms. Furthermore, in Clock ^-/- fibroblasts, knockdown of Npas2 leads to arrhythmicity, suggesting that NPAS2 can compensate for the loss of CLOCK in peripheral cells as well as in SCN. Our data overturn the notion of an SCN-specific role for NPAS2, and instead indicate that the core loops of SCN neuron and peripheral cell circadian clocks are fundamentally similar. This finding redefines a basic principle of molecular circadian clock regulation in peripheral organs which are essential for many metabolic processes. Disturbances of these rhythms lead to disorders like diabetes, obesity, and cancer. Thus, understanding the molecular basis of peripheral circadian oscillators is essential to develop treatments against clock-related disorders.