An alternative mechanism for skeletal muscle dysfunction in long-term post-viral lung disease.

Chronic lung disease is often accompanied by disabling extrapulmonary symptoms, notably skeletal muscle dysfunction and atrophy. Moreover, the severity of respiratory symptoms correlates with decreased muscle mass and in turn lowered physical activity and survival rates. Previous models of muscle atrophy in chronic lung disease often modeled chronic obstructive pulmonary disease (COPD) and relied on cigarette smoke exposure and LPS stimulation, but these conditions independently affect skeletal muscle even without accompanying lung disease. Moreover, there is an emerging and pressing need to understand the extrapulmonary manifestations of long-term post-viral lung disease (PVLD) as found in COVID-19. Here, we examine the development of skeletal muscle dysfunction in the setting of chronic pulmonary disease caused by infection due to the natural pathogen Sendai virus using a mouse model of PVLD. We identify a significant decrease in myofiber size when PVLD is maximal at 49 days after infection. We find no change in the relative types of myofibers, but the greatest decrease in fiber size is localized to fast-twitch-type IIB myofibers based on myosin heavy chain immunostaining. Remarkably, all biomarkers of myocyte protein synthesis and degradation (total RNA, ribosomal abundance, and ubiquitin-proteasome expression) were stable throughout the acute infectious illness and chronic post-viral disease process. Together, the results demonstrate a distinct pattern of skeletal muscle dysfunction in a mouse model of long-term PVLD. The findings thereby provide new insights into prolonged limitations in exercise capacity in patients with chronic lung disease after viral infections and perhaps other types of lung injury.NEW & NOTEWORTHY Our study used a mouse model of post-viral lung disease to study the impact of chronic lung disease on skeletal muscle. The model reveals a decrease in myofiber size that is selective for specific types of myofibers and an alternative mechanism for muscle atrophy that might be independent of the usual markers of protein synthesis and degradation. The findings provide a basis for new therapeutic strategies to correct skeletal muscle dysfunction in chronic respiratory disease.

An alternative mechanism for skeletal muscle dysfunction in long-term post-viral lung disease.

Chronic lung disease is often accompanied by disabling extrapulmonary symptoms, notably skeletal muscle dysfunction and atrophy. Moreover, the severity of respiratory symptoms correlates with decreased muscle mass and in turn lowered physical activity and survival rates. Previous models of muscle atrophy in chronic lung disease often modeled COPD and relied on cigarette smoke exposure and LPS-stimulation, but these conditions independently affect skeletal muscle even without accompanying lung disease. Moreover, there is an emerging and pressing need to understand the extrapulmonary manifestations of long-term post-viral lung disease (PVLD) as found in Covid-19. Here, we examine the development of skeletal muscle dysfunction in the setting of chronic pulmonary disease using a mouse model of PVLD caused by infection due to the natural pathogen Sendai virus. We identify a significant decrease in myofiber size when PVLD is maximal at 49 d after infection. We find no change in the relative types of myofibers, but the greatest decrease in fiber size is localized to fast-twitch type IIB myofibers based on myosin heavy chain immunostaining. Remarkably, all biomarkers of myocyte protein synthesis and degradation (total RNA, ribosomal abundance, and ubiquitin-proteasome expression) were stable throughout the acute infectious illness and chronic post-viral disease process. Together, the results demonstrate a distinct pattern of skeletal muscle dysfunction in a mouse model of long-term PVLD. The findings thereby provide new insight into prolonged limitations in exercise capacity in patients with chronic lung disease after viral infections and perhaps other types of lung injury.

Basal epithelial stem cells cross an alarmin checkpoint for postviral lung disease.

