Obstructive sleep apnea in a mouse model is associated with tissue-specific transcriptomic changes in circadian rhythmicity and mean 24-hour gene expression.

Intermittent hypoxia (IH) is a major clinical feature of obstructive sleep apnea (OSA). The mechanisms that become dysregulated after periods of exposure to IH are unclear, particularly in the early stages of disease. The circadian clock governs a wide array of biological functions and is intimately associated with stabilization of hypoxia-inducible factors (HIFs) under hypoxic conditions. In patients, IH occurs during the sleep phase of the 24-hour sleep-wake cycle, potentially affecting their circadian rhythms. Alterations in the circadian clock have the potential to accelerate pathological processes, including other comorbid conditions that can be associated with chronic, untreated OSA. We hypothesized that changes in the circadian clock would manifest differently in those organs and systems known to be impacted by OSA. Using an IH model to represent OSA, we evaluated circadian rhythmicity and mean 24-hour expression of the transcriptome in 6 different mouse tissues, including the liver, lung, kidney, muscle, heart, and cerebellum, after a 7-day exposure to IH. We found that transcriptomic changes within cardiopulmonary tissues were more affected by IH than other tissues. Also, IH exposure resulted in an overall increase in core body temperature. Our findings demonstrate a relationship between early exposure to IH and changes in specific physiological outcomes. This study provides insight into the early pathophysiological mechanisms associated with IH.

PSMD11 modulates circadian clock function through PER and CRY nuclear translocation.

The molecular circadian clock is regulated by a transcriptional translational feedback loop. However, the post-translational control mechanisms are less understood. The NRON complex is a large ribonucleoprotein complex, consisting of a lncRNA and several proteins. Components of the complex play a distinct role in regulating protein phosphorylation, synthesis, stability, and translocation in cellular processes. This includes the NFAT and the circadian clock pathway. PSMD11 is a component of the NRON complex and a lid component of the 26S proteasome. Among the PSMD family members, PSMD11 has a more specific role in circadian clock function. Here, we used cell and biochemical approaches and characterized the role of PSMD11 in regulating the stability and nuclear translocation of circadian clock proteins. We used size exclusion chromatography to enrich the NRON complex in the cytosolic and nuclear fractions. More specifically, PSMD11 knockdown affected the abundance of PER2 and CRY2 proteins and the nuclear translocation of CRY1. This changed the relative abundance of CRY1 and CRY2 in the nucleus. Thus, this work defines the role of PSMD11 in the NRON complex regulating the nuclear translocation of circadian repressors, thereby enabling cellular circadian oscillations.

Integration of genome-scale data identifies candidate sleep regulators.

STUDY OBJECTIVES: Genetics impacts sleep, yet, the molecular mechanisms underlying sleep regulation remain elusive. In this study, we built machine learning models to predict sleep genes based on their similarity to genes that are known to regulate sleep. METHODS: We trained a prediction model on thousands of published datasets, representing circadian, immune, sleep deprivation, and many other processes, using a manually curated list of 109 sleep genes. RESULTS: Our predictions fit with prior knowledge of sleep regulation and identified key genes and pathways to pursue in follow-up studies. As an example, we focused on the NF-κB pathway and showed that chronic activation of NF-κB in a genetic mouse model impacted the sleep-wake patterns. CONCLUSION: Our study highlights the power of machine learning in integrating prior knowledge and genome-wide data to study genetic regulation of complex behaviors such as sleep.

An in silico genome-wide screen for circadian clock strength in human samples.

