We generated a mouse model system in which hepatocyte circadian oscillators can be attenuated in a conditional fashion. The system is based on the tetracycline-dependent, liver-specific overexpression of the nuclear orphan receptor REV-Erbα, a potent repressor of the essential clock gene Bmal1. Thus, when the tetracycline analog doxycycline was omitted from the food, REV-ERBα accumulated to high levels throughout the day and thereby inhibited Bmal1 transcription constitutively in LAP-tTA/TRE-Rev-erbα mice. As a consequence, the expression of obligatory Bmal1 target genes was decreased to a level that no longer supports local oscillator function. When doxycycline was added to the food, the Rev-erbα transgene was silenced, and hepatocyte oscillator function was reestablished in LAP-tTA/TRE-Rev-erbα mice.
By using this novel mouse model, we were able to discriminate between genes whose cyclic expression is driven either by local hepatocyte oscillators or by systemic circadian cues that are controlled directly or indirectly by the SCN. The transcription of liver genes whose expression displayed daily oscillations in LAP-tTA/TRE-Rev-erbα mice despite arrested hepatocyte clocks are likely under the control of physical and/or chemical cues whose systemic rhythms are driven by the central SCN pacemaker. Such systemically regulated genes are expected to include genes involved in the synchronization of hepatocyte clocks. Genome-wide profiling of the liver circadian transcriptome of LAP-tTA/TRE-Rev-erbα mice fed with Dox-supplemented chow revealed about 350 transcripts with robust circadian accumulation. Less than 10% of these transcripts displayed rhythmic accumulation with high amplitude and magnitude in mice fed with normal chow, suggesting that the cyclic transcription of most circadian genes is influenced by local oscillators. We cannot formally exclude that the cyclic expression of some of the genes resilient to HA-REV-ERBα overexpression was driven by a second, yet unknown and BMAL1-independent oscillator. However, this hypothesis clearly did not apply to mPer2, since mPER2::LUC expression ceased to be rhythmic in liver explants not treated with Dox. We thus consider it more likely that the rhythmic expression of genes in the absence of Dox was governed by systemic cues, which were directly or indirectly controlled by the master pacemaker in the SCN. As illustrated in Figure 7, the system-driven circadian expression of mPer2 is of particular interest with regard to the entrainment of the peripheral oscillators by SCN-borne timing cues. The oscillatory mechanism that is at the center of the circadian clock is thought to involve a negative feedback of CRYs and PERs on their own transcription. An externally driven mPer2 transcription cycle would thus gate the phase of the peripheral clock to that of the systemic signals. Indeed, the system-driven expression of mPer2 provides a direct link between circadian systemic signals and the phase of peripheral oscillators. Although the molecular mechanisms responsible for system-driven mPer2 transcription remain to be identified, the observation that many heat-shock protein genes were found to be expressed in phase with mPer2 suggests that the cyclic transcription of mPer2 and Hsp genes shares certain regulatory mechanisms. Of note, real-time bioluminescence recordings of mPer2::luc-expressing liver and lung explants exposed to a heat shock showed that mPer2 transcription can indeed be influenced by temperature. Moreover, the 5′-flanking region of mPer2 harbors five heat-shock response elements (HSEs) within 1,700 bp, of which one is 100% identical in mouse, rat, man, and dog. The identification of the physiologically relevant HSEs within the mPer2 gene will be particularly important, since the activity of HSF1 can also be influenced by chemical cues (e.g., oxidants) [33]. As feeding cycles are the most dominant Zeitgebers for peripheral clocks thus far identified, it is tempting to speculate that HSF1 senses rhythmic metabolism and thereby synchronizes peripheral clocks by gating mPer2 expression.
