System-Driven and Oscillator-Dependent Circadian Transcription in Mice with a Conditionally Active Liver Clock

Abstract

The mammalian circadian timing system consists of a master pacemaker in neurons of the suprachiasmatic nucleus (SCN) and clocks of a similar molecular makeup in most peripheral body cells. Peripheral oscillators are self-sustained and cell autonomous, but they have to be synchronized by the SCN to ensure phase coherence within the organism. In principle, the rhythmic expression of genes in peripheral organs could thus be driven not only by local oscillators, but also by circadian systemic signals. To discriminate between these mechanisms, we engineered a mouse strain with a conditionally active liver clock, in which REV-ERBα represses the transcription of the essential core clock gene Bmal1 in a doxycycline-dependent manner. We examined circadian liver gene expression genome-wide in mice in which hepatocyte oscillators were either running or arrested, and found that the rhythmic transcription of most genes depended on functional hepatocyte clocks. However, we discovered 31 genes, including the core clock gene mPer2, whose expression oscillated robustly irrespective of whether the liver clock was running or not. By contrast, in liver explants cultured in vitro, circadian cycles of mPer2::luciferase bioluminescence could only be observed when hepatocyte oscillators were operational. Hence, the circadian cycles observed in the liver of intact animals without functional hepatocyte oscillators were likely generated by systemic signals. The finding that rhythmic mPer2 expression can be driven by both systemic cues and local oscillators suggests a plausible mechanism for the phase entrainment of subsidiary clocks in peripheral organs. In contrast to previously held belief, molecular circadian oscillators are not restricted to specialized pacemaker tissues, such as the brain's suprachiasmatic nucleus (SCN), but exist in virtually all body cells. Although the circadian clocks operative in peripheral cell types are as robust as those residing in SCN neurons, they quickly become desynchronized in vitro due to variations in period length. Hence, in intact animals, the phase coherence between peripheral oscillators must be established by daily signals generated by the SCN master clock. Although the hierarchy between master and slave oscillators is now well established, the respective roles of these clocks in governing the circadian transcription program in a given organ have never been examined. In principle, the circadian expression of genes in a peripheral tissue could be driven either by cyclic systemic cues, by peripheral oscillators, or by both. In order to discriminate between genes regulated by local oscillators and systemic cues in liver, we generated mice in which hepatocyte clocks can be turned on and off at will. These studies suggest that 90% of the circadian transcription program in the liver is abolished or strongly attenuated when hepatocyte clocks are turned off, indicating that the expression of most circadian liver genes is orchestrated by local cellular clocks. The remaining 10% of cyclically expressed liver genes continue to be transcribed in a robustly circadian fashion in the absence of functional hepatocyte oscillators. These genes, which unexpectedly include the bona fide clock gene mPer2, must therefore be regulated by oscillating systemic signals, such as hormones, metabolites, or body temperature. Although temperature rhythms display only modest amplitudes, they appear to play a significant role in the phase entrainment of mPer2 transcription.