Ontogeny and function of the circadian clock in intestinal organoids.

Circadian rhythms regulate diverse aspects of gastrointestinal physiology ranging from the composition of microbiota to motility. However, development of the intestinal circadian clock and detailed mechanisms regulating circadian physiology of the intestine remain largely unknown. In this report, we show that both pluripotent stem cell-derived human intestinal organoids engrafted into mice and patient-derived human intestinal enteroids possess circadian rhythms and demonstrate circadian phase-dependent necrotic cell death responses to Clostridium difficile toxin B (TcdB). Intriguingly, mouse and human enteroids demonstrate anti-phasic necrotic cell death responses to TcdB. RNA-Seq analysis shows that ~3-10% of the detectable transcripts are rhythmically expressed in mouse and human enteroids. Remarkably, we observe anti-phasic gene expression of Rac1, a small GTPase directly inactivated by TcdB, between mouse and human enteroids, and disruption of Rac1 abolishes clock-dependent necrotic cell death responses. Our findings uncover robust functions of circadian rhythms regulating clock-controlled genes in both mouse and human enteroids governing organism-specific, circadian phase-dependent necrotic cell death responses, and lay a foundation for human organ- and disease-specific investigation of clock functions using human organoids for translational applications.

DNA Replication Is Required for Circadian Clock Function by Regulating Rhythmic Nucleosome Composition.

Although the coupling between circadian and cell cycles allows circadian clocks to gate cell division and DNA replication in many organisms, circadian clocks were thought to function independently of cell cycle. Here, we show that DNA replication is required for circadian clock function in Neurospora. Genetic and pharmacological inhibition of DNA replication abolished both overt and molecular rhythmicities by repressing frequency (frq) gene transcription. DNA replication is essential for the rhythmic changes of nucleosome composition at the frq promoter. The FACT complex, known to be involved in histone disassembly/reassembly, is required for clock function and is recruited to the frq promoter in a replication-dependent manner to promote replacement of histone H2A.Z by H2A. Finally, deletion of H2A.Z uncoupled the dependence of the circadian clock on DNA replication. Together, these results establish circadian clock and cell cycle as interdependent coupled oscillators and identify DNA replication as a critical process in the circadian mechanism.

Intercellular Coupling of the Cell Cycle and Circadian Clock in Adult Stem Cell Culture.

Circadian clock-gated cell division cycles are observed from cyanobacteria to mammals via intracellular molecular connections between these two oscillators. Here we demonstrate WNT-mediated intercellular coupling between the cell cycle and circadian clock in 3D murine intestinal organoids (enteroids). The circadian clock gates a population of cells with heterogeneous cell-cycle times that emerge as 12-hr synchronized cell division cycles. Remarkably, we observe reduced-amplitude oscillations of circadian rhythms in intestinal stem cells and progenitor cells, indicating an intercellular signal arising from differentiated cells governing circadian clock-dependent synchronized cell division cycles. Stochastic simulations and experimental validations reveal Paneth cell-secreted WNT as the key intercellular coupling component linking the circadian clock and cell cycle in enteroids.

Histone Deacetylase Inhibition by Gut Microbe-Generated Short-Chain Fatty Acids Entrains Intestinal Epithelial Circadian Rhythms.

