Steroid receptor coactivator 3 is a key modulator of regulatory T cell-mediated tumor evasion.

Steroid receptor coactivator 3 (SRC-3) is most strongly expressed in regulatory T cells (Tregs) and B cells, suggesting that it plays an important role in the regulation of Treg function. Using an aggressive E0771 mouse breast cell line syngeneic immune-intact murine model, we observed that breast tumors were "permanently eradicated" in a genetically engineered tamoxifen-inducible Treg-cell-specific SRC-3 knockout (KO) female mouse that does not possess a systemic autoimmune pathological phenotype. A similar eradication of tumor was noted in a syngeneic model of prostate cancer. A subsequent injection of additional E0771 cancer cells into these mice showed continued resistance to tumor development without the need for tamoxifen induction to produce additional SRC-3 KO Tregs. SRC-3 KO Tregs were highly proliferative and preferentially infiltrated into breast tumors by activating the chemokine (C-C motif) ligand (Ccl) 19/Ccl21/chemokine (C-C motif) receptor (Ccr)7 signaling axis, generating antitumor immunity by enhancing the interferon-γ/C-X-C motif chemokine ligand (Cxcl) 9 signaling axis to facilitate the entrance and function of effector T cells and natural killer cells. SRC-3 KO Tregs also show a dominant effect by blocking the immune suppressive function of WT Tregs. Importantly, a single adoptive transfer of SRC-3 KO Tregs into wild-type E0771 tumor-bearing mice can completely abolish preestablished breast tumors by generating potent antitumor immunity with a durable effect that prevents tumor reoccurrence. Therefore, treatment with SRC-3-deleted Tregs represents an approach to completely block tumor growth and recurrence without the autoimmune side effects that typically accompany immune checkpoint modulators.

Transcriptional coactivation of NRF2 signaling in cardiac fibroblasts promotes resistance to oxidative stress.

We recently discovered that steroid receptor coactivators (SRCs) SRCs-1, 2 and 3, are abundantly expressed in cardiac fibroblasts (CFs) and their activation with the SRC small molecule stimulator MCB-613 improves cardiac function and dramatically lowers pro-fibrotic signaling in CFs post-myocardial infarction. These findings suggest that CF-derived SRC activation could be beneficial in the mitigation of chronic heart failure after ischemic insult. However, the cardioprotective mechanisms by which CFs contribute to cardiac pathological remodeling are unclear. Here we present studies designed to identify the molecular and cellular circuitry that governs the anti-fibrotic effects of an MCB-613 derivative, MCB-613-10-1, in CFs. We performed cytokine profiling and whole transcriptome and proteome analyses of CF-derived signals in response to MCB-613-10-1. We identified the NRF2 pathway as a direct MCB-613-10-1 therapeutic target for promoting resistance to oxidative stress in CFs. We show that MCB-613-10-1 promotes cell survival of anti-fibrotic CFs exposed to oxidative stress by suppressing apoptosis. We demonstrate that an increase in HMOX1 expression contributes to CF resistance to oxidative stress-mediated apoptosis via a mechanism involving SRC co-activation of NRF2, hence reducing inflammation and fibrosis. We provide evidence that MCB-613-10-1 acts as a protectant against oxidative stress-induced mitochondrial damage. Our data reveal that SRC stimulation of the NRF2 transcriptional network promotes resistance to oxidative stress and highlights a mechanistic approach toward addressing pathologic cardiac remodeling.

12-h clock regulation of genetic information flow by XBP1s.

