Diurnal Oscillations in Liver Mass and Cell Size Accompany Ribosome Assembly Cycles.

The liver plays a pivotal role in metabolism and xenobiotic detoxification, processes that must be particularly efficient when animals are active and feed. A major question is how the liver adapts to these diurnal changes in physiology. Here, we show that, in mice, liver mass, hepatocyte size, and protein levels follow a daily rhythm, whose amplitude depends on both feeding-fasting and light-dark cycles. Correlative evidence suggests that the daily oscillation in global protein accumulation depends on a similar fluctuation in ribosome number. Whereas rRNA genes are transcribed at similar rates throughout the day, some newly synthesized rRNAs are polyadenylated and degraded in the nucleus in a robustly diurnal fashion with a phase opposite to that of ribosomal protein synthesis. Based on studies with cultured fibroblasts, we propose that rRNAs not packaged into complete ribosomal subunits are polyadenylated by the poly(A) polymerase PAPD5 and degraded by the nuclear exosome.

How the circadian nuclear orphan receptor REV-ERBα represses transcription: Temporal and spatial phase separation combined.

In this issue of Molecular Cell, Zhu et al.1 demonstrate that REV-ERBα and its co-repressor NCOR1 are assembled into daytime-dependent liquid droplets that constitute hubs in which the transcription of multiple REV-ERBα target genes is simultaneously repressed.

PARP-1 drives slumber: A reciprocal relationship between sleep homeostasis and DNA damage repair.

Sleep-wake cycles are orchestrated by the circadian clock and a sleep homeostat that records accumulating sleep pressure. Zada et al. (2021) now show that DNA lesions, recognized by PARP-1, accumulate in neurons of zebrafish larvae and promote homeostatic sleep pressure.

BMAL1 dephosphorylation determines the pace of the circadian clock.

In mammals, virtually all body cells harbor cell-autonomous and self-sustained circadian oscillators that rely on delayed negative feedback loops in gene expression. Transcriptional activation and repression play a major role in keeping these clocks ticking, but numerous post-translational mechanisms-and particularly the phosphorylation of core clock components by protein kinases-are also critically involved in setting the pace of these timekeepers. In this issue of Genes & Development, Klemz and colleagues (pp. 1161-1174) now show how dephosphorylation of BMAL1 by protein phosphatase 4 (PPP4) participates in the modulation of circadian timing.

Circadian hepatocyte clocks keep synchrony in the absence of a master pacemaker in the suprachiasmatic nucleus or other extrahepatic clocks.

It has been assumed that the suprachiasmatic nucleus (SCN) synchronizes peripheral circadian oscillators. However, this has never been convincingly shown, since biochemical time series experiments are not feasible in behaviorally arrhythmic animals. By using long-term bioluminescence recording in freely moving mice, we show that the SCN is indeed required for maintaining synchrony between organs. Surprisingly, however, circadian oscillations persist in the livers of mice devoid of an SCN or oscillators in cells other than hepatocytes. Hence, similar to SCN neurons, hepatocytes can maintain phase coherence in the absence of Zeitgeber signals produced by other organs or environmental cycles.

Glucocorticoid rhythm renders female mice more daring.

Glucocorticoids, which have been implied in mood modulation, display robust diurnal oscillations in the blood. But does their circadian rhythm regulate mood swings? Ikeda et al. now identify a paracrine signaling pathway in the adrenal cortex that potentiates the daily amplitude of plasma glucocorticoids and renders female mice braver.

Blood-borne circadian signal stimulates daily oscillations in actin dynamics and SRF activity.

In peripheral tissues circadian gene expression can be driven either by local oscillators or by cyclic systemic cues controlled by the master clock in the brain's suprachiasmatic nucleus. In the latter case, systemic signals can activate immediate early transcription factors (IETFs) and thereby control rhythmic transcription. In order to identify IETFs induced by diurnal blood-borne signals, we developed an unbiased experimental strategy, dubbed Synthetic TAndem Repeat PROMoter (STAR-PROM) screening. This technique relies on the observation that most transcription factor binding sites exist at a relatively high frequency in random DNA sequences. Using STAR-PROM we identified serum response factor (SRF) as an IETF responding to oscillating signaling proteins present in human and rodent sera. Our data suggest that in mouse liver SRF is regulated via dramatic diurnal changes of actin dynamics, leading to the rhythmic translocation of the SRF coactivator Myocardin-related transcription factor-B (MRTF-B) into the nucleus.

Senescence of Timing Reverted: NAD+ Rejuvenates the Circadian Clock.

The amplitude of circadian rhythms dampens with age, but Levine et al. (2020) now show that nicotinamide adenine dinucleotide (NAD+) can restore robust circadian gene expression and behavior in aged mice through SIRT1-dependent deacetylation of the core clock protein PER2.

