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The mouse Clock mutation reduces circadian pacemaker amplitude and enhances efficacy of resetting stimuli and phase–response curve amplitude

Martha Hotz Vitaterna*, Caroline H. Ko*,, Anne-Marie Chang*,, Ethan D. Buhr*, Ethan M. Fruechte*,, Andrew Schook, Marina P. Antoch,||, Fred W. Turek*, and Joseph S. Takahashi*,**

*Center for Functional Genomics, Center for Sleep and Circadian Biology and Department of Neurobiology and Physiology, Howard Hughes Medical Institute, Northwestern University, 2205 Tech Drive, Evanston, IL 60208-3520; and Department of Psychology, University of Toronto, Toronto, ON, Canada M5S 3G3

Contributed by Joseph S. Takahashi, May 2, 2006


The mouse Clock gene encodes a basic helix–loop–helix-PAStranscription factor, CLOCK, that acts in concert with BMAL1to form the positive elements of the circadian clock mechanismin mammals. The original Clock mutant allele is a dominant negative(antimorphic) mutation that deletes exon 19 and causes an internaldeletion of 51 aa in the C-terminal activation domain of theCLOCK protein. Here we report that heterozygous Clock/+ miceexhibit high-amplitude phase-resetting responses to 6-h lightpulses (Type 0 resetting) as compared with wild-type mice thathave low amplitude (Type 1) phase resetting. The magnitude andtime course of acute light induction in the suprachiasmaticnuclei of the only known light-induced core clock genes, Per1and Per2, are not affected by the Clock/+ mutation. However,the amplitude of the circadian rhythms of Per gene expressionare significantly reduced in Clock homozygous and heterozygousmutants. Rhythms of PER2::LUCIFERASE expression in suprachiasmaticnuclei explant cultures also are reduced in amplitude in Clockheterozygotes. The phase–response curves to changes inculture medium are Type 0 in Clock heterozygotes, but Type 1in wild types, similar to that seen for light in vivo. The increasedefficacy of resetting stimuli and decreased PER expression amplitudecan be explained in a unified manner by a model in which theClock mutation reduces circadian pacemaker amplitude in thesuprachiasmatic nuclei.

circadian clock | Clock gene | entrainment | suprachiasmatic nucleus | Pergenes


The mouse Clock mutation was identified in an N-ethyl-N-nitrosoureamutagenesis screen for circadian variants (1). Clock is a semidominantmutation that lengthens circadian period by 1 h in heterozygotes(Clock/+) and by 4 h in homozygotes (Clock/Clock). With prolongedexposure, Clock homozygotes fail to express persistent circadianrhythms in constant darkness. The lengthened-period and loss-of-rhythmphenotypes are the hallmarks of the original mutant allele.Genetic analysis of the Clock mutant allele over a deletionrevealed that Clock is an antimorph, a type of dominant negativemutation (2). Molecular cloning of Clock, which encodes a basichelix–loop–helix-PAS transcription factor, showedthat the mutant allele is a 5' splice donor mutation skippingexon 19 and causing an internal deletion of 51 aa in the C-terminalactivation domain of the CLOCK protein (35).

In the last decade, significant progress has been made in ourunderstanding of the mechanism of circadian rhythms. An autoregulatorytranscriptional feedback loop forms the core mechanism of thecircadian clock in animals (reviewed in refs. 6 and 7). Thepositive elements of the oscillator are CLOCK and BMAL1, whichform a heterodimeric transcription factor to activate the mammalianPeriod and Cryptochrome genes (specifically, Per1, Per2, Cry1,and Cry2). The negative elements of the oscillator are the geneproducts of Period and Cryptochrome (PERs and CRYs) that accumulate,associate with each other, and translocate into the nucleusto inhibit the CLOCK/BMAL1 activation of their own transcription.As the negative elements turnover, CLOCK and BMAL1 then becomeactive again to begin a new cycle of transcription of the Periodand Cryptochrome genes.

To define the role of Clock more fully within the mammaliancircadian system, we analyzed the entrainment and resettingbehavior of mice to light. In principle, entrainment of thecore circadian clock mechanism described above would be expectedto occur as a consequence of either phasic or tonic changesin the levels of oscillating feedback elements (e.g., the PERor CRY proteins). Within the core circadian clock mechanism,the only elements that have been definitively found to be lightresponsive in the suprachiasmatic nucleus (SCN) are the Per1and Per2 genes (814). Acute light exposure of mice causesphase-dependent elevations of Per1 and Per2 mRNA and proteinlevels in the SCN. We report here that the Clock mutation causesa drastic increase in the phase-resetting effects of light inmice. Both behavioral and molecular analyses of Clock mutantmice demonstrate that Clock alters the resetting effects ofphase-shifting stimuli by reducing pacemaker amplitude ratherthan by changing the strength of light on the inputs to thecircadian pacemaker. These results illustrate that the effectsof Clock on circadian pacemaker amplitude have significant consequenceson the entrainment behavior of mice to light and other resettingstimuli. Given that circadian pacemaker amplitude has been implicatedin the entrainment of human circadian rhythms (15), these resultsprovide a model for studying and manipulating circadian amplitudeto optimize entrainment in humans and other mammals.

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