Our group recently characterized a cell-autonomous mammalian 12-h clock independent from the circadian clock, but its function and mechanism of regulation remain poorly understood. Here, we show that in mouse liver, transcriptional regulation significantly contributes to the establishment of 12-h rhythms of mRNA expression in a manner dependent on Spliced Form of X-box Binding Protein 1 (XBP1s). Mechanistically, the motif stringency of XBP1s promoter binding sites dictates XBP1s's ability to drive 12-h rhythms of nascent mRNA transcription at dawn and dusk, which are enriched for basal transcription regulation, mRNA processing and export, ribosome biogenesis, translation initiation, and protein processing/sorting in the Endoplasmic Reticulum (ER)-Golgi in a temporal order consistent with the progressive molecular processing sequence described by the central dogma information flow (CEDIF). We further identified GA-binding proteins (GABPs) as putative novel transcriptional regulators driving 12-h rhythms of gene expression with more diverse phases. These 12-h rhythms of gene expression are cell autonomous and evolutionarily conserved in marine animals possessing a circatidal clock. Our results demonstrate an evolutionarily conserved, intricate network of transcriptional control of the mammalian 12-h clock that mediates diverse biological pathways. We speculate that the 12-h clock is coopted to accommodate elevated gene expression and processing in mammals at the two rush hours, with the particular genes processed at each rush hour regulated by the circadian and/or tissue-specific pathways.

Coactivator-Dependent Oscillation of Chromatin Accessibility Dictates Circadian Gene Amplitude via REV-ERB Loading.

A central mechanism for controlling circadian gene amplitude remains elusive. We present evidence for a "facilitated repression (FR)" model that functions as an amplitude rheostat for circadian gene oscillation. We demonstrate that ROR and/or BMAL1 promote global chromatin decondensation during the activation phase of the circadian cycle to actively facilitate REV-ERB loading for repression of circadian gene expression. Mechanistically, we found that SRC-2 dictates global circadian chromatin remodeling through spatial and temporal recruitment of PBAF members of the SWI/SNF complex to facilitate loading of REV-ERB in the hepatic genome. Mathematical modeling highlights how the FR model sustains proper circadian rhythm despite fluctuations of REV-ERB levels. Our study not only reveals a mechanism for active communication between the positive and negative limbs of the circadian transcriptional loop but also establishes the concept that clock transcription factor binding dynamics is perhaps a central tenet for fine-tuning circadian rhythm.

A steroid receptor coactivator stimulator (MCB-613) attenuates adverse remodeling after myocardial infarction.

Progressive remodeling of the heart, resulting in cardiomyocyte (CM) loss and increased inflammation, fibrosis, and a progressive decrease in cardiac function, are hallmarks of myocardial infarction (MI)-induced heart failure. We show that MCB-613, a potent small molecule stimulator of steroid receptor coactivators (SRCs) attenuates pathological remodeling post-MI. MCB-613 decreases infarct size, apoptosis, hypertrophy, and fibrosis while maintaining significant cardiac function. MCB-613, when given within hours post MI, induces lasting protection from adverse remodeling concomitant with: 1) inhibition of macrophage inflammatory signaling and interleukin 1 (IL-1) signaling, which attenuates the acute inflammatory response, 2) attenuation of fibroblast differentiation, and 3) promotion of Tsc22d3-expressing macrophages-all of which may limit inflammatory damage. SRC stimulation with MCB-613 (and derivatives) is a potential therapeutic approach for inhibiting cardiac dysfunction after MI.

SRC-2 orchestrates polygenic inputs for fine-tuning glucose homeostasis.

Despite extensive efforts to understand the monogenic contributions to perturbed glucose homeostasis, the complexity of genetic events that fractionally contribute to the spectrum of this pathology remain poorly understood. Proper maintenance of glucose homeostasis is the central feature of a constellation of comorbidities that define the metabolic syndrome. The ability of the liver to balance carbohydrate uptake and release during the feeding-to-fasting transition is essential to the regulation of peripheral glucose availability. The liver coordinates the expression of gene programs that control glucose absorption, storage, and secretion. Herein, we demonstrate that Steroid Receptor Coactivator 2 (SRC-2) orchestrates a hierarchy of nutritionally responsive transcriptional complexes to precisely modulate plasma glucose availability. Using DNA pull-down technology coupled with mass spectrometry, we have identified SRC-2 as an indispensable integrator of transcriptional complexes that control the rate-limiting steps of hepatic glucose release and accretion. Collectively, these findings position SRC-2 as a major regulator of polygenic inputs to metabolic gene regulation and perhaps identify a previously unappreciated model that helps to explain the clinical spectrum of glucose dysregulation.