Transcription factor loading: please take my place!

Interactions of transcription factors with chromatin are highly dynamic. Now Voss et al. (2011) demonstrate that two transcription factors with identical DNA-binding specificities do not compete for occupancy at a given DNA element, but instead, one factor can even facilitate the binding of another. This assisted loading probably involves chromatin-remodeling machines.

Circadian cell-cycle progression: Cracking open the gate.

In cyanobacteria cell division is intimately linked with the circadian cycle. Dong et al. (2010) now identify components of the circadian clock that regulate the formation of the midcell ring for cytokinesis, revealing a critical link between the circadian cycle and the control of cell division.

Poly(ADP-ribose) polymerase 1 participates in the phase entrainment of circadian clocks to feeding.

Circadian clocks in peripheral organs are tightly coupled to cellular metabolism and are readily entrained by feeding-fasting cycles. However, the molecular mechanisms involved are largely unknown. Here we show that in liver the activity of PARP-1, an NAD(+)-dependent ADP-ribosyltransferase, oscillates in a daily manner and is regulated by feeding. We provide biochemical evidence that PARP-1 binds and poly(ADP-ribosyl)ates CLOCK at the beginning of the light phase. The loss of PARP-1 enhances the binding of CLOCK-BMAL1 to DNA and leads to a phase-shift of the interaction of CLOCK-BMAL1 with PER and CRY repressor proteins. As a consequence, CLOCK-BMAL1-dependent gene expression is altered in PARP-1-deficient mice, in particular in response to changes in feeding times. Our results show that Parp-1 knockout mice exhibit impaired food entrainment of peripheral circadian clocks and support a role for PARP-1 in connecting feeding with the mammalian timing system.

Mammalian Circadian Cogwheels Are Parts of Macromolecular Machines.

In this issue of Molecular Cell, two papers address the biochemical structure of a large protein complex containing components of the mammalian circadian clock (Aryal et al., 2017) and a mechanism rendering this molecular timekeeper temperature-compensated (Shinohara et al., 2017).

Steven A. Brown and the synchronization of circadian rhythms by body temperature cycles.

The mammalian circadian timing system has a hierarchical architecture, with a central pacemaker located in the brain's suprachiasmatic nucleus orchestrating rhythms in behaviour and physiology. In cooperation with environmental cycles, it synchronizes the phases of peripheral oscillators operating in most cells of the body. Even cells kept in tissue culture harbour self-sustained and cell-autonomous circadian clocks that keep ticking throughout their lifespan. The master pacemaker in the suprachiasmatic nucleus is synchronized primarily by light-dark cycles, whereas peripheral oscillators are phase entrained by a multitude of systemic signalling pathways. These include pathways depending on feeding-fasting cycles, cellular actin polymerization dynamics, endocrine rhythms and, surprisingly, body temperature oscillations. Using tissue culture and murine models, Steve Brown was the first one to demonstrate that shallow rhythms of mammalian body temperature are timing cues (zeitgebers) for peripheral circadian clocks.

Unbiased identification of signal-activated transcription factors by barcoded synthetic tandem repeat promoter screening (BC-STAR-PROM).

The discovery of transcription factors (TFs) controlling pathways in health and disease is of paramount interest. We designed a widely applicable method, dubbed barcorded synthetic tandem repeat promoter screening (BC-STAR-PROM), to identify signal-activated TFs without any a priori knowledge about their properties. The BC-STAR-PROM library consists of ∼3000 luciferase expression vectors, each harboring a promoter (composed of six tandem repeats of synthetic random DNA) and an associated barcode of 20 base pairs (bp) within the 3' untranslated mRNA region. Together, the promoter sequences encompass >400,000 bp of random DNA, a sequence complexity sufficient to capture most TFs. Cells transfected with the library are exposed to a signal, and the mRNAs that it encodes are counted by next-generation sequencing of the barcodes. This allows the simultaneous activity tracking of each of the ∼3000 synthetic promoters in a single experiment. Here we establish proof of concept for BC-STAR-PROM by applying it to the identification of TFs induced by drugs affecting actin and tubulin cytoskeleton dynamics. BC-STAR-PROM revealed that serum response factor (SRF) is the only immediate early TF induced by both actin polymerization and microtubule depolymerization. Such changes in cytoskeleton dynamics are known to occur during the cell division cycle, and real-time bioluminescence microscopy indeed revealed cell-autonomous SRF-myocardin-related TF (MRTF) activity bouts in proliferating cells.

Temperature regulates splicing efficiency of the cold-inducible RNA-binding protein gene Cirbp.