MOTIVATION: Years of time-series gene expression studies have built a strong understanding of clock-controlled pathways across species. However, comparatively little is known about how 'non-clock' pathways influence clock function. We need a strong understanding of clock-coupled pathways in human tissues to better appreciate the links between disease and clock function. RESULTS: We developed a new computational approach to explore candidate pathways coupled to the clock in human tissues. This method, termed LTM, is an in silico screen to infer genetic influences on circadian clock function. LTM uses natural variation in gene expression in human data and directly links gene expression variation to clock strength independent of longitudinal data. We applied LTM to three human skin and one melanoma datasets and found that the cell cycle is the top candidate clock-coupled pathway in healthy skin. In addition, we applied LTM to thousands of tumor samples from 11 cancer types in the TCGA database and found that extracellular matrix organization-related pathways are tightly associated with the clock strength in humans. Further analysis shows that clock strength in tumor samples is correlated with the proportion of cancer-associated fibroblasts and endothelial cells. Therefore, we show both the power of LTM in predicting clock-coupled pathways and classify factors associated with clock strength in human tissues. AVAILABILITY AND IMPLEMENTATION: LTM is available on GitHub (https://github.com/gangwug/LTMR) and figshare (https://figshare.com/articles/software/LTMR/21217604) to facilitate its use. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

duper is a null mutation of Cryptochrome 1 in Syrian hamsters.

The duper mutation is a recessive mutation that shortens the period length of the circadian rhythm in Syrian hamsters. These animals show a large phase shift when responding to light pulses. Limited genetic resources for the Syrian hamster (Mesocricetus auratus) presented a major obstacle to cloning duper. This caused the duper mutation to remain unknown for over a decade. In this study, we did a de novo genome assembly of Syrian hamsters with long-read sequencing data from two different platforms, Pacific Biosciences and Oxford Nanopore Technologies. Using two distinct ecotypes and a fast homozygosity mapping strategy, we identified duper as an early nonsense allele of Cryptochrome 1 (Cry1) leading to a short, unstable protein. CRY1 is known as a highly conserved component of the repressive limb of the core circadian clock. The genome assembly and other genomic datasets generated in this study will facilitate the use of the Syrian hamster in biomedical research.

NF-κB modifies the mammalian circadian clock through interaction with the core clock protein BMAL1.

In mammals, the circadian clock coordinates cell physiological processes including inflammation. Recent studies suggested a crosstalk between these two pathways. However, the mechanism of how inflammation affects the clock is not well understood. Here, we investigated the role of the proinflammatory transcription factor NF-κB in regulating clock function. Using a combination of genetic and pharmacological approaches, we show that perturbation of the canonical NF-κB subunit RELA in the human U2OS cellular model altered core clock gene expression. While RELA activation shortened period length and dampened amplitude, its inhibition lengthened period length and caused amplitude phenotypes. NF-κB perturbation also altered circadian rhythms in the master suprachiasmatic nucleus (SCN) clock and locomotor activity behavior under different light/dark conditions. We show that RELA, like the clock repressor CRY1, repressed the transcriptional activity of BMAL1/CLOCK at the circadian E-box cis-element. Biochemical and biophysical analysis showed that RELA binds to the transactivation domain of BMAL1. These data support a model in which NF-kB competes with CRY1 and coactivator CBP/p300 for BMAL1 binding to affect circadian transcription. This is further supported by chromatin immunoprecipitation analysis showing that binding of RELA, BMAL1 and CLOCK converges on the E-boxes of clock genes. Taken together, these data support a significant role for NF-κB in directly regulating the circadian clock and highlight mutual regulation between the circadian and inflammatory pathways.

Normalized coefficient of variation (nCV): a method to evaluate circadian clock robustness in population scale data.

SUMMARY: Robust oscillation of clock genes is a core feature of the circadian system. Relative amplitude (rAMP) measures the robustness of clock gene oscillations but only works for longitudinal samples. We lack a method for estimating robust oscillations from human samples without labeled time. We show that the normalized coefficient of variation (nCV) of 10 clock genes is linearly correlated with their normalized rAMP, independent of time labels. We found that the mean nCV of clock genes are consistently decreased in tumors compared to nontumors, suggesting a new therapeutic target in cancer treatment by enhancing clock robustness. nCV can provide a simple measure of the clock robustness in population-level datasets. AVAILABILITY AND IMPLEMENTATION: The nCV package (https://github.com/gangwug/nCV) and web application (https://github.com/gangwug/nCVapp) are available on the GitHub repository. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Intermittent Hypoxia Alters the Circadian Expression of Clock Genes in Mouse Brain and Liver.