A multi-layer monitoring system for clinical management of Congestive Heart Failure.

BACKGROUND: Congestive Heart Failure (CHF) is a serious cardiac condition that brings high risks of urgent hospitalization and death. Remote monitoring systems are well-suited to managing patients suffering from CHF, and can reduce deaths and re-hospitalizations, as shown by the literature, including multiple systematic reviews. METHODS: The monitoring system proposed in this paper aims at helping CHF stakeholders make appropriate decisions in managing the disease and preventing cardiac events, such as decompensation, which can lead to hospitalization or death. Monitoring activities are stratified into three layers: scheduled visits to a hospital following up on a cardiac event, home monitoring visits by nurses, and patient's self-monitoring performed at home using specialized equipment. Appropriate hardware, desktop and mobile software applications were developed to enable a patient's monitoring by all stakeholders. For the first two layers, we designed and implemented a Decision Support System (DSS) using machine learning (Random Forest algorithm) to predict the number of decompensations per year and to assess the heart failure severity based on a variety of clinical data. For the third layer, custom-designed sensors (the Blue Scale system) for electrocardiogram (EKG), pulse transit times, bio-impedance and weight allowed frequent collection of CHF-related data in the comfort of the patient's home. We also performed a short-term Heart Rate Variability (HRV) analysis on electrocardiograms self-acquired by 15 healthy volunteers and compared the obtained parameters with those of 15 CHF patients from PhysioNet's PhysioBank archives. RESULTS: We report numerical performances of the DSS, calculated as multiclass accuracy, sensitivity and specificity in a 10-fold cross-validation. The obtained average accuracies are: 71.9% in predicting the number of decompensations and 81.3% in severity assessment. The most serious class in severity assessment is detected with good sensitivity and specificity (0.87 / 0.95), while, in predicting decompensation, high specificity combined with good sensitivity prevents false alarms. The HRV parameters extracted from the self-measured EKG using the Blue Scale system of sensors are comparable with those reported in the literature about healthy people. CONCLUSIONS: The performance of DSSs trained with new patients confirmed the results of previous work, and emphasizes the strong correlation between some CHF markers, such as brain natriuretic peptide (BNP) and ejection fraction (EF), with the outputs of interest. Comparing HRV parameters from healthy volunteers with HRV parameters obtained from PhysioBank archives, we confirm the literature that considers the HRV a promising method for distinguishing healthy from CHF patients.

Interpreting PPV and NPV of Diagnostic Tests with Uncertain Prevalence.

OBJECTIVE: Medical decision-making is often uncertain. The positive predictive value (PPV) and negative predictive value (NPV) are conditional probabilities characterizing diagnostic tests and assessing diagnostic interventions in clinical medicine and epidemiology. The PPV is the probability that a patient has a specified disease, given a positive test result for that disease. The NPV is the probability that a patient does not have the disease, given a negative test result for that disease. Both values depend on disease incidence or prevalence, which may be highly uncertain for unfamiliar diseases, epidemics, etc. Probability distributions for this uncertainty are usually unavailable. We develop a non-probabilistic method for interpreting PPV and NPV with uncertain prevalence. METHODS: Uncertainty in PPV and NPV is managed with the non-probabilistic concept of robustness in info-gap theory. Robustness of PPV or NPV estimates is the greatest uncertainty (in prevalence) at which the estimate's error is acceptable. RESULTS: Four properties are demonstrated. Zeroing: best estimates of PPV or NPV have no robustness to uncertain prevalence; best estimates are unreliable for interpreting diagnostic tests. Trade-off: robustness increases as error increases; this trade-off identifies robustly reliable error in PPV or NPV. Preference reversal: sometimes sub-optimal PPV or NPV estimates are more robust to uncertain incidence or prevalence than optimal estimates, motivating reversal of preference from the putative optimum to the sub-optimal estimate. Trade-off between specificity and robustness to uncertainty: the robustness increases as test-specificity decreases. These four properties underlie the interpretation of PPV and NPV. CONCLUSIONS: The PPV and NPV assess diagnostic tests, but are sensitive to lack of knowledge that generates non-probabilistic uncertain prevalence and must be supplemented with robustness analysis. When uncertainties abound, as with unfamiliar diseases, assessing robustness is critical to avoiding erroneous decisions.