BACKGROUND & AIMS: The circadian clock orchestrates ∼24-hour oscillations of gastrointestinal epithelial structure and function that drive diurnal rhythms in gut microbiota. Here, we use experimental and computational approaches in intestinal organoids to reveal reciprocal effects of gut microbial metabolites on epithelial timekeeping by an epigenetic mechanism. METHODS: We cultured enteroids in media supplemented with sterile supernatants from the altered Schaedler Flora (ASF), a defined murine microbiota. Circadian oscillations of bioluminescent PER2 and Bmal1 were measured in the presence or absence of individual ASF supernatants. Separately, we applied machine learning to ASF metabolomics to identify phase-shifting metabolites. RESULTS: Sterile filtrates from 3 of 7 ASF species (ASF360 Lactobacillus intestinalis, ASF361 Ligilactobacillus murinus, and ASF502 Clostridium species) induced minimal alterations in circadian rhythms, whereas filtrates from 4 ASF species (ASF356 Clostridium species, ASF492 Eubacterium plexicaudatum, ASF500 Pseudoflavonifactor species, and ASF519 Parabacteroides goldsteinii) induced profound, concentration-dependent phase shifts. Random forest classification identified short-chain fatty acid (SCFA) (butyrate, propionate, acetate, and isovalerate) production as a discriminating feature of ASF "shifters." Experiments with SCFAs confirmed machine learning predictions, with a median phase shift of 6.2 hours in murine enteroids. Pharmacologic or botanical histone deacetylase (HDAC) inhibitors yielded similar findings. Further, mithramycin A, an inhibitor of HDAC inhibition, reduced SCFA-induced phase shifts by 20% (P < .05) and conditional knockout of HDAC3 in enteroids abrogated butyrate effects on Per2 expression. Key findings were reproducible in human Bmal1-luciferase enteroids, colonoids, and Per2-luciferase Caco-2 cells. CONCLUSIONS: Gut microbe-generated SCFAs entrain intestinal epithelial circadian rhythms by an HDACi-dependent mechanism, with critical implications for understanding microbial and circadian network regulation of intestinal epithelial homeostasis.

Circadian clock regulation of the glycogen synthase (gsn) gene by WCC is critical for rhythmic glycogen metabolism in Neurospora crassa.

Circadian clocks generate rhythms in cellular functions, including metabolism, to align biological processes with the 24-hour environment. Disruption of this alignment by shift work alters glucose homeostasis. Glucose homeostasis depends on signaling and allosteric control; however, the molecular mechanisms linking the clock to glucose homeostasis remain largely unknown. We investigated the molecular links between the clock and glycogen metabolism, a conserved glucose homeostatic process, in Neurospora crassa We find that glycogen synthase (gsn) mRNA, glycogen phosphorylase (gpn) mRNA, and glycogen levels, accumulate with a daily rhythm controlled by the circadian clock. Because the synthase and phosphorylase are critical to homeostasis, their roles in generating glycogen rhythms were investigated. We demonstrate that while gsn was necessary for glycogen production, constitutive gsn expression resulted in high and arrhythmic glycogen levels, and deletion of gpn abolished gsn mRNA rhythms and rhythmic glycogen accumulation. Furthermore, we show that gsn promoter activity is rhythmic and is directly controlled by core clock component white collar complex (WCC). We also discovered that WCC-regulated transcription factors, VOS-1 and CSP-1, modulate the phase and amplitude of rhythmic gsn mRNA, and these changes are similarly reflected in glycogen oscillations. Together, these data indicate the importance of clock-regulated gsn transcription over signaling or allosteric control of glycogen rhythms, a mechanism that is potentially conserved in mammals and critical to metabolic homeostasis.

Modeling Reveals a Key Mechanism for Light-Dependent Phase Shifts of Neurospora Circadian Rhythms.

Light shifts and synchronizes the phase of the circadian clock to daily environments, which is critical for maintaining the daily activities of an organism. It has been proposed that such light-dependent phase shifts are triggered by light-induced upregulation of a negative element of the core circadian clock (i.e., frq, Per1/2) in many organisms, including fungi. However, we find, using systematic mathematical modeling of the Neurospora crassa circadian clock, that the upregulation of the frq gene expression alone is unable to reproduce the observed light-dependent phase responses. Indeed, we find that the depression of the transcriptional activator white-collar-1, previously shown to be promoted by FRQ and VVD, is a key molecular mechanism for accurately simulating light-induced phase response curves for wild-type and mutant strains of Neurospora. Our findings elucidate specific molecular pathways that can be utilized to control phase resetting of circadian rhythms.

