3.3 The output pathway
3.3.2 Outputs from the periphery
Most aspects of mammalian physiology are under circadian control, and many organs that are known to harbor circadian oscillators are subject to circadian regulation. The detoxifying function of the liver, the urine production by the kidneys, or the heart beating rate are examples of circadian physiological functions that are performed by such specialized organs. These circadian functions can in principle be influenced either by the local peripheral oscillators or by systemic cues, such as circulating hormones or neural signals, emitted by exogenous oscillators. Whether a given circadian output of an organ is due to the action of endogenous or exogenous oscillators has mainly been addressed by searching for targets of the clock machinery. It has thus been shown that the transcription factor DBP was circadianly expressed in the liver, due to the periodic binding of BMAL1 and CLOCK to its promoter (Ripperger et al., 2000; Ripperger and Schibler, 2006). In turn, DBP is implicated in regulating many aspects of the physiology, such as the production of detoxifying enzymes (Lavery et al., 1999) and the regulation of the pool of pyridoxal phosphate (Gachon et al., 2004a). Thus the peripheral oscillator can lead to the circadian transcription of clock-output genes that might in turn activate physiological pathways in a circadian manner. On the other hand, it is also known that a wild-type SCN graft can restore rhythmic behavior in a recipient mouse that does not have functional peripheral oscillators (Sujino et al., 2003). Many circadian aspects of the physiology are expected to be recovered in this case. For example, feeding is expected to follow the locomotor activity and thus, all the food-induced physiological processes are expected to recover rhythmicity. However, our knowledge of molecular circadian output
functions regulated by either endogenous or exogenous oscillators in a given organ is very limited. For example, we are completely ignorant about the fraction and identity of liver genes whose circadian transcription is driven by the hepatocyte oscillators or, alternatively, by rhythmic signals emanating from the SCN master pacemaker.
Circadian transcription
Many known clock genes encode transcription factors, and the currently publicized models for the mammalian oscillator mechanism involve a transcriptional feedback loop. It is then not surprising that considerable efforts have been committed to the search of clock-controlled genes whose expression is regulated at the level of transcription (Kornmann et al., 2001, Akhtar et al., 2002, Panda et al., 2002a, Storch et al., 2002, Ueda et al., 2002). Many studies on the circadian
transcriptomes of several organs have been performed using DNA microarray hybridization. We (Kornmann et al., 2001) used an enhanced differential display method that we developed in order to optimize detection of low abundance transcripts. These transcriptome profiling endeavors helped addressing a number of central issues:
1. As stated above many clock outputs are probably driven by
transcriptional mechanisms. Thus, identifying clock-controlled genes (CCGs) might help in providing a link between core clock mechanisms and circadian physiological outputs.
2. Many of the core clock genes, e.g. Per1, Per2, Cry1, Rev-Erbα, and Bmal1, dispay circadian mRNA accumulation cycles. It is thus possible that the identification of genes with circadian expression in all cell types may reveal additional core clock components.
3. The peripheral oscillators are supplied daily with environmental and systemic information arising form the SCN. These cues from the master clock might play a central role in the input pathway of peripheral clocks and must eventually act on circadian gene expression. It is thus possible that genes involved in clock resetting can be identified among the genes revealed by circadian transcriptome profiling.
All in all, these studies have revealed that in a given tissue between 1 and 10% of
the transcriptome was under circadian control but that only a few transcripts were reproducibly circadian in all examined tissues. Indeed, most of the circadian mRNA seem to be expressed in a tissue-specific manner (Duffield, 2003). The liver is by far the most extensively studied organ in terms of circadian
transcription. Three independent microarrays studies (Ueda et al., 2002, Storch et al., 2002, Panda et al., 2002a) have revealed close to 1000 circadian transcripts in the liver. However, the lists reported in these papers probably contain many false positives and false negatives, since only 81 genes are shared between the three studies (Duffield, 2003). Many of the genes that are circadian in the liver are related to metabolic functions, energy homeostasis and food-processing. Among these genes, one might expect to find genes that are controlled by the local hepatic oscillators, as is the case for Dbp and Rev-erbα. The daily transcription cycles of some other genes may be influenced by cues arising from rhythmic behavior, such as locomotor activity and feeding cycles. Finally, one expects to find genes whose circadian expression is directly regulated by hormone rhythms driven by the SCN.
For example, given that corticosterone oscillates with a 24-hour cycle in the blood plasma, glucocorticoid responsive liver genes would be expected to be transcribed in a circadian manner.
For several reasons, it would be interesting to identify genes whose circadian expression is controlled by systemic cues, since such genes are good candidates for assuming functions in the synchronization of peripheral circadian oscillators.
Moreover, the discrimination between genes whose cyclic activity depends on oscillators versus those whose transcription is directly driven by systemic signals will yield information on the extent to which each of these pathways is involved in circadian physiology.
Chapter 4
Thesis project
During my thesis work, I first characterized many hepatic clock controlled genes.