MYC Induces Oncogenic Stress through RNA Decay and Ribonucleotide Catabolism in Breast Cancer.

Upregulation of MYC is a hallmark of cancer, wherein MYC drives oncogenic gene expression and elevates total RNA synthesis across cancer cell transcriptomes. Although this transcriptional anabolism fuels cancer growth and survival, the consequences and metabolic stresses induced by excess cellular RNA are poorly understood. Herein, we discover that RNA degradation and downstream ribonucleotide catabolism is a novel mechanism of MYC-induced cancer cell death. Combining genetics and metabolomics, we find that MYC increases RNA decay through the cytoplasmic exosome, resulting in the accumulation of cytotoxic RNA catabolites and reactive oxygen species. Notably, tumor-derived exosome mutations abrogate MYC-induced cell death, suggesting excess RNA decay may be toxic to human cancers. In agreement, purine salvage acts as a compensatory pathway that mitigates MYC-induced ribonucleotide catabolism, and inhibitors of purine salvage impair MYC+ tumor progression. Together, these data suggest that MYC-induced RNA decay is an oncogenic stress that can be exploited therapeutically. Significance: MYC is the most common oncogenic driver of poor-prognosis cancers but has been recalcitrant to therapeutic inhibition. We discovered a new vulnerability in MYC+ cancer where MYC induces cell death through excess RNA decay. Therapeutics that exacerbate downstream ribonucleotide catabolism provide a therapeutically tractable approach to TNBC (Triple-negative Breast Cancer) and other MYC-driven cancers.

Steroid Receptor Coactivator-3 is a Key Modulator of Regulatory T Cell-Mediated Tumor Evasion.

Steroid receptor coactivator 3 (SRC-3) is most strongly expressed in regulatory T cells (Tregs) and B cells, suggesting that it plays an important role in the regulation of Treg function. Using an aggressive E0771 mouse breast cell line syngeneic immune-intact murine model, we observed that breast tumors were 'permanently eradicated' in a genetically engineered tamoxifen-inducible Treg-cell specific SRC-3 knockout (KO) female mouse that does not possess a systemic autoimmune pathological phenotype. A similar eradication of tumor was noted in a syngeneic model of prostate cancer. A subsequent injection of additional E0771 cancer cells into these mice showed continued resistance to tumor development without the need for tamoxifen induction to produce additional SRC-3 KO Tregs. SRC-3 KO Tregs were highly proliferative and preferentially infiltrated into breast tumors by activating the Chemokine (C-C motif) ligand (Ccl) 19/Ccl21/ Chemokine (C-C motif) Receptor (Ccr)7 signaling axis, generating antitumor immunity by enhancing the interferon-γ/C-X-C Motif Chemokine Ligand (Cxcl) 9 signaling axis to facilitate the entrance and function of effector T cells and Natural Killer cells. SRC-3 KO Tregs also show a dominant effect by blocking the immune suppressive function of WT Tregs. Importantly, a single adoptive transfer of SRC-3 KO Tregs into wild-type E0771 tumor-bearing mice can completely abolish pre-established breast tumors by generating potent antitumor immunity with a durable effect that prevents tumor reoccurrence. Therefore, treatment with SRC-3 deleted Tregs represents a novel approach to completely block tumor growth and recurrence without the autoimmune side-effects that typically accompany immune checkpoint modulators. Significance statement: Tregs are essential in restraining immune responses for immune homeostasis. SRC-3 is a pleiotropic coactivator, the second-most highly expressed transcriptional coactivator in Tregs, and a suspect in Treg function. The disruption of SRC-3 expression in Tregs leads to a 'complete lifetime eradication' of tumors in aggressive syngeneic breast cancer mouse models because deletion of SRC-3 alters the expression of a wide range of key genes involved in efferent and afferent Treg signaling. SRC-3KO Tregs confer this long-lasting protection against cancer recurrence in mice without an apparent systemic autoimmune pathological phenotype. Therefore, treatment with SRC-3 deleted Tregs could represent a novel and efficient future target for eliminating tumor growth and recurrence without the autoimmune side-effects that typically accompany immune checkpoint modulators.