In mammals, body temperature fluctuates diurnally around a mean value of 36°C-37°C. Despite the small differences between minimal and maximal values, body temperature rhythms can drive robust cycles in gene expression in cultured cells and, likely, animals. Here we studied the mechanisms responsible for the temperature-dependent expression of cold-inducible RNA-binding protein (CIRBP). In NIH3T3 fibroblasts exposed to simulated mouse body temperature cycles, Cirbp mRNA oscillates about threefold in abundance, as it does in mouse livers. This daily mRNA accumulation cycle is directly controlled by temperature oscillations and does not depend on the cells' circadian clocks. Here we show that the temperature-dependent accumulation of Cirbp mRNA is controlled primarily by the regulation of splicing efficiency, defined as the fraction of Cirbp pre-mRNA processed into mature mRNA. As revealed by genome-wide "approach to steady-state" kinetics, this post-transcriptional mechanism is widespread in the temperature-dependent control of gene expression.

SIRT1 regulates circadian clock gene expression through PER2 deacetylation.

The mammalian circadian timing system is composed of a central pacemaker in the suprachiasmatic nucleus of the brain that synchronizes countless subsidiary oscillators in peripheral tissues. The rhythm-generating mechanism is thought to rely on a feedback loop involving positively and negatively acting transcription factors. BMAL1 and CLOCK activate the expression of Period (Per) and Cryptochrome (Cry) genes, and once PER and CRY proteins accumulate to a critical level they form complexes with BMAL1-CLOCK heterodimers and thereby repress the transcription of their own genes. Here, we show that SIRT1, an NAD(+)-dependent protein deacetylase, is required for high-magnitude circadian transcription of several core clock genes, including Bmal1, Rorgamma, Per2, and Cry1. SIRT1 binds CLOCK-BMAL1 in a circadian manner and promotes the deacetylation and degradation of PER2. Given the NAD(+) dependence of SIRT1 deacetylase activity, it is likely that SIRT1 connects cellular metabolism to the circadian core clockwork circuitry.

Real-time recording of circadian liver gene expression in freely moving mice reveals the phase-setting behavior of hepatocyte clocks.

The mammalian circadian timing system consists of a master pacemaker in the suprachiasmatic nucleus (SCN) in the hypothalamus, which is thought to set the phase of slave oscillators in virtually all body cells. However, due to the lack of appropriate in vivo recording technologies, it has been difficult to study how the SCN synchronizes oscillators in peripheral tissues. Here we describe the real-time recording of bioluminescence emitted by hepatocytes expressing circadian luciferase reporter genes in freely moving mice. The technology employs a device dubbed RT-Biolumicorder, which consists of a cylindrical cage with reflecting conical walls that channel photons toward a photomultiplier tube. The monitoring of circadian liver gene expression revealed that hepatocyte oscillators of SCN-lesioned mice synchronized more rapidly to feeding cycles than hepatocyte clocks of intact mice. Hence, the SCN uses signaling pathways that counteract those of feeding rhythms when their phase is in conflict with its own phase.

Transcriptional regulatory logic of the diurnal cycle in the mouse liver.

Many organisms exhibit temporal rhythms in gene expression that propel diurnal cycles in physiology. In the liver of mammals, these rhythms are controlled by transcription-translation feedback loops of the core circadian clock and by feeding-fasting cycles. To better understand the regulatory interplay between the circadian clock and feeding rhythms, we mapped DNase I hypersensitive sites (DHSs) in the mouse liver during a diurnal cycle. The intensity of DNase I cleavages cycled at a substantial fraction of all DHSs, suggesting that DHSs harbor regulatory elements that control rhythmic transcription. Using chromatin immunoprecipitation followed by DNA sequencing (ChIP-seq), we found that hypersensitivity cycled in phase with RNA polymerase II (Pol II) loading and H3K27ac histone marks. We then combined the DHSs with temporal Pol II profiles in wild-type (WT) and Bmal1-/- livers to computationally identify transcription factors through which the core clock and feeding-fasting cycles control diurnal rhythms in transcription. While a similar number of mRNAs accumulated rhythmically in Bmal1-/- compared to WT livers, the amplitudes in Bmal1-/- were generally lower. The residual rhythms in Bmal1-/- reflected transcriptional regulators mediating feeding-fasting responses as well as responses to rhythmic systemic signals. Finally, the analysis of DNase I cuts at nucleotide resolution showed dynamically changing footprints consistent with dynamic binding of CLOCK:BMAL1 complexes. Structural modeling suggested that these footprints are driven by a transient heterotetramer binding configuration at peak activity. Together, our temporal DNase I mappings allowed us to decipher the global regulation of diurnal transcription rhythms in the mouse liver.