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Pulmonary inflammatory responses lie under circadian control; however, the importance of circadian mechanisms in the underlying fibrotic phenotype is not understood. Here, we identify a striking change to these mechanisms resulting in a gain of amplitude and lack of synchrony within pulmonary fibrotic tissue. These changes result from an infiltration of mesenchymal cells, an important cell type in the pathogenesis of pulmonary fibrosis. Mutation of the core clock protein REVERBα in these cells exacerbated the development of bleomycin-induced fibrosis, whereas mutation of REVERBα in club or myeloid cells had no effect on the bleomycin phenotype. Knockdown of REVERBα revealed regulation of the little-understood transcription factor TBPL1. Both REVERBα and TBPL1 altered integrinβ1 focal-adhesion formation, resulting in increased myofibroblast activation. The translational importance of our findings was established through analysis of 2 human cohorts. In the UK Biobank, circadian strain markers (sleep length, chronotype, and shift work) are associated with pulmonary fibrosis, making them risk factors. In a separate cohort, REVERBα expression was increased in human idiopathic pulmonary fibrosis (IPF) lung tissue. Pharmacological targeting of REVERBα inhibited myofibroblast activation in IPF fibroblasts and collagen secretion in organotypic cultures from IPF patients, thus suggesting that targeting of REVERBα could be a viable therapeutic approach.

Organismal physiology is widely regulated by the molecular circadian clock, a feedback loop composed of protein complexes whose members are enriched in intrinsically disordered regions. These regions can mediate protein-protein interactions via SLiMs, but the contribution of these disordered regions to clock protein interactions had not been elucidated. To determine the functionality of these disordered regions, we applied a synthetic peptide microarray approach to the disordered clock protein FRQ in Neurospora crassa . We identified residues required for FRQ’s interaction with its partner protein FRH, the mutation of which demonstrated FRH is necessary for persistent clock oscillations but not repression of transcriptional activity. Additionally, the microarray demonstrated an enrichment of FRH binding to FRQ peptides with a net positive charge. We found that positively charged residues occurred in significant “blocks” within the amino acid sequence of FRQ and that ablation of one of these blocks affected both core clock timing and physiological clock output. Finally, we found positive charge clusters were a commonly shared molecular feature in repressive circadian clock proteins. Overall, our study suggests a mechanistic purpose for positive charge blocks and yielded insights into repressive arm protein roles in clock function. Many clock proteins contain intrinsically disordered regions, but how these regions mediate protein interactions is poorly understood. Here, the authors identify charge blocks within a disordered clock protein that regulate circadian timing.

The circadian clock in mammals is driven by an autoregulatory transcriptional feedback mechanism that takes approximately 24 hours to complete. A key component of this mechanism is a heterodimeric transcriptional activator consisting of two basic helix-loop-helix PER-ARNT-SIM (bHLH-PAS) domain protein subunits, CLOCK and BMAL1. Here, we report the crystal structure of a complex containing the mouse CLOCK:BMAL1 bHLH-PAS domains at 2.3 Å resolution. The structure reveals an unusual asymmetric heterodimer with the three domains in each of the two subunits—bHLH, PAS-A, and PAS-B—tightly intertwined and involved in dimerization interactions, resulting in three distinct protein interfaces. Mutations that perturb the observed heterodimer interfaces affect the stability and activity of the CLOCK:BMAL1 complex as well as the periodicity of the circadian oscillator. The structure of the CLOCK:BMAL1 complex is a starting point for understanding at an atomic level the mechanism driving the mammalian circadian clock.