Info-gap management of public health Policy for TB with HIV-prevalence and epidemiological uncertainty.

BACKGROUND: Formulation and evaluation of public health policy commonly employs science-based mathematical models. For instance, epidemiological dynamics of TB is dominated, in general, by flow between actively and latently infected populations. Thus modelling is central in planning public health intervention. However, models are highly uncertain because they are based on observations that are geographically and temporally distinct from the population to which they are applied. AIMS: We aim to demonstrate the advantages of info-gap theory, a non-probabilistic approach to severe uncertainty when worst cases cannot be reliably identified and probability distributions are unreliable or unavailable. Info-gap is applied here to mathematical modelling of epidemics and analysis of public health decision-making. METHODS: Applying info-gap robustness analysis to tuberculosis/HIV (TB/HIV) epidemics, we illustrate the critical role of incorporating uncertainty in formulating recommendations for interventions. Robustness is assessed as the magnitude of uncertainty that can be tolerated by a given intervention. We illustrate the methodology by exploring interventions that alter the rates of diagnosis, cure, relapse and HIV infection. RESULTS: We demonstrate several policy implications. Equivalence among alternative rates of diagnosis and relapse are identified. The impact of initial TB and HIV prevalence on the robustness to uncertainty is quantified. In some configurations, increased aggressiveness of intervention improves the predicted outcome but also reduces the robustness to uncertainty. Similarly, predicted outcomes may be better at larger target times, but may also be more vulnerable to model error. CONCLUSIONS: The info-gap framework is useful for managing model uncertainty and is attractive when uncertainties on model parameters are extreme. When a public health model underlies guidelines, info-gap decision theory provides valuable insight into the confidence of achieving agreed-upon goals.

A steroid receptor coactivator small molecule "stimulator" attenuates post-stroke ischemic brain injury.

Introduction: Pathologic remodeling of the brain following ischemic stroke results in neuronal loss, increased inflammation, oxidative stress, astrogliosis, and a progressive decrease in brain function. We recently demonstrated that stimulation of steroid receptor coactivator 3 with the small-molecule stimulator MCB-613 improves cardiac function in a mouse model of myocardial ischemia. Since steroid receptor coactivators are ubiquitously expressed in the brain, we reasoned that an MCB-613 derivative (MCB-10-1), could protect the brain following ischemic injury. To test this, we administered MCB-10-1 to rats following middle cerebral artery occlusion and reperfusion. Methods: Neurologic impairment and tissue damage responses were evaluated on day 1 and day 4 following injury in rats treated with control or 10-1. Results: We show that 10-1 attenuates injury post-stroke. 10-1 decreases infarct size and mitigates neurologic impairment. When given within 30 min post middle cerebral artery occlusion and reperfusion, 10-1 induces lasting protection from tissue damage in the ischemic penumbra concomitant with: (1) promotion of reparative microglia; (2) an increase in astrocyte NRF2 and GLT-1 expression; (3) early microglia activation; and (4) attenuation of astrogliosis. Discussion: Steroid receptor coactivator stimulation with MCB-10-1 is a potential therapeutic strategy for reducing inflammation and oxidative damage that cause neurologic impairment following an acute ischemic stroke.

Defining the mammalian coactivation of hepatic 12-h clock and lipid metabolism.

