Feedback Loops of the Mammalian Circadian Clock Constitute Repressilator.

Mammals evolved an endogenous timing system to coordinate their physiology and behaviour to the 24h period of the solar day. While it is well accepted that circadian rhythms are generated by intracellular transcriptional feedback loops, it is still debated which network motifs are necessary and sufficient for generating self-sustained oscillations. Here, we systematically explore a data-based circadian oscillator model with multiple negative and positive feedback loops and identify a series of three subsequent inhibitions known as "repressilator" as a core element of the mammalian circadian oscillator. The central role of the repressilator motif is consistent with time-resolved ChIP-seq experiments of circadian clock transcription factors and loss of rhythmicity in core clock gene knockouts.

Inducible cAMP early repressor regulates the Period 1 gene of the hepatic and adrenal clocks.

Light, restricted feeding, and hormonal inputs may operate as time givers (zeitgebers) for the circadian clock within peripheral organs through the activation of tissue-specific signaling cascades. cAMP signaling through CREM (cAMP-responsive element modulator) and its variant ICER (inducible cAMP early repressor) is linked to the circadian regulation of pineal melatonin synthesis, although little is known about its influence in other organs. We performed experiments in the absence of light and feeding-time cues to test which core clock genes are controlled by CREM/ICER in the liver and adrenal gland. In vivo, Crem loss-of-function mutation resulted in fine-tuning of all measured adrenal clock genes (Per1/2/3, Cry1/2, Bmal1, and Rev-erbα), whereas only Per1 and Cry1 were affected in the liver. Icer expression was circadian in the adrenal gland, with peak gene expression at zeitgeber 12 and the highest protein levels at zeitgeber ∼20. The expression of both Icer and Per1 genes responded to cAMP stimuli in an immediate-early fashion. In immortal cells, forskolin induced expression of Per1 after 2 h, and de novo protein synthesis led to Per1 attenuation. We show that the de novo synthesized protein responsible for Per1 attenuation is ICER. Indeed, Per1 expression is up-regulated in cells ectopically expressing antisense Icer, and mobility shift experiments identified ICER binding to cAMP-responsive elements of the Per1 promoter. We propose that ICER acts as a noise filter for different signals that could affect transcription in the adrenal gland. Because ICER is an immediate-early repressor, the circadian nature of adrenal Icer expression could serve a role in a time-dependent gating mechanism.

Mouse genotypes drive the liver and adrenal gland clocks.

Circadian rhythms regulate a plethora of physiological processes. Perturbations of the rhythm can result in pathologies which are frequently studied in inbred mouse strains. We show that the genotype of mouse lines defines the circadian gene expression patterns. Expression of majority of core clock and output metabolic genes are phase delayed in the C56BL/6J line compared to 129S2 in the adrenal glands and the liver. Circadian amplitudes are generally higher in the 129S2 line. Experiments in dark - dark (DD) and light - dark conditions (LD), exome sequencing and data mining proposed that mouse lines differ in single nucleotide variants in the binding regions of clock related transcription factors in open chromatin regions. A possible mechanisms of differential circadian expression could be the entrainment and transmission of the light signal to peripheral organs. This is supported by the genotype effect in adrenal glands that is largest under LD, and by the high number of single nucleotide variants in the Receptor, Kinase and G-protein coupled receptor Panther molecular function categories. Different phenotypes of the two mouse lines and changed amino acid sequence of the Period 2 protein possibly contribute further to the observed differences in circadian gene expression.

Timing of circadian genes in mammalian tissues.

Circadian clocks are endogenous oscillators driving daily rhythms in physiology. The cell-autonomous clock is governed by an interlocked network of transcriptional feedback loops. Hundreds of clock-controlled genes (CCGs) regulate tissue specific functions. Transcriptome studies reveal that different organs (e.g. liver, heart, adrenal gland) feature substantially varying sets of CCGs with different peak phase distributions. To study the phase variability of CCGs in mammalian peripheral tissues, we develop a core clock model for mouse liver and adrenal gland based on expression profiles and known cis-regulatory sites. 'Modulation factors' associated with E-boxes, ROR-elements, and D-boxes can explain variable rhythms of CCGs, which is demonstrated for differential regulation of cytochromes P450 and 12 h harmonics. By varying model parameters we explore how tissue-specific peak phase distributions can be generated. The central role of E-boxes and ROR-elements is confirmed by analysing ChIP-seq data of BMAL1 and REV-ERB transcription factors.

The interplay of cis-regulatory elements rules circadian rhythms in mouse liver.

The mammalian circadian clock is driven by cell-autonomous transcriptional feedback loops that involve E-boxes, D-boxes, and ROR-elements. In peripheral organs, circadian rhythms are additionally affected by systemic factors. We show that intrinsic combinatorial gene regulation governs the liver clock. With a temporal resolution of 2 h, we measured the expression of 21 clock genes in mouse liver under constant darkness and equinoctial light-dark cycles. Based on these data and known transcription factor binding sites, we develop a six-variable gene regulatory network. The transcriptional feedback loops are represented by equations with time-delayed variables, which substantially simplifies modelling of intermediate protein dynamics. Our model accurately reproduces measured phases, amplitudes, and waveforms of clock genes. Analysis of the network reveals properties of the clock: overcritical delays generate oscillations; synergy of inhibition and activation enhances amplitudes; and combinatorial modulation of transcription controls the phases. The agreement of measurements and simulations suggests that the intrinsic gene regulatory network primarily determines the circadian clock in liver, whereas systemic cues such as light-dark cycles serve to fine-tune the rhythms.

Energetics of MazG unfolding in correlation with its structural features.

MazG is a homodimeric alpha-helical protein that belongs to the superfamily of all-alpha NTP pyrophosphatases. Its function has been connected to the regulation of the toxin-antitoxin module mazEF, implicated in programmed growth arrest/cell death of Escherichia coli cells under conditions of amino acid starvation. The goal of the first detailed biophysical study of a member of the all-alpha NTP pyrophosphatase superfamily, presented here, is to improve molecular understanding of the unfolding of this type of proteins. Thermal unfolding of MazG monitored by differential scanning calorimetry, circular dichroism spectroscopy, and fluorimetry at neutral pH in the presence of a reducing agent (dithiothreitol) can be successfully described as a reversible four-state transition between a dimeric native state, two dimeric intermediate states, and a monomeric denatured state. The first intermediate state appears to have a structure similar to that of the native state while the final thermally denatured monomeric state is not fully unfolded and contains a significant fraction of residual alpha-helical structure. In the absence of dithiothreitol, disulfide cross-linking causes misfolding of MazG that appears to be responsible for the formation of multimeric aggregates. MazG is most stable at pH 7-8, while at pH <6, it exists in a molten-globule-like state. The thermodynamic parameters characterizing each step of MazG denaturation transition obtained by global fitting of the four-state model to differential scanning calorimetry, circular dichroism, and fluorimetry temperature profiles are in agreement with the observed structural characteristics of the MazG conformational states and their assumed functional role.