Epithelial cells are charged with protection at barrier sites, but whether this normally beneficial response might sometimes become dysfunctional still needs definition. Here, we recognized a pattern of imbalance marked by basal epithelial cell growth and differentiation that replaced normal airspaces in a mouse model of progressive postviral lung disease due to the Sendai virus. Single-cell and lineage-tracing technologies identified a distinct subset of basal epithelial stem cells (basal ESCs) that extended into gas-exchange tissue to form long-term bronchiolar-alveolar remodeling regions. Moreover, this cell subset was selectively expanded by crossing a cell-growth and survival checkpoint linked to the nuclear-localized alarmin IL-33 that was independent of IL-33 receptor signaling and instead connected to autocrine chromatin accessibility. This mechanism creates an activated stem-progenitor cell lineage with potential for physiological or pathological function. Thus, conditional loss of Il33 gene function in basal epithelial cells disrupted the homeostasis of the epithelial barrier at skin and gut sites but also markedly attenuated postviral disease in the lung based on the downregulation of remodeling and inflammation. Thus, we define a basal ESC strategy to deploy innate immune machinery that appears to overshoot the primordial goal of self-defense. Our findings reveal new targets to stratify and correct chronic and often deadly postviral disease.

Achilles-Mediated and Sex-Specific Regulation of Circadian mRNA Rhythms in Drosophila.

The circadian clock is an evolutionarily conserved mechanism that generates the rhythmic expression of downstream genes. The core circadian clock drives the expression of clock-controlled genes, which in turn play critical roles in carrying out many rhythmic physiological processes. Nevertheless, the molecular mechanisms by which clock output genes orchestrate rhythmic signals from the brain to peripheral tissues are largely unknown. Here we explored the role of one rhythmic gene, Achilles, in regulating the rhythmic transcriptome in the fly head. Achilles is a clock-controlled gene in Drosophila that encodes a putative RNA-binding protein. Achilles expression is found in neurons throughout the fly brain using fluorescence in situ hybridization (FISH), and legacy data suggest it is not expressed in core clock neurons. Together, these observations argue against a role for Achilles in regulating the core clock. To assess its impact on circadian mRNA rhythms, we performed RNA sequencing (RNAseq) to compare the rhythmic transcriptomes of control flies and those with diminished Achilles expression in all neurons. Consistent with previous studies, we observe dramatic upregulation of immune response genes upon knock-down of Achilles. Furthermore, many circadian mRNAs lose their rhythmicity in Achilles knock-down flies, suggesting that a subset of the rhythmic transcriptome is regulated either directly or indirectly by Achilles. These Achilles-mediated rhythms are observed in genes involved in immune function and in neuronal signaling, including Prosap, Nemy and Jhl-21. A comparison of RNAseq data from control flies reveals that only 42.7% of clock-controlled genes in the fly brain are rhythmic in both males and females. As mRNA rhythms of core clock genes are largely invariant between the sexes, this observation suggests that sex-specific mechanisms are an important, and heretofore under-appreciated, regulator of the rhythmic transcriptome.

Daily oscillations in expression and responsiveness of Toll-like receptors in splenic immune cells.

Circadian rhythms refer to biologic processes that oscillate with an approximate 24-h period. These rhythms direct nearly all aspects of animal behavior and physiology. The aim of our study was to determine if Toll-like receptor (TLR) expression and responsiveness exhibit time-of-day dependent differences. Therefore, we isolated an adherent splenocyte population, which consisted primarily of B cells, dendritic cells, and macrophages, over the course of a 24-h light-dark period and measured daily changes in Tlr1-8 mRNA levels and cytokine expression after cells were challenged at Zeitgeber time (ZT) 1 or ZT13 with a TLR ligand. In addition, we assessed TLR3 protein levels in adherent splenocytes over the 24-h light-dark period and challenged mice at ZT1 or ZT13 with poly(I:C), the TLR3 ligand. Our study revealed that in this adherent cell population, all Tlrs exhibited rhythmic expression except Tlr2 and Tlr5, and all TLRs, except TLR8, demonstrated daily variations in responsiveness after challenge with their respective ligand. We also revealed that TLR3 protein levels fluctuate over the daily light-dark cycle in adherent splenocytes and mice exhibit a time-of-day dependent immune response when challenged with poly(I:C). Finally, we demonstrated that mRNA levels of Tlr2 and Tlr6 display rhythmic expression in splenic macrophages. Taken together, these findings could have important implications for TLR-directed therapeutics.