At least one-third of adults in the United States experience intermittent hypoxia (IH) due to health or living conditions. The majority of these adults suffer with sleep breathing conditions and associated circadian rhythm disorders. The impact of IH on the circadian clock is not well characterized. In the current study, we used an IH mouse model to understand the impact of IH on the circadian gene expression of the canonical clock genes in the central (the brain) and peripheral (the liver) tissues. Gene expression was measured using a Quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR). CircaCompare was used to evaluate the differential rhythmicity between normoxia and IH. Our observations suggested that the circadian clock in the liver was less sensitive to IH compared to the circadian clock in the brain.

Short-term exposure to intermittent hypoxia leads to changes in gene expression seen in chronic pulmonary disease.

Obstructive sleep apnea (OSA) results from episodes of airway collapse and intermittent hypoxia (IH) and is associated with a host of health complications. Although the lung is the first organ to sense changes in oxygen levels, little is known about the consequences of IH to the lung hypoxia-inducible factor-responsive pathways. We hypothesized that exposure to IH would lead to cell-specific up- and downregulation of diverse expression pathways. We identified changes in circadian and immune pathways in lungs from mice exposed to IH. Among all cell types, endothelial cells showed the most prominent transcriptional changes. Upregulated genes in myofibroblast cells were enriched for genes associated with pulmonary hypertension and included targets of several drugs currently used to treat chronic pulmonary diseases. A better understanding of the pathophysiologic mechanisms underlying diseases associated with OSA could improve our therapeutic approaches, directing therapies to the most relevant cells and molecular pathways.

CRY1-CBS binding regulates circadian clock function and metabolism.

Circadian disruption influences metabolic health. Metabolism modulates circadian function. However, the mechanisms coupling circadian rhythms and metabolism remain poorly understood. Here, we report that cystathionine ß-synthase (CBS), a central enzyme in one-carbon metabolism, functionally interacts with the core circadian protein cryptochrome 1 (CRY1). In cells, CBS augments CRY1-mediated repression of the CLOCK/BMAL1 complex and shortens circadian period. Notably, we find that mutant CBS-I278T protein, the most common cause of homocystinuria, does not bind CRY1 or regulate its repressor activity. Transgenic CbsZn/Zn  mice, while maintaining circadian locomotor activity period, exhibit reduced circadian power and increased expression of E-BOX outputs. CBS function is reciprocally influenced by CRY1 binding. CRY1 modulates enzymatic activity of the CBS. Liver extracts from Cry1-/- mice show reduced CBS activity that normalizes after the addition of exogenous wild-type (WT) CRY1. Metabolomic analysis of WT, CbsZn/Zn , Cry1-/- , and Cry2-/- samples highlights the metabolic importance of endogenous CRY1. We observed temporal variation in one-carbon and transsulfuration pathways attributable to CRY1-induced CBS activation. CBS-CRY1 binding provides a post-translational switch to modulate cellular circadian physiology and metabolic control.

A population-based gene expression signature of molecular clock phase from a single epidermal sample.