Unraveling the Control of Cell Cycle Periods during Intestinal Stem Cell Differentiation.

During differentiation, intestinal stem cells (ISCs), a prototypical adult stem cell pool, become either secretory transit-amplifying cells, which give rise to all secretory cell types, or absorptive transit-amplifying cells, which give rise to enterocytes. These cells exhibit distinct cell cycle dynamics: ISCs cycle with a period of 24 h and absorptive transit-amplifying cells cycle with a period of ∼12 h, whereas secretory transit-amplifying cells arrest their cycle. The cell cycle dynamics of ISCs and their progeny are a systems-level property that emerges from interactions between the cell cycle control machinery and multiple regulatory pathways. Although many mathematical models have been developed to study the details of the cell cycle and related regulatory pathways, few models have been constructed to unravel the dynamic consequences of their interactions. To fill this gap, we present a simplified model focusing on the interaction between four key regulatory pathways (STAT, Wnt, Notch, and MAPK) and cell cycle control. After experimentally validating a model prediction, which showed that the Notch pathway can fine-tune the cell cycle period, we perform further model analysis that reveals that the change of cell cycle period accompanying ISC differentiation may be controlled by a design principle that has been well studied in dynamical systems theory-a saddle node on invariant circle bifurcation. Given that the mechanisms that control the cell cycle are conserved in most eukaryotic cell types, this general principle potentially controls the interplay between proliferation and differentiation for a broad range of stem cells.

Circadian rhythms synchronize mitosis in Neurospora crassa.

The cell cycle and the circadian clock communicate with each other, resulting in circadian-gated cell division cycles. Alterations in this network may lead to diseases such as cancer. Therefore, it is critical to identify molecular components that connect these two oscillators. However, molecular mechanisms between the clock and the cell cycle remain largely unknown. A model filamentous fungus, Neurospora crassa, is a multinucleate system used to elucidate molecular mechanisms of circadian rhythms, but not used to investigate the molecular coupling between these two oscillators. In this report, we show that a conserved coupling between the circadian clock and the cell cycle exists via serine/threonine protein kinase-29 (STK-29), the Neurospora homolog of mammalian WEE1 kinase. Based on this finding, we established a mathematical model that predicts circadian oscillations of cell cycle components and circadian clock-dependent synchronized nuclear divisions. We experimentally demonstrate that G1 and G2 cyclins, CLN-1 and CLB-1, respectively, oscillate in a circadian manner with bioluminescence reporters. The oscillations of clb-1 and stk-29 gene expression are abolished in a circadian arrhythmic frq(ko) mutant. Additionally, we show the light-induced phase shifts of a core circadian component, frq, as well as the gene expression of the cell cycle components clb-1 and stk-29, which may alter the timing of divisions. We then used a histone hH1-GFP reporter to observe nuclear divisions over time, and show that a large number of nuclear divisions occur in the evening. Our findings demonstrate the circadian clock-dependent molecular dynamics of cell cycle components that result in synchronized nuclear divisions in Neurospora.

Spiral-based microfluidic device for long-term time course imaging of Neurospora crassa with single nucleus resolution.

Real-time imaging of fluorescent reporters plays a critical role in elucidating fundamental molecular mechanisms including circadian rhythms in the model filamentous fungus, Neurospora crassa. However, monitoring N. crassa for an extended period of time with single nucleus resolution is a technically challenging task due to hyphal growth that rapidly moves beyond a region of interest during microscopy experiments. In this report, we have proposed a two-dimensional spiral-based microfluidic platform and applied for monitoring the single-nucleus dynamics in N. crassa for long-term time course experiments.

Rhythm and bugs: circadian clocks, gut microbiota, and enteric infections.