Circadian Rhythms Influence the Severity of Sepsis in Mice via a TLR2-Dependent, Leukocyte-Intrinsic Mechanism.

Circadian rhythms coordinate an organism's activities and biological processes to the optimal time in the 24-h daylight cycle. We previously demonstrated that male C57BL/6 mice develop sepsis more rapidly when the disease is induced in the nighttime versus the daytime. In this report, we elucidate the mechanism of this diurnal difference. Sepsis was induced via cecal ligation and puncture (CLP) at zeitgeber time (ZT)-19 (2 am) or ZT-7 (2 pm). Like the males used in our prior study, female C57BL/6 mice had a worse outcome when CLP was induced at ZT-19 versus ZT-7, and these effects persisted when we pooled the data from both sexes. In contrast, mice with a mutated Period 2 (Per2) gene had a similar outcome when CLP was induced at ZT-19 versus ZT-7. Bone marrow chimeras reconstituted with C57BL/6 immune cells exhibited a worse outcome when sepsis was induced at ZT-19 versus ZT-7, whereas chimeras with Per2-mutated immune cells did not. Next, murine macrophages were subjected to serum shock to synchronize circadian rhythms and exposed to bacteria cultured from the mouse cecum at 4-h intervals for 48 h. We observed that IL-6 production oscillated with a 24-h period in C57BL/6 cells exposed to cecal bacteria. Interestingly, we observed a similar pattern when cells were exposed to the TLR2 agonist lipoteichoic acid. Furthermore, TLR2-knockout mice exhibited a similar sepsis phenotype when CLP was induced at ZT-19 versus ZT-7. Together, these data suggest that circadian rhythms in immune cells mediate diurnal variations in murine sepsis severity via a TLR2-dependent mechanism.

Transcriptional profiling reveals extraordinary diversity among skeletal muscle tissues.

Skeletal muscle comprises a family of diverse tissues with highly specialized functions. Many acquired diseases, including HIV and COPD, affect specific muscles while sparing others. Even monogenic muscular dystrophies selectively affect certain muscle groups. These observations suggest that factors intrinsic to muscle tissues influence their resistance to disease. Nevertheless, most studies have not addressed transcriptional diversity among skeletal muscles. Here we use RNAseq to profile mRNA expression in skeletal, smooth, and cardiac muscle tissues from mice and rats. Our data set, MuscleDB, reveals extensive transcriptional diversity, with greater than 50% of transcripts differentially expressed among skeletal muscle tissues. We detect mRNA expression of hundreds of putative myokines that may underlie the endocrine functions of skeletal muscle. We identify candidate genes that may drive tissue specialization, including Smarca4, Vegfa, and Myostatin. By demonstrating the intrinsic diversity of skeletal muscles, these data provide a resource for studying the mechanisms of tissue specialization.

Guidelines for Genome-Scale Analysis of Biological Rhythms.

Genome biology approaches have made enormous contributions to our understanding of biological rhythms, particularly in identifying outputs of the clock, including RNAs, proteins, and metabolites, whose abundance oscillates throughout the day. These methods hold significant promise for future discovery, particularly when combined with computational modeling. However, genome-scale experiments are costly and laborious, yielding "big data" that are conceptually and statistically difficult to analyze. There is no obvious consensus regarding design or analysis. Here we discuss the relevant technical considerations to generate reproducible, statistically sound, and broadly useful genome-scale data. Rather than suggest a set of rigid rules, we aim to codify principles by which investigators, reviewers, and readers of the primary literature can evaluate the suitability of different experimental designs for measuring different aspects of biological rhythms. We introduce CircaInSilico, a web-based application for generating synthetic genome biology data to benchmark statistical methods for studying biological rhythms. Finally, we discuss several unmet analytical needs, including applications to clinical medicine, and suggest productive avenues to address them.

ExpressionDB: An open source platform for distributing genome-scale datasets.