Similar to certain nuclear hormone receptors, HSF1 forms functionally inert cytoplasmic complexes with chaperones and co-chaperones in the absence of activating cues [36]. Upon exposure to elevated temperature, oxidative stress, heavy metals, or endobiotic substances (e.g., arachidonic acid), HSF1 gets activated in multiple consecutive steps [37]. These comprise: release from chaperones and co-chaperones, trimerization via an unmasked coiled-coil domain, binding to its cognate DNA sequences in regulatory regions of target genes, and stimulation of the transactivation potential via the calcium/calmodulin-dependent kinase II (CAMKII)-mediated phosphorylation of a serine residue within the HSF1 regulatory domain [38]. Of note, CamkIIb mRNA is among the transcripts whose diurnal accumulation is governed by systemic cues and whose phase is in keeping with a role of CAMKIIb in the circadian activation of HSF1 (see Figure 5A). In addition, CAMKIIb might also participate in the synchronization of peripheral clocks by a more direct mechanism. Thus, in fruit fly cells, CAMKII phosphorylates CLK, the Drosophila ortholog of CLOCK, and in cotransfection experiments. this enhances the stimulation of CLK-CYC target genes [39].
Nocturnin and Cirbp are two systemically driven genes encoding proteins potentially involved in mRNA metabolism and/or activity. Nocturnin, the vertebrate homolog of yeast CCR4, is an mRNA deadenylase [40] with rhythmic expression in many mouse tissues [41]. As both mRNA stability and translation efficiency can depend on poly(A) length, Nocturnin could influence the rhythmic accumulation of circadian proteins by post-transcriptional mechanisms. Likewise, CIRBP, a nuclear, ubiquitously expressed RNA-binding protein [42], could affect the cyclic accumulation or translation of target mRNAs in a temperature-dependent fashion, as diurnal Cirbp expression correlates negatively with body temperature rhythms. In addition, CIRBP has been demonstrated to activate the extracellular signal-regulated kinase (ERK) pathway in NIH3T3 fibroblasts [43]. Similar to CAMKII, Drosophila ERK2 can phosphorylate CLK and thereby increase the transactivation potential of this transcription factor. Cirbp mRNA and protein levels have previously been found to oscillate in brain, but not in liver [32]. However, in the latter tissue, the accumulation of Cirbp transcripts has been determined only for two time points, and the cyclic accumulation of Cirbp mRNA in liver (see Figure 5B) may thus have escaped this analysis. The similarly high amplitude of diurnal Cirbp mRNA accumulation in brain [32] and liver (this study) is somewhat surprising. In fact, most brain areas display much shallower accumulation cycles for clock and clock-controlled mRNAs than the liver. For example, circadian mPer1 and Dbp mRNA levels oscillate about 13-fold and 100-fold, respectively, in liver, but only about 1.4-fold and 2-fold, respectively, in brain [44]. The low-amplitude rhythms in the latter tissue may be the consequence of an incomplete phase entrainment of local oscillators in brain neurons by the SCN, perhaps because the transport of chemical timing cues across the blood-brain barrier is inefficient. The high amplitude of Cirbp expression in the brain may thus be caused by daily temperature fluctuations, which have similar amplitudes in the brain and peripheral organs [45].
In conclusion, we have established a transgenic mouse model that allowed us to study rhythmic liver gene expression genome-wide in the presence and absence of functional hepatocyte oscillators. The identification of genes whose amplitude and phase are nearly identical under these two conditions revealed possible mechanisms by which peripheral oscillators could be entrained. The observation that in liver the circadian expression of mPer2 can be governed by both systemic cues and hepatocyte oscillators provides a plausible mechanism for the phase entrainment of molecular oscillators in peripheral tissues. Strikingly, heat-induced and cold-induced genes were also identified among the genes whose rhythmic expression is driven by systemic cues. Of note, body temperature rhythms have previously been shown to contribute to the phase entrainment of peripheral clocks [8], and it is thus tempting to speculate that the molecular mechanism governing temperature-dependent Hsp and/or Cirbp expression are involved in this process. We feel confident that the in-depth analysis of cis-acting regulatory elements and transcription factors participating in the systemic control of circadian gene transcription will provide valuable information on the phase-entrainment pathways operative in peripheral tissues.
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