For this purpose I developed a novel method for the differential display of mRNAs. This method, dubbed ADDER (for Amplification of Double-stranded cDNA End Restriction fragments), has the advantage that it does not depend on a previous identification of genes and has been proven particularly useful in the
detection of some low abundance transcripts17 . Obviously, it has the shortcoming of being labor intensive, in that it involves conducting of thousands of PCR reactions and the identification of differentially expressed transcripts by gel purification, molecular cloning, and sequencing. The results of our ADDER analysis are described in the joined publication (Kornmann et al., 2001). 51 hepatic transcript displaying different expressions around the clock were cloned and sequenced. Most of these encode functions that deal with food-processing, energy homeostasis and detoxification. These results strongly suggest that the circadian clock plays a pivotal role in metabolic reactions associated with food processing.
The recent improvements of the microarray technology allowed to confirm and complement these results (Akhtar et al., 2002, Panda et al., 2002a, Storch et al., 2002, Ueda et al., 2002).
I also used the ADDER technique as a mean to ascertain the fact that a restricted feeding regimen primarily influences circadian gene expression. To this end, I compared the liver transcriptomes of mice fed ad libitum throughout the day with those of mice subjected to daytime-restricted feeding regimen at six four-hour intervals around the clock. While most transcripts were unaffected by the restricted feeding regimen, every observed circadian gene was phase-inverted. These results were included in (Damiola et al., 2000).
In order to get insight into the relative importance of the central SCN pacemaker and peripheral clocks in driving circadian gene expression, I generated double-transgenic mice in which the liver oscillator has been specifically
inactivated. These mice express the orphan nuclear receptor REV-ERBα in an inducible and tissue specific fashion. Since REV-ERBα is a strong repressor of the essential clock gene Bmal1, the overexpression of REV-ERBα from a transgene attenuates Bmal1 transcription, which in turn should arrest the oscillator. To engineer these transgenic mice, I used the tet-off regulatory system designed by Bujard and coworkers (Gossen and Bujard, 1992, Gossen et al., 1994). A REV-ERBα cDNA sequence has been placed under the control of a minimal CMV promoter, downstream of seven tetracycline responsive DNA elements (TREs). In
17 Indeed, circadian transcript cannot have a long half-life, they are thus likely not to accumulate to great amounts.
parallel, the tetracycline dependent trans-activator gene (tTA)18 is expressed from the C/ebpβ-LAP LCR/promoter (Talbot et al., 1994). In transgenic mice, the C/ebpβ-LAP locus control region (LCR) drives the constitutive expression of the tTA gene in a nearly hepatocyte-specific fashion (Kistner et al., 1996). In wild-type mice the circadian accumulation of the REV-ERBα repressor allows the Bmal1 gene to be expressed during a sufficient time window , and BMAL1 protein can thus reach the concentration required for normal circadian rhythm generation.
In untreated double transgenic LAP-tTA, TRE-Rev-erbα mice, however, transgene-encoded REV-ERBα levels are always above the concentration required to repress Bmal1 transcription, and BMAL1 can thus not accumulate to functionally relevant levels. This arrests the circadian clock in hepatocytes without affecting the one operative in other cell types19 . Importantly, TRE-Rev-erbα transgene expression can be inhibited completely by providing doxycycline20 through the food, which reanimates the hepatocyte oscillator in vivo and in vitro. Surprisingly, while the expression of bona fide targets of BMAL1, such as Dbp or endogenous Rev-erbα are greatly downregulated in hepatocytes when their oscillator is inactivated, circadian mPer2 expression persists at nearly normal levels. This result challenges the concept that BMAL1 is required for mPer2 expression. It also suggests that rhythmic mPer2 expression can be driven by exogenous circadian cues. Hence, mPer2 is an excellent candidate for an input gene involved in the synchronization of hepatocyte oscillators.
In order to identify additional genes whose circadian expression is regulated by systemic cues, we profiled the circadian liver transcriptomes for mice treated with doxycycline, in which the hepatocyte oscillators were operative, and untreated mice, in which the hepatocyte oscillators were arrested. We thus found 31 gene whose circadian expression was nearly indistinguishable in phase and amplitude between the doxycycline treated and untreated mice. These included genes that were quite unexpected, such as the tubulin-α or the nocturnin. Interestingly, however, this group of circadian genes also comprised several genes encoding
18 tTA encodes a fusion protein between the bacterial tetracycline operon repressor and the viral VP16 activation domain. This protein binds the TREs and activates the transcription
19 Indeed, the locomotor activity of these mice is normal and the Bmal1 gene expression is indistinguishable in kidney, lung, heart or spleen of mice that were fed with and without doxycycline.
20 Doxycycline is a tetracycline analog with reduced toxicity
various heat shock proteins, and most of these are well known targets of the heat-shock transcription factor HSF1. It is thus tempting to speculate that feeding time or any other systemic circadian cue periodically activates the heat-shock factor, and that periodically activated HSF1 drives genes involved in the phase
entrainment of peripheral clocks, perhaps even that of mPer2. We wish to
emphasize that, unlike of what we and others have found for the bulk of circadian liver genes, a high fraction of the subset of systemically regulated circadian genes are expressed in most if not all tissues and cell types. This would be expected for genes implicated in the synchronization of peripheral clocks.
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