RNA-sequencing (RNA-seq) and microarrays are methods for measuring gene expression across the entire transcriptome. Recent advances have made these techniques practical and affordable for essentially any laboratory with experience in molecular biology. A variety of computational methods have been developed to decrease the amount of bioinformatics expertise necessary to analyze these data. Nevertheless, many barriers persist which discourage new labs from using functional genomics approaches. Since high-quality gene expression studies have enduring value as resources to the entire research community, it is of particular importance that small labs have the capacity to share their analyzed datasets with the research community. Here we introduce ExpressionDB, an open source platform for visualizing RNA-seq and microarray data accommodating virtually any number of different samples. ExpressionDB is based on Shiny, a customizable web application which allows data sharing locally and online with customizable code written in R. ExpressionDB allows intuitive searches based on gene symbols, descriptions, or gene ontology terms, and it includes tools for dynamically filtering results based on expression level, fold change, and false-discovery rates. Built-in visualization tools include heatmaps, volcano plots, and principal component analysis, ensuring streamlined and consistent visualization to all users. All of the scripts for building an ExpressionDB with user-supplied data are freely available on GitHub, and the Creative Commons license allows fully open customization by end-users. We estimate that a demo database can be created in under one hour with minimal programming experience, and that a new database with user-supplied expression data can be completed and online in less than one day.

Achilles is a circadian clock-controlled gene that regulates immune function in Drosophila.

The circadian clock is a transcriptional/translational feedback loop that drives the rhythmic expression of downstream mRNAs. Termed "clock-controlled genes," these molecular outputs of the circadian clock orchestrate cellular, metabolic, and behavioral rhythms. As part of our on-going work to characterize key upstream regulators of circadian mRNA expression, we have identified a novel clock-controlled gene in Drosophila melanogaster, Achilles (Achl), which is rhythmic at the mRNA level in the brain and which represses expression of antimicrobial peptides in the immune system. Achilles knock-down in neurons dramatically elevates expression of crucial immune response genes, including IM1 (Immune induced molecule 1), Mtk (Metchnikowin), and Drs (Drosomysin). As a result, flies with knocked-down Achilles expression are resistant to bacterial challenges. Meanwhile, no significant change in core clock gene expression and locomotor activity is observed, suggesting that Achilles influences rhythmic mRNA outputs rather than directly regulating the core timekeeping mechanism. Notably, Achilles knock-down in the absence of immune challenge significantly diminishes the fly's overall lifespan, indicating a behavioral or metabolic cost of constitutively activating this pathway. Together, our data demonstrate that (1) Achilles is a novel clock-controlled gene that (2) regulates the immune system, and (3) participates in signaling from neurons to immunological tissues.

MetaCycle: an integrated R package to evaluate periodicity in large scale data.

Detecting periodicity in large scale data remains a challenge. While efforts have been made to identify best of breed algorithms, relatively little research has gone into integrating these methods in a generalizable method. Here, we present MetaCycle, an R package that incorporates ARSER, JTK_CYCLE and Lomb-Scargle to conveniently evaluate periodicity in time-series data. MetaCycle has two functions, meta2d and meta3d, designed to analyze two-dimensional and three-dimensional time-series datasets, respectively. Meta2d implements N-version programming concepts using a suite of algorithms and integrating their results. AVAILABILITY AND IMPLEMENTATION: MetaCycle package is available on the CRAN repository (https://cran.r-project.org/web/packages/MetaCycle/index.html) and GitHub (https://github.com/gangwug/MetaCycle). CONTACT: hogenesch@gmail.comSupplementary information: Supplementary data are available at Bioinformatics online.

The daily rhythms of mitochondrial gene expression and oxidative stress regulation are altered by aging in the mouse liver.