PURPOSE OF REVIEW: To highlight recent developments in understanding the dynamic relationship between circadian rhythms, the gut microbiome, and gastrointestinal infections. RECENT FINDINGS: In humans and mice, the composition and functions of the intestinal microbiome display diurnal rhythms orchestrated by feeding behaviors and host circadian gene expression. Jet lag, or circadian disruption, perturbs these rhythms to produce gut dysbiosis. When mice are orally infected with Salmonella typhimurium in the morning (the beginning of their rest period) they show higher levels of colonization and gut inflammation vs. infection at other times of day. At the cellular level, recent studies highlight circadian regulation of innate and adaptive gut immunity in coordination with the microbiome, as well as intestinal stem cell growth and regeneration. SUMMARY: Taken together, these reports support a key role for circadian rhythms in regulating the gut microbiome and host responses to gastrointestinal pathogens. Further research is needed to translate these findings to improving outcomes for patients with gastrointestinal infections by guiding the right interventions for the right patients at the right time.

Mathematical modeling and validation of glucose compensation of the neurospora circadian clock.

Autonomous circadian oscillations arise from transcriptional-translational feedback loops of core clock components. The period of a circadian oscillator is relatively insensitive to changes in nutrients (e.g., glucose), which is referred to as "nutrient compensation". Recently, a transcription repressor, CSP-1, was identified as a component of the circadian system in Neurospora crassa. The transcription of csp-1 is under the circadian regulation. Intriguingly, CSP-1 represses the circadian transcription factor, WC-1, forming a negative feedback loop that can influence the core oscillator. This feedback mechanism is suggested to maintain the circadian period in a wide range of glucose concentrations. In this report, we constructed a mathematical model of the Neurospora circadian clock incorporating the above WC-1/CSP-1 feedback loop, and investigated molecular mechanisms of glucose compensation. Our model shows that glucose compensation exists within a narrow range of parameter space where the activation rates of csp-1 and wc-1 are balanced with each other, and simulates loss of glucose compensation in csp-1 mutants. More importantly, we experimentally validated rhythmic oscillations of the wc-1 gene expression and loss of glucose compensation in the wc-1(ov) mutant as predicted in the model. Furthermore, our stochastic simulations demonstrate that the CSP-1-dependent negative feedback loop functions in glucose compensation, but does not enhance the overall robustness of oscillations against molecular noise. Our work highlights predictive modeling of circadian clock machinery and experimental validations employing Neurospora and brings a deeper understanding of molecular mechanisms of glucose compensation.

Neurospora crassa as a model organism to explore the interconnected network of the cell cycle and the circadian clock.

Budding and fission yeast pioneered uncovering molecular mechanisms of eukaryotic cell division cycles. However, they do not possess canonical circadian clock machinery that regulates physiological processes with a period of about 24h. On the other hand, Neurospora crassa played a critical role in elucidating molecular mechanisms of circadian rhythms, but have not been utilized frequently for cell cycle studies. Recent findings demonstrate that there exists a conserved coupling between the cell cycle and the circadian clock from N.crassa to Mus musculus, which poses Neurospora as an ideal model organism to investigate molecular mechanisms and emerging behavior of the coupled network of the cell cycle and circadian rhythms. In this review, we briefly describe generic eukaryotic cell cycle regulation focusing on G1/S and G2/M transitions, and highlight that these transitions may be targeted for the circadian clock to influence timing of cell division cycles.

Monitoring Circadian Oscillations with a Bioluminescence Reporter in Organoids.

Most living organisms possess circadian rhythms, which are biological processes that occur within a period of approximately 24 h and regulate a diverse repertoire of cellular and physiological processes ranging from sleep-wake cycles to metabolism. This clock mechanism entrains the organism based on environmental changes and coordinates the temporal regulation of molecular and physiological events. Previously, it was demonstrated that autonomous circadian rhythms are maintained even at the single-cell level using cell lines such as NIH3T3 fibroblasts, which were instrumental in uncovering the mechanisms of circadian rhythms. However, these cell lines are homogeneous cultures lacking multicellularity and robust intercellular communications. In the past decade, extensive work has been performed on the development, characterization, and application of 3D organoids, which are in vitro multicellular systems that resemble in vivo morphological structures and functions. This paper describes a protocol for detecting circadian rhythms using a bioluminescent reporter in human intestinal enteroids, which enables the investigation of circadian rhythms in multicellular systems in vitro.

