In mammals, virtually all body cells possess self-sustained, cell-autonomous circadian clocks [1–3]. The oscillators in peripheral organs are entrained by a master pacemaker residing in the suprachiasmatic nucleus (SCN) of the brain's hypothalamus, which is itself synchronized by daily light–dark cycles [4]. The molecular details of the signaling pathways used by the SCN to phase-entrain peripheral clocks are still obscure; however, daily feeding–fasting cycles, circadian hormones, and body temperature appear to play pivotal roles in this process [5–9]. The accumulation of mPER1 and/or mPER2, two integral clock components, is altered upon the administration of phase-shifting cues. Hence, these proteins are likely to be involved in the synchronization of circadian clocks [10,11].
On the molecular level, mammalian circadian oscillators are thought to rely on two interconnected negative loops of clock gene expression [12,13]. According to this model, the principal feedback loop is driven by the repressors PER1, PER2, CRY1, and CRY2 and the PAS-domain basic helix-loop-helix (PAS-bHLH) transcription factors BMAL1, CLOCK, and probably NPAS2 [14]. The transcription of the repressor-encoding genes is activated by these PAS-bHLH transcription factors until the PER-CRY complexes reach critical concentrations at which they annul the transactivation potential of the PAS-bHLH proteins and thereby inhibit transcription of their own genes. The concentration of PAS-bHLH activators is adjusted by an accessory feedback loop in which the orphan nuclear receptor REV-ERBα (and, probably to a lesser extent, its paralog REV-ERBβ) periodically represses Bmal1 transcription. The inhibitory activity of REV-ERBα counteracts the transactivation activity of ROR nuclear orphan receptors, which bind to the same RORE elements within the Bmal1 promoter [15]. The cyclic expression of REV-ERBα is itself governed by the PAS-bHLH activators and CRY-PER repressors of the principal negative feedback loop, thereby interconnecting the Rev-erbα-Bmal1 feedback loop directly to the principal feedback loop [16,17]. Since BMAL1 and CLOCK are metabolically more stable than CRY and PER proteins, their abundance varies only slightly throughout the day [16,18,19].
Post-translational protein modifications are also believed to play important roles in the modulation of PER and CRY activities [18,20,21]. However, to date, Bmal1 is the only known clock gene whose inactivation immediately leads to arrhythmicity of behavior and to the ablation of mPer1 and mPer2 mRNA accumulation cycles in the SCN [22].
Transcriptome profiling studies have uncovered a large number of cyclically expressed genes [23–27]. Although most of these genes appear to be involved in output functions, some may also serve as input regulators participating in the synchronization of local clocks. The oscillating activity of these latter genes would be expected to integrate systemic cues such as circadian hormones, metabolites, or body temperature rhythms, into the clockwork circuitry of peripheral cell types. In the intact organism, the cyclic expression of such genes should not necessarily depend upon functional local clocks.
In this study, we describe our attempt to engineer a mouse strain with conditionally active hepatocyte circadian clocks. We used these mice to classify mRNAs into transcripts whose circadian accumulation in the liver do or do not require local circadian oscillators. The identification of systemically driven and oscillator-driven genes not only provides insight into the structural organization of the mammalian circadian timing system, but should also open new avenues to study the phase entrainment mechanisms of peripheral clocks.
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