The circadian clock regulates many cellular processes, notably including the cell cycle, metabolism and aging. Mitochondria play essential roles in metabolism and are the major sites of reactive oxygen species (ROS) production in the cell. The clock regulates mitochondrial functions by driving daily changes in NAD(+) levels and Sirt3 activity. In addition to this central route, in the present study, we find that the expression of some mitochondrial genes is also rhythmic in the liver, and that there rhythms are disrupted by the Clock(Δ19) mutation in young mice, suggesting that they are regulated by the core circadian oscillator. Related to this observation, we also find that the regulation of oxidative stress is rhythmic in the liver. Since mitochondria and ROS play important roles in aging, and mitochondrial functions are also disturbed by aging, these related observations prompt the compelling hypothesis that circadian oscillators influence aging by regulating ROS in mitochondria. During aging, the expression rhythms of some mitochondrial genes were altered in the liver and the temporal regulation over the dynamics of mitochondrial oxidative stress was disrupted. However, the expression of clock genes was not affected. Our results suggested that mitochondrial functions are combinatorially regulated by the clock and other age-dependent mechanism(s), and that aging disrupts mitochondrial rhythms through mechanisms downstream of the clock.

Physical activity, and not fat mass is a primary predictor of circadian parameters in young men.

Circadian rhythms are ≈24 h oscillations in physiology and behavior, and disruptions have been shown to have negative effects on health. Wrist skin temperature has been used by several groups as a valid method of assessing circadian rhythms in humans. We tested the hypothesis that circadian temperature amplitude (TempAmp) and stability (TempStab) would significantly differ among groups of healthy young men of varying adiposities, and that we could identify physiological and behavioral measures that were significantly associated with these temperature parameters. Wrist skin temperatures taken at 10 min intervals for 7 consecutive days were determined in 18 optimal (OGroup), 20 fair (FGroup) and 21 poor (PGroup) %Fat grouped young men and subsequently analyzed using available validated software. Body composition, cardiorespiratory fitness, actigraphy, daily nutritional and sleep data, and fasting lipid, insulin and glucose concentration measures were also determined. Significant changes in TempAmp and TempStab parameters in subjects with a single metabolic syndrome (MetS) risk factor compared to those with no MetS factors was observed. In addition, stepwise multivariate regression analyses showed that 50% of the variance in TempAmp was explained by actigraphy (mean steps taken per day; MSTPD), cardiorespiratory fitness, and late night eating per week (#LNE); and 57% in TempStab by MSTPD, time spent in moderate-to-vigorous activity per day, fat mass, and #LNE. Overwhelmingly, physical activity was the most important measure associated with the differences in circadian rhythm parameters. Further research is warranted to determine the effects of increasing the amount and timing of physical activity on the status of the circadian system in a variety of populations.

Considerations for RNA-seq analysis of circadian rhythms.

Circadian rhythms are daily endogenous oscillations of behavior, metabolism, and physiology. At a molecular level, these oscillations are generated by transcriptional-translational feedback loops composed of core clock genes. In turn, core clock genes drive the rhythmic accumulation of downstream outputs-termed clock-controlled genes (CCGs)-whose rhythmic translation and function ultimately underlie daily oscillations at a cellular and organismal level. Given the circadian clock's profound influence on human health and behavior, considerable efforts have been made to systematically identify CCGs. The recent development of next-generation sequencing has dramatically expanded our ability to study the expression, processing, and stability of rhythmically expressed mRNAs. Nevertheless, like any new technology, there are many technical issues to be addressed. Here, we discuss considerations for studying circadian rhythms using genome scale transcriptional profiling, with a particular emphasis on RNA sequencing. We make a number of practical recommendations-including the choice of sampling density, read depth, alignment algorithms, read-depth normalization, and cycling detection algorithms-based on computational simulations and our experience from previous studies. We believe that these results will be of interest to the circadian field and help investigators design experiments to derive most values from these large and complex data sets.

Calcitonin gene-related peptide neurons mediate sleep-specific circadian output in Drosophila.