The 12-h clock coordinates lipid homeostasis, energy metabolism, and stress rhythms via the transcriptional regulator XBP1. However, the biochemical and physiological bases for integrated control of the 12-h clock and diverse metabolic pathways remain unclear. Here, we show that steroid receptor coactivator SRC-3 coactivates XBP1 transcription and regulates hepatic 12-h cistrome and gene rhythmicity. Mice lacking SRC-3 show abnormal 12-h rhythms in hepatic transcription, metabolic functions, systemic energetics, and rate-limiting lipid metabolic processes, including triglyceride, phospholipid, and cardiolipin pathways. Notably, 12-h clock coactivation is not only preserved, with its cistromic activation priming ahead of the zeitgeber cue of light, but concomitant with rhythmic remodeling in the absence of food. These findings reveal that SRC-3 integrates the mammalian 12-h clock, energy metabolism, and membrane and lipid homeostasis and demonstrates a role for the 12-h clock machinery as an active transcriptional mechanism in anticipating physiological and metabolic energy needs and stresses.

Assessing the acceptability and usability of an interactive serious game in aiding treatment decisions for patients with localized prostate cancer.

BACKGROUND: Men diagnosed with localized prostate cancer face a potentially life-altering treatment decision that can be overwhelming. Enhancing patient knowledge through education can significantly reduce feelings of uncertainty while simultaneously increasing confidence in decision making. Serious games have been shown in other populations to increase health knowledge and assist with the health decision-making process. We developed an interactive serious game, Time After Time, which translates evidence-based treatment outcome data into an accessible and understandable format that men can utilize in their prostate cancer treatment decision-making process. The game specifically aims to raise men's awareness and understanding of the impact of health-related quality of life issues associated with the major treatment options and to enrich their conversations with their health care providers. OBJECTIVE: This study determined the acceptability and usability of the alpha version of Time After Time, an interactive decision aid for men diagnosed with localized prostate cancer, in order to inform future iterations of the serious game. METHODS: The study employed a mixed methods approach to assess the acceptability and usability of the Time After Time serious game using qualitative focus groups and a quantitative Likert scale survey. RESULTS: A total of 13 men who had already completed treatment for localized prostate cancer completed the survey and participated in focus group meetings. The majority of the study participants rated Time After Time as an appropriate decision tool for localized prostate cancer and verified that it meets its goals of increasing focus on side effects and generating questions for the patient's health care team. However, participants also expressed concerns about game usability and the diversity of information covered regarding treatment options and potential treatment outcomes. CONCLUSIONS: Serious games are a promising approach to health education and decision support for older men. Participants were receptive to the idea of a serious game as a decision aid in localized prostate cancer. However, usability issues are a major concern for this demographic, as is clarity and transparency of data sources.

XBP1 links the 12-hour clock to NAFLD and regulation of membrane fluidity and lipid homeostasis.

A distinct 12-hour clock exists in addition to the 24-hour circadian clock to coordinate metabolic and stress rhythms. Here, we show that liver-specific ablation of X-box binding protein 1 (XBP1) disrupts the hepatic 12-hour clock and promotes spontaneous non-alcoholic fatty liver disease (NAFLD). We show that hepatic XBP1 predominantly regulates the 12-hour rhythmicity of gene transcription in the mouse liver and demonstrate that perturbation of the 12-hour clock, but not the core circadian clock, is associated with the onset and progression of this NAFLD phenotype. Mechanistically, we provide evidence that the spliced form of XBP1 (XBP1s) binds to the hepatic 12-hour cistrome to directly regulate the 12-hour clock, with a periodicity paralleling the harmonic activation of the 12-hour oscillatory transcription of many rate-limiting metabolic genes known to have perturbations in human metabolic disease. Functionally, we show that Xbp1 ablation significantly reduces cellular membrane fluidity and impairs lipid homeostasis via rate-limiting metabolic processes in fatty acid monounsaturated and phospholipid remodeling pathways. These findings reveal that genetic disruption of the hepatic 12-hour clock links to the onset and progression of NAFLD development via transcriptional regulator XBP1, and demonstrate a role for XBP1 and the 12-hour clock in the modulation of phospholipid composition and the maintenance of lipid homeostasis.

Unveiling "Musica Universalis" of the Cell: A Brief History of Biological 12-Hour Rhythms.

