Jürgen A. Ripperger

Rhythmic CLOCK-BMAL1 binding to multiple E-box motifs drives circadian Dbp transcription and chromatin transitions

Mammalian circadian rhythms are based on transcriptional and post-translational feedback loops. Essentially, the activity of the transcription factors BMAL1 (also known as MOP3) and CLOCK is rhythmically counterbalanced by Period (PER) and Cryptochrome (CRY) proteins to govern time of day–dependent gene expression. Here we show that circadian regulation of the mouse albumin D element–binding protein (Dbp) gene involves rhythmic binding of BMAL1 and CLOCK and marked daily chromatin transitions. Thus, the Dbp transcription cycle is paralleled by binding of BMAL1 and CLOCK to multiple extra- and intragenic E boxes, acetylation of Lys9 of histone H3, trimethylation of Lys4 of histone H3 and a reduction of histone density. In contrast, the antiphasic daily repression cycle is accompanied by dimethylation of Lys9 of histone H3, the binding of heterochromatin protein 1α and an increase in histone density. The rhythmic conversion of transcriptionally permissive chromatin to facultative heterochromatin relies on the presence of functional BMAL1-CLOCK binding sites.

Regulation of Monoamine Oxidase A by Circadian-Clock Components Implies Clock Influence on Mood

The circadian clock has been implicated in addiction and several forms of depression [1, 2], indicating interactions between the circadian and the reward systems in the brain [3-5]. Rewards such as food, sex, and drugs influence this system in part by modulating dopamine neurotransmission in the mesolimbic dopamine reward circuit, including the ventral tegmental area (VTA) and the ventral striatum (NAc). Hence, changes in dopamine levels in these brain areas are proposed to influence mood in humans and mice [6-10]. To establish a molecular link between the circadian-clock mechanism and dopamine metabolism, we analyzed the murine promoters of genes encoding key enzymes important in dopamine metabolism. We find that transcription of the monoamine oxidase A (Maoa) promoter is regulated by the clock components BMAL1, NPAS2, and PER2. A mutation in the clock gene Per2 in mice leads to reduced expression and activity of MAOA in the mesolimbic dopaminergic system. Furthermore, we observe increased levels of dopamine and altered neuronal activity in the striatum, and these results probably lead to behavioral alterations observed in Per2 mutant mice in despair-based tests. These findings suggest a role of circadian-clock components in dopamine metabolism highlighting a role of the clock in regulating mood-related behaviors.

The mammalian clock component PERIOD2 coordinates circadian output by interaction with nuclear receptors

Mammalian circadian clocks provide a temporal framework to synchronize biological functions. To obtain robust rhythms with a periodicity of about a day, these clocks use molecular oscillators consisting of two interlocked feedback loops. The core loop generates rhythms by transcriptional repression via the Period (PER) and Cryptochrome (CRY) proteins, whereas the stabilizing loop establishes roughly antiphasic rhythms via nuclear receptors. Nuclear receptors also govern many pathways that affect metabolism and physiology. Here we show that the core loop component PER2 can coordinate circadian output with the circadian oscillator. PER2 interacts with nuclear receptors including PPARalpha and REV-ERBalpha and serves as a coregulator of nuclear receptor-mediated transcription. Consequently, PER2 is rhythmically bound at the promoters of nuclear receptor target genes in vivo. In this way, the circadian oscillator can modulate the expression of nuclear receptor target genes like Bmal1, Hnf1alpha, and Glucose-6-phosphatase. The concept that PER2 may propagate clock information to metabolic pathways via nuclear receptors adds an important facet to the clock-dependent regulation of biological networks.

Lack of food anticipation in Per2 mutant mice.

Predicting time of food availability is key for survival in most animals. Under restricted feeding conditions, this prediction is manifested in anticipatory bouts of locomotor activity and body temperature. This process seems to be driven by a food-entrainable oscillator independent of the main, light-entrainable clock located in the suprachiasmatic nucleus (SCN) of the hypothalamus . Although the SCN clockwork involves self-sustaining transcriptional and translational feedback loops based on rhythmic expression of mRNA and proteins of clock genes , the molecular mechanisms responsible for food anticipation are not well understood. Period genes Per1 and Per2 are crucial for the SCNs resetting to light . Here, we investigated the role of these genes in circadian anticipatory behavior by studying rest-activity and body-temperature rhythms of Per1 and Per2 mutant mice under restricted feeding conditions. We also monitored expression of clock genes in the SCN and peripheral tissues. Whereas wild-type and Per1 mutant mice expressed regular food-anticipatory activity, Per2 mutant mice did not show food anticipation. In peripheral tissues, however, phase shifts of clock-gene expression in response to timed food restriction were comparable in all genotypes. In conclusion, a mutation in Per2 abolishes anticipation of mealtime, without interfering with peripheral synchronization by feeding cycles.