BACKGROUND: Imbalances in amount and timing of sleep are harmful to physical and mental health. Therefore, the study of the underlying mechanisms is of great biological importance. Proper timing and amount of sleep are regulated by both the circadian clock and homeostatic sleep drive. However, very little is known about the cellular and molecular mechanisms by which the circadian clock regulates sleep. In this study, we describe a novel role for diuretic hormone 31 (DH31), the fly homolog of the vertebrate neuropeptide calcitonin gene-related peptide, as a circadian wake-promoting signal that awakens the fly in anticipation of dawn. RESULTS: Analysis of loss-of-function and gain-of-function Drosophila mutants demonstrates that DH31 suppresses sleep late at night. DH31 is expressed by a subset of dorsal circadian clock neurons that also express the receptor for the circadian neuropeptide pigment-dispersing factor (PDF). PDF secreted by the ventral pacemaker subset of circadian clock neurons acts on PDF receptors in the DH31-expressing dorsal clock neurons to increase DH31 secretion before dawn. Activation of PDF receptors in DH31-positive DN1 specifically affects sleep and has no effect on circadian rhythms, thus constituting a dedicated locus for circadian regulation of sleep. CONCLUSIONS: We identified a novel signaling molecule (DH31) as part of a neuropeptide relay mechanism for circadian control of sleep. Our results indicate that outputs of the clock controlling sleep and locomotor rhythms are mediated via distinct neuronal pathways.

A circadian gene expression atlas in mammals: implications for biology and medicine.

To characterize the role of the circadian clock in mouse physiology and behavior, we used RNA-seq and DNA arrays to quantify the transcriptomes of 12 mouse organs over time. We found 43% of all protein coding genes showed circadian rhythms in transcription somewhere in the body, largely in an organ-specific manner. In most organs, we noticed the expression of many oscillating genes peaked during transcriptional "rush hours" preceding dawn and dusk. Looking at the genomic landscape of rhythmic genes, we saw that they clustered together, were longer, and had more spliceforms than nonoscillating genes. Systems-level analysis revealed intricate rhythmic orchestration of gene pathways throughout the body. We also found oscillations in the expression of more than 1,000 known and novel noncoding RNAs (ncRNAs). Supporting their potential role in mediating clock function, ncRNAs conserved between mouse and human showed rhythmic expression in similar proportions as protein coding genes. Importantly, we also found that the majority of best-selling drugs and World Health Organization essential medicines directly target the products of rhythmic genes. Many of these drugs have short half-lives and may benefit from timed dosage. In sum, this study highlights critical, systemic, and surprising roles of the mammalian circadian clock and provides a blueprint for advancement in chronotherapy.

Machine learning helps identify CHRONO as a circadian clock component.

Over the last decades, researchers have characterized a set of "clock genes" that drive daily rhythms in physiology and behavior. This arduous work has yielded results with far-reaching consequences in metabolic, psychiatric, and neoplastic disorders. Recent attempts to expand our understanding of circadian regulation have moved beyond the mutagenesis screens that identified the first clock components, employing higher throughput genomic and proteomic techniques. In order to further accelerate clock gene discovery, we utilized a computer-assisted approach to identify and prioritize candidate clock components. We used a simple form of probabilistic machine learning to integrate biologically relevant, genome-scale data and ranked genes on their similarity to known clock components. We then used a secondary experimental screen to characterize the top candidates. We found that several physically interact with known clock components in a mammalian two-hybrid screen and modulate in vitro cellular rhythms in an immortalized mouse fibroblast line (NIH 3T3). One candidate, Gene Model 129, interacts with BMAL1 and functionally represses the key driver of molecular rhythms, the BMAL1/CLOCK transcriptional complex. Given these results, we have renamed the gene CHRONO (computationally highlighted repressor of the network oscillator). Bi-molecular fluorescence complementation and co-immunoprecipitation demonstrate that CHRONO represses by abrogating the binding of BMAL1 to its transcriptional co-activator CBP. Most importantly, CHRONO knockout mice display a prolonged free-running circadian period similar to, or more drastic than, six other clock components. We conclude that CHRONO is a functional clock component providing a new layer of control on circadian molecular dynamics.

The growth and impact of Alzheimer disease centers as measured by social network analysis.

