Final Report Summary - TIMESIGNAL (Signalling within the mammalian circadian timing system)
In mammals, virtually all physiological processes are subject to daily oscillations. The rhythmic fluctuations in physiology are coordinated by a complex circadian timing system, composed of a master pacemaker residing in the suprachiasmatic nucleus (SCN) of the brain, and subsidiary clocks operative in nearly all body cells. In the absence of external timing cues the SCN generates rhythms with a period length of approximately - but not exactly - 24 hours, and it must therefore be synchronized every day by light-dark cycles in order to stay in resonance with geophysical time. The SCN clock has a limited phase-shifting capacity. In other words, it can adjust time by only one to two hours per day. Therefore, west- and east-bound transatlantic flights (associated with rapid and large changes of time zones) provoke jet lags during several consecutive days. This temporary disruption of circadian physiology also interferes with functions of peripheral organs, such as metabolism and detoxification in the liver. Hence, many frequent travelers suffer from indigestion during the first days in the new time zone in addition to experiencing problems with their sleep-wake cycles.
The ERC AdG research project TimeSignal was dedicated to the identification and characterization of signaling pathways involved in the systemic regulation of diurnal gene expression in mice and cultured cells. By studying such pathways we hoped to elucidate mechanisms involved in the synchronization of circadian oscillators in peripheral organs. To this end we developed novel tools allowing us to identify and study signals depending on feeding-fasting rhythms, previously shown to be the dominant Zeitgebers (“time givers”) for circadian clocks in most tissues, blood-borne signals and body temperature-dependent cues. The redundancy in Zeitgeber cues and the associated signaling pathways pose a major challenge. For example, the steady-state phase of circadian gene expression in the liver is not significantly affected by the elimination of a single pathway. However, the recording of phase-shifting kinetics is much more sensitive to the disruption of individual regulators involved in the phase resetting of peripheral clocks. If performed by conventional methods, i.e. the temporal recording of mRNA and protein accumulation patterns, such experiments are extremely labor-intensive, require unduly large numbers of mice and provide only a poor temporal resolution. To overcome these problems we engineered a device, dubbed RT-Biolumicorder, which allows us to monitor the expression of circadian luciferase reporter genes in real-time and for extended time periods in peripheral organs of freely moving mice. Using this novel technology we could shed light on several important facets of the circadian timing system. Thus, we could show that the SCN is indeed required for the synchronization of circadian clocks in peripheral organs. While this has always been assumed, it had never been documented by compelling in vivo experiments. Surprisingly, however, the phase coherence is maintained within the liver of SCN-lesioned mice, suggesting that within some organs the phases are coupled between cells. Moreover, using the RT-Biolumicorder technology we could demonstrate that there is no inertia between the synchronization of the SCN and that of peripheral clocks.
The activation of an immediate early transcription factor is often the last step in a signal transduction cascade. Synthetic Tandem Repeat Promoter screening (STAR-PROM), another novel technology established in the framework of TimeSignal, enabled us to screen for immediate transcription factors activated by diurnally active blood-borne signals. This project resulted in the discovery that daily oscillations in actin and tubulin cytoskeleton dynamics participate in the synchronization of circadian clocks. In liver, spleen, and possibly other organs, blood-borne factors activate the GTPase RhoA in a rhythmic fashion, thereby eliciting daily polymerization cycles of globular into fibrillary actin. This leads to activity rhythms of Myocardin-Related Transcription Factors (MRTFs), which serve as coactivators of Serum Response Factor (SRF). SRF is a transcription factor binding to enhancer elements within the core clock gene Per2 and thus participates in the system-driven phase-resetting of peripheral clocks. In dividing cells, surges of microtubule dissociation and actin polymerization induce bursts of MRTF activation, possibly accounting for the synchronization of circadian oscillators by the cell division cycle.
The ERC AdG research project TimeSignal was dedicated to the identification and characterization of signaling pathways involved in the systemic regulation of diurnal gene expression in mice and cultured cells. By studying such pathways we hoped to elucidate mechanisms involved in the synchronization of circadian oscillators in peripheral organs. To this end we developed novel tools allowing us to identify and study signals depending on feeding-fasting rhythms, previously shown to be the dominant Zeitgebers (“time givers”) for circadian clocks in most tissues, blood-borne signals and body temperature-dependent cues. The redundancy in Zeitgeber cues and the associated signaling pathways pose a major challenge. For example, the steady-state phase of circadian gene expression in the liver is not significantly affected by the elimination of a single pathway. However, the recording of phase-shifting kinetics is much more sensitive to the disruption of individual regulators involved in the phase resetting of peripheral clocks. If performed by conventional methods, i.e. the temporal recording of mRNA and protein accumulation patterns, such experiments are extremely labor-intensive, require unduly large numbers of mice and provide only a poor temporal resolution. To overcome these problems we engineered a device, dubbed RT-Biolumicorder, which allows us to monitor the expression of circadian luciferase reporter genes in real-time and for extended time periods in peripheral organs of freely moving mice. Using this novel technology we could shed light on several important facets of the circadian timing system. Thus, we could show that the SCN is indeed required for the synchronization of circadian clocks in peripheral organs. While this has always been assumed, it had never been documented by compelling in vivo experiments. Surprisingly, however, the phase coherence is maintained within the liver of SCN-lesioned mice, suggesting that within some organs the phases are coupled between cells. Moreover, using the RT-Biolumicorder technology we could demonstrate that there is no inertia between the synchronization of the SCN and that of peripheral clocks.
The activation of an immediate early transcription factor is often the last step in a signal transduction cascade. Synthetic Tandem Repeat Promoter screening (STAR-PROM), another novel technology established in the framework of TimeSignal, enabled us to screen for immediate transcription factors activated by diurnally active blood-borne signals. This project resulted in the discovery that daily oscillations in actin and tubulin cytoskeleton dynamics participate in the synchronization of circadian clocks. In liver, spleen, and possibly other organs, blood-borne factors activate the GTPase RhoA in a rhythmic fashion, thereby eliciting daily polymerization cycles of globular into fibrillary actin. This leads to activity rhythms of Myocardin-Related Transcription Factors (MRTFs), which serve as coactivators of Serum Response Factor (SRF). SRF is a transcription factor binding to enhancer elements within the core clock gene Per2 and thus participates in the system-driven phase-resetting of peripheral clocks. In dividing cells, surges of microtubule dissociation and actin polymerization induce bursts of MRTF activation, possibly accounting for the synchronization of circadian oscillators by the cell division cycle.