Guangsen Shi

PER1 Phosphorylation Specifies Feeding Rhythm in Mice

Organization of circadian behavior, physiology, and metabolism is important for human health. An S662G mutation in hPER2 has been linked to familial advanced sleep-phase syndrome (FASPS). Although the paralogous phosphorylation site S714 in PER1 is conserved in mice, its specific function in circadian organization remains unknown. Here, we find that the PER1S714G mutation accelerates the molecular feedback loop. Furthermore, hPER1S714G mice, but not hPER2S662G mice, exhibit peak time of food intake that is several hours before daily energy expenditure peaks. Both the advanced feeding behavior and the accelerated clock disrupt the phase of expression of several key metabolic regulators in the liver and adipose tissue. Consequently, hPER1S714G mice rapidly develop obesity on a high-fat diet. Our studies demonstrate that PER1 and PER2 are linked to different downstream pathways and that PER1 maintains coherence between the circadian clock and energy metabolism.

Dual roles of FBXL3 in the mammalian circadian feedback loops are important for period determination and robustness of the clock

The mammalian circadian clock is composed of interlocking feedback loops. Cryptochrome is a central component in the core negative feedback loop, whereas Rev-Erbα, a member of the nuclear receptor family, is an essential component of the interlocking loop. To understand the roles of different clock genes, we conducted a genetic interaction screen by generating single- and double-mutant mice. We found that the deletion of Rev-erbα in F-box/leucine rich-repeat protein (Fbxl3)-deficient mice rescued its long-circadian period phenotype, and our results further revealed that FBXL3 regulates Rev-Erb/retinoic acid receptor-related orphan receptor-binding element (RRE)-mediated transcription by inactivating the Rev-Erbα:histone deacetylase 3 corepressor complex. By analyzing the Fbxl3 and Cryptochrome 1 double-mutant mice, we found that FBXL3 also regulates the amplitudes of E-box–driven gene expression. These two separate roles of FBXL3 in circadian feedback loops provide a mechanism that contributes to the period determination and robustness of the clock.

Interaction of MAGED1 with nuclear receptors affects circadian clock function

The circadian clock has a central role in physiological adaption and anticipation of day/night changes. In a genetic screen for novel regulators of circadian rhythms, we found that mice lacking MAGED1 (Melanoma Antigen Family D1) exhibit a shortened period and altered rest–activity bouts. These circadian phenotypes are proposed to be caused by a direct effect on the core molecular clock network that reduces the robustness of the circadian clock. We provide in vitro and in vivo evidence indicating that MAGED1 binds to RORα to bring about positive and negative effects on core clock genes of Bmal1, Rev‐erbα and E4bp4 expression through the Rev‐Erbα/ROR responsive elements (RORE). Maged1 is a non‐rhythmic gene that, by binding RORα in non‐circadian way, enhances rhythmic input and buffers the circadian system from irrelevant, perturbing stimuli or noise. We have thus identified and defined a novel circadian regulator, Maged1, which is indispensable for the robustness of the circadian clock to better serve the organism.

A NANOS3 mutation linked to protein degradation causes premature ovarian insufficiency

Primary ovarian insufficiency (POI), or premature ovarian failure, is defined as the cessation of ovarian function before the age of 40. An insufficient ovarian follicle pool derived from primordial germ cells (PGCs) is an important cause of POI. Although the Nanos gene family is known to be required for PGC development and maintenance in diverse model organisms, the relevance of this information to human biology is not yet clear. In this study, we screened the coding regions of the NANOS1, NANOS2 and NANOS3 genes in 100 Chinese POI patients and identified four variants in the coding regions of these three genes, including one synonymous variant in NANOS3, one missense variant in each of NANOS1 and NANOS2 and one potentially relevant mutation (c.457C>T; p.Arg153Trp, heterozygous) in NANOS3. We demonstrated that the p.Arg153Trp substitution decreases the stability of NANOS3, potentially resulting in a hypomorph. Furthermore, an investigation of the relationship between the number of PGCs and the dosage of NANOS3 in mouse models showed that the population of PGCs is controlled by the level of NANOS3 protein. Taken together, our results provide new insight into the properties of the NANOS3 protein and establish that NANOS3 mutation is one possible cause of POI.

SWItch/sucrose nonfermentable (SWI/SNF) complex subunit BAF60a integrates hepatic circadian clock and energy metabolism

Many aspects of energy metabolism, including glucose and lipid homeostasis and mitochondrial oxidative metabolism, are under precise control by the mammalian circadian clock. However, the molecular mechanism for coordinate integration of the circadian clock and various metabolic pathways is poorly understood. Here we show that BAF60a, a chromatin‐remodeling complex subunit, is rhythmically expressed in the liver of mice. Mice with liver‐specific knockdown of BAF60a show abnormalities in the rhythmic expression pattern of clock and metabolic genes and in the circulating metabolite profile. Consistently, knockdown of BAF60a impairs the oscillation of clock genes in serum‐shocked HepG2 cells. At the molecular level, BAF60a activates Bmal1 and G6Pase transcription by way of the coactivation of retinoid‐related orphan receptor alpha (RORα). In addition, BAF60a is present near ROR response elements (RORE) on the proximal Bmal1 and G6Pase promoters and turns the chromatin structure into the active state. Conclusion: Our data suggest a critical role for BAF60a in the coordinated regulation of hepatic circadian clock and energy metabolism in mammals. (HEPATOLOGY 2011;)