"Musica universalis" is an ancient philosophical concept claiming the movements of celestial bodies follow mathematical equations and resonate to produce an inaudible harmony of music, and the harmonious sounds that humans make were an approximation of this larger harmony of the universe. Besides music, electromagnetic waves such as light and electric signals also are presented as harmonic resonances. Despite the seemingly universal theme of harmonic resonance in various disciplines, it was not until recently that the same harmonic resonance was discovered also to exist in biological systems. Contrary to traditional belief that a biological system is either at stead-state or cycles with a single frequency, it is now appreciated that most biological systems have no homeostatic "set point," but rather oscillate as composite rhythms consisting of superimposed oscillations. These oscillations often cycle at different harmonics of the circadian rhythm, and among these, the ~12-hour oscillation is most prevalent. In this review, we focus on these 12-hour oscillations, with special attention to their evolutionary origin, regulation, and functions in mammals, as well as their relationship to the circadian rhythm. We further discuss the potential roles of the 12-hour clock in regulating hepatic steatosis, aging, and the possibility of 12-hour clock-based chronotherapy. Finally, we posit that biological rhythms are also musica universalis: whereas the circadian rhythm is synchronized to the 24-hour light/dark cycle coinciding with the Earth's rotation, the mammalian 12-hour clock may have evolved from the circatidal clock, which is entrained by the 12-hour tidal cues orchestrated by the moon.

A novel mathematical method for disclosing oscillations in gene transcription: A comparative study.

Circadian rhythmicity, the 24-hour cycle responsive to light and dark, is determined by periodic oscillations in gene transcription. This phenomenon has broad ramifications in physiologic function. Recent work has disclosed more cycles in gene transcription, and to the uncovering of these we apply a novel signal processing methodology known as the pencil method and compare it to conventional parametric, nonparametric, and statistical methods. METHODS: In order to assess periodicity of gene expression over time, we analyzed a database derived from livers of mice entrained to a 12-hour light/12-hour dark cycle. We also analyzed artificially generated signals to identify differences between the pencil decomposition and other alternative methods. RESULTS: The pencil decomposition revealed hitherto-unsuspected oscillations in gene transcription with 12-hour periodicity. The pencil method was robust in detecting the 24-hour circadian cycle that was known to exist, as well as confirming the existence of shorter-period oscillations. A key consequence of this approach is that orthogonality of the different oscillatory components can be demonstrated. thus indicating a biological independence of these oscillations, that has been subsequently confirmed empirically by knocking out the gene responsible for the 24-hour clock. CONCLUSION: System identification techniques can be applied to biological systems and can uncover important characteristics that may elude visual inspection of the data. SIGNIFICANCE: The pencil method provides new insights on the essence of gene expression and discloses a wide variety of oscillations in addition to the well-studied circadian pattern. This insight opens the door to the study of novel mechanisms by which oscillatory gene expression signals exert their regulatory effect on cells to influence human diseases.

A Cell-Autonomous Mammalian 12 hr Clock Coordinates Metabolic and Stress Rhythms.

Besides circadian rhythms, oscillations cycling with a 12 hr period exist. However, the prevalence, origin, regulation, and function of mammalian 12 hr rhythms remain elusive. Utilizing an unbiased mathematical approach identifying all superimposed oscillations, we uncovered prevalent 12 hr gene expression and metabolic rhythms in mouse liver, coupled with a physiological 12 hr unfolded protein response oscillation. The mammalian 12 hr rhythm is cell autonomous, driven by a dedicated 12 hr pacemaker distinct from the circadian clock, and can be entrained in vitro by metabolic and ER stress cues. Mechanistically, we identified XBP1s as a transcriptional regulator of the mammalian 12 hr clock. Downregulation of the 12 hr gene expression strongly correlates with human hepatic steatosis and steatohepatitis, implying its importance in maintaining metabolic homeostasis. The mammalian 12 hr rhythm of gene expression also is conserved in nematodes and crustaceans, indicating an ancient origin of the 12 hr clock. Our work sheds new light on how perturbed biological rhythms contribute to human disease.