BACKGROUND: For circadian medicine to influence health, such as when to take a drug or undergo a procedure, a biomarker of molecular clock phase is required--one that is easily measured and generalizable across a broad population. It is not clear that any circadian biomarker yet satisfies these criteria. METHODS: We analyzed 24-h molecular rhythms in human dermis and epidermis at three distinct body sites, leveraging both longitudinal (n = 20) and population (n = 154) data. We applied cyclic ordering by periodic structure (CYCLOPS) to order the population samples where biopsy time was not recorded. With CYCLOPS-predicted phases, we used ZeitZeiger to discover potential biomarkers of clock phase. RESULTS: Circadian clock function was strongest in the epidermis, regardless of body site. We identified a 12-gene expression signature that reported molecular clock phase to within 3 h (mean error = 2.5 h) from a single sample of epidermis--the skin's most superficial layer. This set performed well across body sites, ages, sexes, and detection platforms. CONCLUSIONS: This research shows that the clock in epidermis is more robust than dermis regardless of body site. To encourage ongoing validation of this putative biomarker in diverse populations, diseases, and experimental designs, we developed SkinPhaser--a user-friendly app to test biomarker performance in datasets ( https://github.com/gangwug/SkinPhaser ).

A large-scale study reveals 24-h operational rhythms in hospital treatment.

Hospitals operate 24 h a day, and it is assumed that important clinical decisions occur continuously around the clock. However, many aspects of hospital operation occur at specific times of day, including medical team rounding and shift changes. It is unclear whether this impacts patient care, as no studies have addressed this. We analyzed the daily distribution of ∼500,000 doses of 12 separate drugs in 1,546 inpatients at a major children's hospital in the United States from 2010 to 2017. We tracked both order time (when a care provider places an electronic request for a drug) and dosing time (when the patient receives the drug). Order times were time-of-day-dependent, marked by distinct morning-time surges and overnight lulls. Nearly one-third of all 103,847 orders for treatment were placed between 8:00 AM and 12:00 PM. First doses from each order were also rhythmic but shifted by 2 h. These 24-h rhythms in orders and first doses were remarkably consistent across drugs, diagnosis, and hospital units. This rhythm in hospital medicine coincided with medical team rounding time, not necessarily immediate medical need. Lastly, we show that the clinical response to hydralazine, an acute antihypertensive, is dosing time-dependent and greatest at night, when the fewest doses were administered. The prevailing dogma is that hospital treatment is administered as needed regardless of time of day. Our findings challenge this notion and reveal a potential operational barrier to best clinical care.

The NRON complex controls circadian clock function through regulated PER and CRY nuclear translocation.

Post-translational regulation plays a central role in the circadian clock mechanism. However, nucleocytoplasmic translocation of core clock proteins, a key step in circadian timekeeping, is not fully understood. Earlier we found that the NRON scaffolding complex regulates nuclear translocation of NFAT and its signaling. Here, we show that components of the NRON complex also regulate the circadian clock. In peripheral cell clock models, genetic perturbation of the NRON complex affects PER and CRY protein nuclear translocation, dampens amplitude, and alters period length. Further, we show small molecules targeting the NFAT pathway alter nuclear translocation of PER and CRY proteins and impact circadian rhythms in peripheral cells and tissue explants of the master clock in the suprachiasmatic nucleus. Taken together, these studies highlight a key role for the NRON complex in regulating PER/CRY subcellular localization and circadian timekeeping.

Population-level rhythms in human skin with implications for circadian medicine.

Skin is the largest organ in the body and serves important barrier, regulatory, and sensory functions. The epidermal layer shows rhythmic physiological responses to daily environmental variation (e.g., DNA repair). We investigated the role of the circadian clock in the transcriptional regulation of epidermis using a hybrid experimental design, in which a limited set of human subjects (n = 20) were sampled throughout the 24-h cycle and a larger population (n = 219) were sampled once. We found a robust circadian oscillator in human epidermis at the population level using pairwise correlations of clock and clock-associated genes in 298 epidermis samples. We then used CYCLOPS to reconstruct the temporal order of all samples, and identified hundreds of rhythmically expressed genes at the population level in human epidermis. We compared these results with published time-series skin data from mice and found a strong concordance in circadian phase across species for both transcripts and pathways. Furthermore, like blood, epidermis is readily accessible and a potential source of biomarkers. Using ZeitZeiger, we identified a biomarker set for human epidermis that is capable of reporting circadian phase to within 3 hours from a single sample. In summary, we show rhythms in human epidermis that persist at the population scale and describe a path to develop robust single-sample circadian biomarkers.