A Cryptochrome 2 mutation yields advanced sleep phase in humans

Familial Advanced Sleep Phase (FASP) is a heritable human sleep phenotype characterized by very early sleep and wake times. We identified a missense mutation in the human Cryptochrome 2 (CRY2) gene that co-segregates with FASP in one family. The mutation leads to replacement of an alanine residue at position 260 with a threonine (A260T). In mice, the CRY2 mutation causes a shortened circadian period and reduced phase-shift to early-night light pulse associated with phase-advanced behavioral rhythms in the light-dark cycle. The A260T mutation is located in the phosphate loop of the flavin adenine dinucleotide (FAD) binding domain of CRY2. The mutation alters the conformation of CRY2, increasing its accessibility and affinity for FBXL3 (an E3 ubiquitin ligase), thus promoting its degradation. These results demonstrate that CRY2 stability controlled by FBXL3 plays a key role in the regulation of human sleep wake behavior. DOI: http://dx.doi.org/10.7554/eLife.16695.001

An intensity ratio of interlocking loops determines circadian period length

Circadian clocks allow organisms to orchestrate the daily rhythms in physiology and behaviors, and disruption of circadian rhythmicity can profoundly affect fitness. The mammalian circadian oscillator consists of a negative primary feedback loop and is associated with some ‘auxiliary’ loops. This raises the questions of how these interlocking loops coordinate to regulate the period and maintain its robustness. Here, we focused on the REV-ERBα/Cry1 auxiliary loop, consisting of Rev-Erbα/ROR-binding elements (RORE) mediated Cry1 transcription, coordinates with the negative primary feedback loop to modulate the mammalian circadian period. The silicon simulation revealed an unexpected rule: the intensity ratio of the primary loop to the auxiliary loop is inversely related to the period length, even when post-translational feedback is fixed. Then we measured the mRNA levels from two loops in 10-mutant mice and observed the similar monotonic relationship. Additionally, our simulation and the experimental results in human osteosarcoma cells suggest that a coupling effect between the numerator and denominator of this intensity ratio ensures the robustness of circadian period and, therefore, provides an efficient means of correcting circadian disorders. This ratio rule highlights the contribution of the transcriptional architecture to the period dynamics and might be helpful in the construction of synthetic oscillators.

The circadian clock influences heart performance.

Circadian clocks are believed to provide the selective advantage of anticipation, thus allowing organisms to respond efficiently to stimuli at the appropriate moment. Disrupted circadian rhythms have been found to affect a variety of basic physiological processes. However, the importance of the circadian clock in regulating heart performance remains undetermined. We hypothesized that the circadian clock plays a crucial role in heart performance through the anticipation of daily workload. Echocardiography was employed to monitor heart function and structure in mice in a noninvasive, real-time manner. In wild-type mice, both the ejection fraction (EF) and the shortening fraction (FS), two important markers of cardiac function, show diurnal variation. In addition, the amplitude of the EF and the FS enlarges in response to forced exercise in a time-dependent manner. The diurnal variations in EF and FS are altered in mice with disruptions in circadian clock genes and are significantly attenuated under an imposed light regimen. Furthermore, it shows that the overexpression of peroxisome proliferator–activated receptor gamma coactivator 1 alpha (Pgc1α) under control of the muscle creatine kinase (MCK) promoter inhibited clock gene expression in the heart and muscle and decreased the expression of peroxisome proliferator–activated receptor alpha (Pparα), metabolic genes glucose transporter (Glut4), and acetyl-coA synthetase (Acs1). Pgc1α overexpression abolished the diurnal variation of EF. We thus propose that PGC1α might play an important role in circadian-mediated, impaired cardiac function by regulating the circadian rhythm of metabolic genes.

Human genetics and sleep behavior

Why we sleep remains one of the greatest mysteries in science. In the past few years, great advances have been made to better understand this phenomenon. Human genetics has contributed significantly to this movement, as many features of sleep have been found to be heritable. Discoveries about these genetic variations that affect human sleep will aid us in understanding the underlying mechanism of sleep. Here we summarize recent discoveries about the genetic variations affecting the timing of sleep, duration of sleep and EEG patterns. To conclude, we also discuss some of the sleep-related neurological disorders such as Autism Spectrum Disorder (ASD) and Alzheimers Disease (AD) and the potential challenges and future directions of human genetics in sleep research.

Loss of ZBTB20 impairs circadian output and leads to unimodal behavioral rhythms

Many animals display morning and evening bimodal activities in the day/night cycle. However, little is known regarding the potential components involved in the regulation of bimodal behavioral rhythms in mammals. Here, we identified that the zinc finger protein gene Zbtb20 plays a crucial role in the regulation of bimodal activities in mice. Depletion of Zbtb20 in nerve system resulted in the loss of early evening activity, but the increase of morning activity. We found that Zbtb20-deficient mice exhibited a pronounced decrease in the expression of Prokr2 and resembled phenotypes of Prok2 and Prokr2-knockout mice. Injection of adeno-associated virus-double-floxed Prokr2 in suprachiasmatic nucleus could partly restore evening activity in Nestin-Cre; Zbtb20fl/fl (NS-ZB20KO) mice. Furthermore, loss of Zbtb20 in Foxg1 loci, but intact in the suprachiasmatic nucleus, was not responsible for the unimodal activity of NS-ZB20KO mice. Our study provides evidence that ZBTB20-mediated PROKR2 signaling is critical for the evening behavioral rhythms. DOI: http://dx.doi.org/10.7554/eLife.17171.001