IMPORTANCE: Alzheimer disease (AD) is a neurodegenerative disorder with no effective therapies. In 1984, the National Institute on Aging created the first 5 AD centers (ADCs) in an effort to coordinate research efforts into the pathology and treatment of the disease. Since that time, the ADC program has expanded to include 27 centers in major medical schools throughout the United States. A major aim of ADCs is to develop shared resources, such as tissue samples and patient populations, and thereby promote large-scale, high-impact studies that go beyond the capabilities of any single investigator or institution working in isolation. OBJECTIVE: To quantitatively evaluate the performance of the ADC program over the past 25 years. DESIGN AND SETTING: We systematically harvested every article published by ADC investigators and used social network analysis to analyze copublication networks. RESULTS: A total of 12170 ADC papers were published from 1985 through 2012. The frequency of collaborations has increased greatly from the time that the ADCs were started until the present, even after the expansion of ADCs and the recruitment of new investigators plateaued. Moreover, the collaborations established within the context of the ADC program are increasingly interinstitutional, consistent with the overall goal of the program to catalyze multicenter research teams. Most important, we determined that collaborative multi-ADC research articles are consistently of higher impact than AD articles as a whole. CONCLUSIONS AND RELEVANCE: The ADC program has successfully fostered high-impact, multiuniversity collaborations; we suggest that its structural and administrative features could be replicated in other fields of patient-oriented research.

O-GlcNAc signaling entrains the circadian clock by inhibiting BMAL1/CLOCK ubiquitination.

Circadian clocks are coupled to metabolic oscillations through nutrient-sensing pathways. Nutrient flux into the hexosamine biosynthesis pathway triggers covalent protein modification by O-linked ß-D-N-acetylglucosamine (O-GlcNAc). Here we show that the hexosamine/O-GlcNAc pathway modulates peripheral clock oscillation. O-GlcNAc transferase (OGT) promotes expression of BMAL1/CLOCK target genes and affects circadian oscillation of clock genes in vitro and in vivo. Both BMAL1 and CLOCK are rhythmically O-GlcNAcylated, and this protein modification stabilizes BMAL1 and CLOCK by inhibiting their ubiquitination. In vivo analysis of genetically modified mice with perturbed hepatic OGT expression shows aberrant circadian rhythms of glucose homeostasis. These results establish the counteraction between O-GlcNAcylation and ubiquitination as a key mechanism that regulates the circadian clock and suggest a crucial role for O-GlcNAc signaling in transducing nutritional signals to the core circadian timing machinery.

Brain-specific rescue of Clock reveals system-driven transcriptional rhythms in peripheral tissue.

The circadian regulatory network is organized in a hierarchical fashion, with a central oscillator in the suprachiasmatic nuclei (SCN) orchestrating circadian oscillations in peripheral tissues. The nature of the relationship between central and peripheral oscillators, however, is poorly understood. We used the tetOFF expression system to specifically restore Clock function in the brains of Clock(Δ19) mice, which have compromised circadian clocks. Rescued mice showed normal locomotor rhythms in constant darkness, with activity period lengths approximating wildtype controls. We used microarray analysis to assess whether brain-specific rescue of circadian rhythmicity was sufficient to restore circadian transcriptional output in the liver. Compared to Clock mutants, Clock-rescue mice showed significantly larger numbers of cycling transcripts with appropriate phase and period lengths, including many components of the core circadian oscillator. This indicates that the SCN oscillator overcomes local circadian defects and signals directly to the molecular clock. Interestingly, the vast majority of core clock genes in liver were responsive to Clock expression in the SCN, suggesting that core clock genes in peripheral tissues are intrinsically sensitive to SCN cues. Nevertheless, most circadian output in the liver was absent or severely low-amplitude in Clock-rescue animals, demonstrating that the majority of peripheral transcriptional rhythms depend on a fully functional local circadian oscillator. We identified several new system-driven rhythmic genes in the liver, including Alas1 and Mfsd2. Finally, we show that 12-hour transcriptional rhythms (i.e., circadian "harmonics") are disrupted by Clock loss-of-function. Brain-specific rescue of Clock converted 12-hour rhythms into 24-hour rhythms, suggesting that signaling via the central circadian oscillator is required to generate one of the two daily peaks of expression. Based on these data, we conclude that 12-hour rhythms are driven by interactions between central and peripheral circadian oscillators.