The circadian molecular clock creates epidermal stem cell heterogeneity

Murine epidermal stem cells undergo alternate cycles of dormancy and activation, fuelling tissue renewal. However, only a subset of stem cells becomes active during each round of morphogenesis, indicating that stem cells coexist in heterogeneous responsive states. Using a circadian-clock reporter-mouse model, here we show that the dormant hair-follicle stem cell niche contains coexisting populations of cells at opposite phases of the clock, which are differentially predisposed to respond to homeostatic cues. The core clock protein Bmal1 modulates the expression of stem cell regulatory genes in an oscillatory manner, to create populations that are either predisposed, or less prone, to activation. Disrupting this clock equilibrium, through deletion of Bmal1 (also known as Arntl) or Per1/2, resulted in a progressive accumulation or depletion of dormant stem cells, respectively. Stem cell arrhythmia also led to premature epidermal ageing, and a reduction in the development of squamous tumours. Our results indicate that the circadian clock fine-tunes the temporal behaviour of epidermal stem cells, and that its perturbation affects homeostasis and the predisposition to tumorigenesis.

The Multiple Facets of Per2

The Period 2 (Per2) gene is an important component of the circadian system. It appears to be not only part of the core oscillator mechanism, but also part of the input and output pathways of the clock. Because of its involvement at multiple levels of the circadian system, Per2 needs to meet a variety of different demands. We discuss how Per2 might be able to fulfill multiple functions by reviewing known facts and combine this with speculations based on these facts. This might provide new views about Per2 function and help to better understand diseases that are rooted in the circadian system.

The daily rhythm of mice

The house mouse Mus musculus represents a valuable tool for the analysis and the understanding of the mammalian circadian oscillator. Forward and reverse genetics allowed the identification of clock components and the verification of their function within the circadian clockwork. In many cases unforeseen links were discovered between a particular circadian regulatory protein and various diseases or syndromes. Thus, this model system is not only perfectly suited to pinpoint the components of the mammalian circadian clock, but also to unravel metabolic, physiological, and pathological processes linked to the circadian timing system.

The role of clock genes and rhythmicity in the liver

The liver is the important organ to maintain energy homeostasis of an organism. To achieve this, many biochemical reactions run in this organ in a rhythmic fashion. An elegant way to coordinate the temporal expression of genes for metabolic enzymes relies in the link to the circadian timing system. In this fashion not only a maximum of synchronization is achieved, but also anticipation of daily recurring events is possible. Here we will focus on the input and output pathways of the hepatic circadian oscillator and discuss the recently found flexibility of its circadian transcriptional networks.

Role of the circadian clock gene Per2 in adaptation to cold temperature

Adaptive thermogenesis allows mammals to resist to cold. For instance, in brown adipose tissue (BAT) the facultative uncoupling of the proton gradient from ATP synthesis in mitochondria is used to generate systemic heat. However, this system necessitates an increase of the Uncoupling protein 1 (Ucp1) and its activation by free fatty acids. Here we show that mice without functional Period2 (Per2) were cold sensitive because their adaptive thermogenesis system was less efficient. Upon cold-exposure, Heat shock factor 1 (HSF1) induced Per2 in the BAT. Subsequently, PER2 as a co-activator of PPARα increased expression of Ucp1. PER2 also increased Fatty acid binding protein 3 (Fabp3), a protein important to transport free fatty acids from the plasma to mitochondria to activate UCP1. Hence, in BAT PER2 is important for the coordination of the molecular response of mice exposed to cold by synchronizing UCP1 expression and its activation.

Flexible phase adjustment of circadian albumin D site-binding protein (Dbp) gene expression by CRYPTOCHROME1

The albumin D site-binding protein (DBP) governs circadian transcription of a number of hepatic detoxification and metabolic enzymes prior to the activity phase and subsequent food intake of mice. However, the behavior of mice is drastically affected by the photoperiod. Therefore, continuous adjustment of the phase of circadian Dbp expression is required in the liver. Here we describe a direct impact of CRYPTOCHROME1 (CRY1) on the phase of Dbp expression. Dbp and the nuclear receptor Rev-Erbalpha are circadian target genes of BMAL1 and CLOCK. Surprisingly, dynamic CRY1 binding to the Dbp promoter region delayed BMAL1 and CLOCK-mediated transcription of Dbp compared with Rev-Erbalpha. Extended presence of CRY1 in the nucleus enabled continuous uncoupling of the phase of Dbp from Rev-Erbalpha expression upon change from short to longer photoperiods. CRY1 thus maintained the peak of DBP accumulation close to the activity phase. In contrast, Rev-Erbalpha expression was phase-locked to the circadian oscillator and shaped by accumulation of its own gene product. Our data indicate that fine-tuning of circadian transcription in the liver is even more sophisticated than expected.