Systematic analysis of negative and positive feedback loops for robustness and temperature compensation in circadian rhythms.

Temperature compensation and robustness to biological noise are two key characteristics of the circadian clock. These features allow the circadian pacemaker to maintain a steady oscillation in a wide range of environmental conditions. The presence of a time-delayed negative feedback loop in the regulatory network generates autonomous circadian oscillations in eukaryotic systems. In comparison, the circadian clock of cyanobacteria is controlled by a strong positive feedback loop. Positive feedback loops with substrate depletion can also generate oscillations, inspiring other circadian clock models. What makes a circadian oscillatory network robust to extrinsic noise is unclear. We investigated four basic circadian oscillators with negative, positive, and combinations of positive and negative feedback loops to explore network features necessary for circadian clock resilience. We discovered that the negative feedback loop system performs the best in compensating temperature changes. We also show that a positive feedback loop can reduce extrinsic noise in periods of circadian oscillators, while intrinsic noise is reduced by negative feedback loops.

Organoids as Model Systems to Investigate Circadian Clock-Related Diseases and Treatments.

Circadian rhythms exist in most cell types in mammals regulating temporal organization of numerous cellular and physiological processes ranging from cell cycle to metabolism. The master clock, suprachiasmatic nucleus (SCN) in the hypothalamus, processes light input and coordinates peripheral clocks optimizing organisms' survival and functions aligning with external conditions. Intriguingly, it was demonstrated that circadian rhythms in the mouse liver can be decoupled from the master clock under time-restricted feeding regimen when food was provided during their inactive phase. Furthermore, mouse liver showed clock-controlled gene expression even in the absence of the master clock demonstrating independent functions of peripheral clocks apart from the SCN. These findings suggest a dynamic relationship between the master and peripheral clocks and highlight potential functions of peripheral clocks independent of the master clock. Importantly, disruption of circadian rhythms correlates with numerous human ailments including cancer and metabolic diseases, suggesting that diseases may be exacerbated by disruption of circadian rhythms in the SCN and/or peripheral clocks. However, molecular mechanisms providing causative links between circadian rhythms and human diseases remain largely unknown. Recent technical advances highlighted PCS- and tissue-derived 3-dimensional organoids as in vitro organs that possess numerous applications ranging from disease modeling to drug screening. In this mini-review, we highlight recent findings on the importance and contributions of peripheral clocks and potential uses of 3D organoids investigating complex circadian clock-related diseases.

Methods for Assessing Circadian Rhythms and Cell Cycle in Intestinal Enteroids.

Endogenous circadian clocks play a key role in regulating a vast array of biological processes from cell cycle to metabolism, and disruption of circadian rhythms exacerbates a range of human ailments including cardiovascular, metabolic, and gastrointestinal diseases. Determining the state of a patient's circadian rhythms and clock-controlled signaling pathways has important implications for precision and personalized medicine, from improving the diagnosis of circadian-related disorders to optimizing the timing of drug delivery. Patient-derived 3-dimensional enteroids or in vitro "mini gut" is an attractive model uncovering human- and patient-specific circadian target genes that may be critical for personalized medicine. Here, we introduce several procedures to assess circadian rhythms and cell cycle dynamics in enteroids through time course sample collection methods and assay techniques including immunofluorescence, live cell confocal microscopy, and bioluminescence. These methods can be applied to evaluate the state of circadian rhythms and circadian clock-gated cell division cycles using mouse and human intestinal enteroids.

An integrated microfluidic bubble pocket for long-term perfused three-dimensional intestine-on-a-chip model.