Computational and experimental insights into the circadian effects of SIRT1.

The circadian clock orchestrates 24-h rhythms in physiology in most living organisms. At the molecular level, the dogma is that circadian oscillations are based on a negative transcriptional feedback loop. Recent studies found the NAD+-dependent histone deacetylase, SIRT1, directly regulates acetylation status of clock components and influences circadian amplitude in cells. While Nakahata et al. [Nakahata Y, Kaluzova M (2008) Cell 134:329-340] reported that loss of SIRT1 increases amplitude through BMAL1 acetylation, Asher et al. [Asher G, Gatfield D (2008) Cell 134:317-328] reported that loss of SIRT1 decreases amplitude through an increase in acetylated PER2. To address this SIRT1 paradox, we developed a circadian enzymatic model. Predictions from this model and experimental validation strongly align with the findings of Asher et al., with PER2 as the primary target of SIRT1. Further, the model suggested SIRT1 influences BMAL1 expression through actions on PGC1α. We validated this finding experimentally. Thus, our computational and experimental approaches suggest SIRT1 positively regulates clock function through actions on PER2 and PGC1α.

A database of tissue-specific rhythmically expressed human genes has potential applications in circadian medicine.

The discovery that half of the mammalian protein-coding genome is regulated by the circadian clock has clear implications for medicine. Recent studies demonstrated that the circadian clock influences therapeutic outcomes in human heart disease and cancer. However, biological time is rarely given clinical consideration. A key barrier is the absence of information on tissue-specific molecular rhythms in the human body. We have applied the cyclic ordering by periodic structure (CYCLOPS) algorithm, designed to reconstruct sample temporal order in the absence of time-of-day information, to the gene expression collection of 13 tissues from 632 human donors. We identified rhythms in gene expression across the body; nearly half of protein-coding genes were shown to be cycling in at least 1 of the 13 tissues analyzed. One thousand of these cycling genes encode proteins that either transport or metabolize drugs or are themselves drug targets. These results provide a useful resource for studying the role of circadian rhythms in medicine and support the idea that biological time might play a role in determining drug response.

Circadian Dysregulation: The Next Frontier in Obstructive Sleep Apnea Research.

OBJECTIVE: To review the effects of the circadian clock on homeostasis, the functional interaction between the circadian clock and hypoxia-inducible factors, and the role of circadian dysregulation in the progression of cardiopulmonary disease in obstructive sleep apnea (OSA). DATA SOURCES: The MEDLINE database was accessed through PubMed. REVIEW METHODS: A general review is presented on molecular pathways disrupted in OSA, circadian rhythms and the role of the circadian clock, hypoxia signaling, crosstalk between the circadian and hypoxia systems, the role of the circadian clock in cardiovascular disease, and implications for practice. Studies included in this State of the Art Review demonstrate the potential contribution of the circadian clock and hypoxia in animal models or human disease. CONCLUSIONS: Molecular crosstalk between the circadian clock and hypoxia-inducible factors has not been evaluated in disease models of OSA. IMPLICATIONS FOR PRACTICE: Pediatric OSA is highly prevalent and, if left untreated, may lead to cardiopulmonary sequelae. Changes in inflammatory markers that normally demonstrate circadian rhythmicity are also seen among patients with OSA. Hypoxia-inducible transcription factors interact with core circadian clock transcription factors; however, the interplay between these pathways has not been elucidated in the cardiopulmonary system. This gap in knowledge hinders our ability to identify potential biomarkers of OSA and develop alternative therapeutic strategies. A deeper understanding of the mechanisms by which OSA impinges on clock function and the impact of clock dysregulation on the cardiopulmonary system may lead to future advancements for the care of patients with OSA. The aim of this review is to shed light on this important clinical topic.

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.