The Mammalian Circadian Timing System: Synchronization of Peripheral Clocks C. SAINI 1 , D.M. SUTER 1,2 , A. LIANI 1 ,P.GOS 1 , AND U. SCHIBLER 1 1 Department of Molecular Biology and National CCR Frontiers in Genetics, Sciences III, University of Geneva, 1211 Geneva-4, Switzerland Correspondence: ueli.schibler@unige.ch Mammalian physiology has to adapt to daily alternating periods during which animals either forage and feed or sleep and fast. The adaptation of physiology to these oscillations is controlled by a circadian timekeeping system, in which a master pace- maker in the suprachiasmatic nucleus (SCN) synchronizes slave clocks in peripheral organs. Because the temporal coordina- tion of metabolism is a major purpose of clocks in many tissues, it is important that metabolic and circadian cycles are tightly coordinated. Recent studies have revealed a multitude of signaling components that possibly link metabolism to circadian gene expression. Owing to this redundancy, the implication of any single signaling pathway in the synchronization of periph- eral oscillators cannot be assessed by determining the steady-state phase, but instead requires the monitoring of phase-shifting kinetics at a high temporal resolution. In mammals, most physiological processes undergo daily oscillations that are controlled by an endogenous circadian timing system and/or daytime-dependent envi- ronmental changes (Fig. 1) (Dibner et al. 2010). The term “circadian” comes from the Latin words “circa diem” (around a day) and indicates that circadian time is similar but not identical to geophysical time. For example, the clocks of mice and humans free-run with period lengths of slightly under and over 24 h, respectively. Hence, their oscillators must be readjusted every day by a few minutes to stay in resonance with geophysical time. This is achieved primarily by daily variations in light intensities that are perceived by the retina and conveyed via the retino-hypothalamic tract to neurons of suprachiasmatic nuclei. The suprachiasmatic nucleus (SCN), composed of two small groups of neurons located in the ventral hypothalamus right above the optical chiasma, has been identified as the central circadian pacemaker by elegant lesion and transplantation experiments (Ralph et al. 1990; Silver et al. 1996). Meanwhile, self-sustained and cell-autonomous circadian oscillators have been discov- ered in cultured cells (Balsalobre et al. 1998) and in explants of several peripheral tissues (Yamazaki et al. 2000; Yoo et al. 2004). The molecular makeup of cellular clocks is virtually identical in SCN neurons and periph- eral cell types. In both of them, the circadian rhythm generation is thought to be based on interlocked negative feedback loops in gene expression (Ko and Takahashi 2006; Dibner et al. 2010). The major loop consists of two period genes, Per1 and Per2, and two cryptochrome genes, Cry1 and Cry2. These genes are activated by heterodimers of the PAS domain helix-loop-helix tran- scription factors BMAL1 and CLOCK (and its closely related paralog NPAS2 in the brain). PER and CRY pro- teins form heteropolymeric complexes with additional proteins, such as the casein kinases 1 1 (CK11) and 1 d (CK1d), the nucleic-acid-binding proteins NONO and PSF, the SIN3– HDAC complex, and the histone methyl- transferase adaptor protein WDR5 (Brown et al. 2005; Duong et al. 2011). Once these protein complexes have reached a critical activity, they inhibit Per and Cry gene expression by attenuating the activity of BMAL1- CLOCK/NPAS2 heterodimers. As a consequence, the levels of PER and CRY messenger RNA (mRNA) and proteins decrease, and a new cycle of CRY and PER expression can ensue. This feedback circuitry also affects the expression of BMAL1 and, to a lesser extent, that of CLOCK, through an accessory loop involving transcrip- tional activators of the retinoic acid-related orphan re- ceptors (RORa, RORb, and RORg) and repressors of the REV-ERB orphan receptor family (REV-ERBa and REV-ERBb). Both RORs and REV-ERBs bind to two ROR-binding elements (ROREs) within the Bmal1 pro- moter (Preitner et al. 2002) and the first Clock intron (Crumbley and Burris 2011), respectively, but whereas RORs activate transcription, REV-ERBs recruit the NCOR1–HDAC3 corepressor complex and thereby re- duce transcription (Feng et al. 2011). In addition to tran- scriptional mechanisms, posttranslational modifications of core clock proteins also play crucial roles in the circa- dian clockwork circuitry (Vanselow and Kramer 2007; Virshup et al. 2007; Reischl and Kramer 2011). Thus, phosphorylation of PER proteins by CK1 and other kinases tune the period length of the oscillations by affecting PER protein stability and activity. Moreover, CLOCK, BMAL1, CRYs, and REV-ERBa are all phos- phoproteins, although many of the relevant kinases re- main to be identified. Other posttranslational modifica- tions have also been found on core clock proteins. Thus, CLOCK can be sumoylated (Cardone et al. 2005) 2 Present address: Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 02138. Copyright # 2011 Cold Spring Harbor Laboratory Press; all rights reserved; doi: 10.1101/sqb.2011.76.010918 Cold Spring Harbor Symposia on Quantitative Biology, Volume LXXVI 39