Perfused three-dimensional (3D) cultures enable long-term in situ growth and monitoring of 3D organoids making them well-suited for investigating organoid development, growth, and function. One of the limitations of this long-term on-chip perfused 3D culture is unintended and disruptive air bubbles. To overcome this obstacle, we invented an imaging platform that integrates an innovative microfluidic bubble pocket for long-term perfused 3D culture of gastrointestinal (GI) organoids. We successfully applied 3D printing technology to create polymer molds that cast polydimethylsiloxane (PDMS) culture chambers in addition to bubble pockets. Our developed platform traps unintended, or induced, air bubbles in an integrated PDMS pocket chamber, where the bubbles diffuse out across the gas permeable PDMS or an outlet tube. We demonstrated that our robust platform integrated with the novel bubble pocket effectively circumvents the development of bubbles into human and mouse GI organoid cultures during long-term perfused time-course imaging. Our platform with the innovative integrated bubble pocket is ideally suited for studies requiring long-term perfusion monitoring of organ growth and morphogenesis as well as function.

Minimum criteria for DNA damage-induced phase advances in circadian rhythms.

Robust oscillatory behaviors are common features of circadian and cell cycle rhythms. These cyclic processes, however, behave distinctively in terms of their periods and phases in response to external influences such as light, temperature, nutrients, etc. Nevertheless, several links have been found between these two oscillators. Cell division cycles gated by the circadian clock have been observed since the late 1950s. On the other hand, ionizing radiation (IR) treatments cause cells to undergo a DNA damage response, which leads to phase shifts (mostly advances) in circadian rhythms. Circadian gating of the cell cycle can be attributed to the cell cycle inhibitor kinase Wee1 (which is regulated by the heterodimeric circadian clock transcription factor, BMAL1/CLK), and possibly in conjunction with other cell cycle components that are known to be regulated by the circadian clock (i.e., c-Myc and cyclin D1). It has also been shown that DNA damage-induced activation of the cell cycle regulator, Chk2, leads to phosphorylation and destruction of a circadian clock component (i.e., PER1 in Mus or FRQ in Neurospora crassa). However, the molecular mechanism underlying how DNA damage causes predominantly phase advances in the circadian clock remains unknown. In order to address this question, we employ mathematical modeling to simulate different phase response curves (PRCs) from either dexamethasone (Dex) or IR treatment experiments. Dex is known to synchronize circadian rhythms in cell culture and may generate both phase advances and delays. We observe unique phase responses with minimum delays of the circadian clock upon DNA damage when two criteria are met: (1) existence of an autocatalytic positive feedback mechanism in addition to the time-delayed negative feedback loop in the clock system and (2) Chk2-dependent phosphorylation and degradation of PERs that are not bound to BMAL1/CLK.

Author Correction: FRQ-CK1 interaction determines the period of circadian rhythms in Neurospora.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

FRQ-CK1 interaction determines the period of circadian rhythms in Neurospora.

Circadian clock mechanisms have been extensively investigated but the main rate-limiting step that determines circadian period remains unclear. Formation of a stable complex between clock proteins and CK1 is a conserved feature in eukaryotic circadian mechanisms. Here we show that the FRQ-CK1 interaction, but not FRQ stability, correlates with circadian period in Neurospora circadian clock mutants. Mutations that specifically affect the FRQ-CK1 interaction lead to severe alterations in circadian period. The FRQ-CK1 interaction has two roles in the circadian negative feedback loop. First, it determines the FRQ phosphorylation profile, which regulates FRQ stability and also feeds back to either promote or reduce the interaction itself. Second, it determines the efficiency of circadian negative feedback process by mediating FRQ-dependent WC phosphorylation. Our conclusions are further supported by mathematical modeling and in silico experiments. Together, these results suggest that the FRQ-CK1 interaction is a major rate-limiting step in circadian period determination.