The frq gene, essential for circadian clock function, is shown to differ from most other genes in Neurospora by exhibiting non-optimal codon usage; by contrast, optimization of codon usage is unexpectedly found to affect the structure and function of the coded protein, subsequently impairing circadian feedback loops. Time for non-optimal codons Many biological processes are rhythmically regulated on a daily, or circadian, cycle. Highly expressed genes, such as those regulating the circadian rhythm, normally contain optimal codons, to allow efficient expression. Two studies, from the laboratories of Carl Johnson and Yi Liu, now find that central circadian proteins in cyanobacteria and Neurospora unexpectedly use non-optimal codons, and that optimizing their codes causes a change in an adaptive response in cyanobacteria, and compromises function of the Neurospora clock. These works highlight an unanticipated selection against optimal codon usage. Codon-usage bias has been observed in almost all genomes and is thought to result from selection for efficient and accurate translation of highly expressed genes 1 , 2 , 3 . Codon usage is also implicated in the control of transcription, splicing and RNA structure 4 , 5 , 6 . Many genes exhibit little codon-usage bias, which is thought to reflect a lack of selection for messenger RNA translation. Alternatively, however, non-optimal codon usage may be of biological importance. The rhythmic expression and the proper function of the Neurospora FREQUENCY (FRQ) protein are essential for circadian clock function. Here we show that, unlike most genes in Neurospora , frq exhibits non-optimal codon usage across its entire open reading frame. Optimization of frq codon usage abolishes both overt and molecular circadian rhythms. Codon optimization not only increases FRQ levels but, unexpectedly, also results in conformational changes in FRQ protein, altered FRQ phosphorylation profile and stability, and impaired functions in the circadian feedback loops. These results indicate that non-optimal codon usage of frq is essential for its circadian clock function. Our study provides an example of how non-optimal codon usage functions to regulate protein expression and to achieve optimal protein structure and function.

Ubiquitin ligases control the degradation of core clock proteins to govern the speed and resetting properties of the circadian pacemaker. However, few studies have addressed their potential to regulate other cellular events within clock neurons beyond clock protein turnover. Here, we report that the ubiquitin ligase, UBR4/POE, strengthens the central pacemaker by facilitating neuropeptide trafficking in clock neurons and promoting network synchrony. Ubr4 -deficient mice are resistant to jetlag, whereas poe knockdown flies are prone to arrhythmicity, behaviors reflective of the reduced axonal trafficking of circadian neuropeptides. At the cellular level, Ubr4 ablation impairs the export of secreted proteins from the Golgi apparatus by reducing the expression of Coronin 7, which is required for budding of Golgi-derived transport vesicles. In summary, UBR4/POE fulfills a conserved and unexpected role in the vesicular trafficking of neuropeptides, a function that has important implications for circadian clock synchrony and circuit-level signal processing. Although ubiquitin ligases are known to control clock protein degradation, their other roles in clock neurons are unclear. Here the authors report that UBR4 promotes export of neuropeptides from the Golgi for axonal trafficking, which is important for circadian clock synchrony in mice and flies.

The circadian clock drives daily rhythms in behavior and physiology. A recent study suggests that intestinal permeability is also under control of the circadian clock. However, the precise mechanisms remain largely unknown. Because intestinal permeability depends on tight junction (TJ) that regulates the epithelial paracellular pathway, this study investigated whether the circadian clock regulates the expression levels of TJ proteins in the intestine. The expression levels of TJ proteins in the large intestinal epithelium and colonic permeability were analyzed every 4, 6, or 12 hours between wild-type mice and mice with a mutation of a key clock gene Period2 (Per2; mPer2(m/m). In addition, the susceptibility to dextran sodium sulfate (DSS)-induced colitis was compared between wild-type mice and mPer2(m/m) mice. The mRNA and protein expression levels of Occludin and Claudin-1 exhibited daily variations in the colonic epithelium in wild-type mice, whereas they were constitutively high in mPer2(m/m) mice. Colonic permeability in wild-type mice exhibited daily variations, which was inversely associated with the expression levels of Occludin and Claudin-1 proteins, whereas it was constitutively low in mPer2(m/m) mice. mPer2(m/m) mice were more resistant to the colonic injury induced by DSS than wild-type mice. Occludin and Claudin-1 expressions in the large intestine are under the circadian control, which is associated with temporal regulation of colonic permeability and also susceptibility to colitis.

Either expression level or transcriptional activity of various nuclear receptors (NRs) have been demonstrated to be under circadian control. With a few exceptions, little is known about the roles of NRs as direct regulators of the circadian circuitry. Here we show that the nuclear receptor HNF4A strongly transrepresses the transcriptional activity of the CLOCK:BMAL1 heterodimer. We define a central role for HNF4A in maintaining cell-autonomous circadian oscillations in a tissue-specific manner in liver and colon cells. Not only transcript level but also genome-wide chromosome binding of HNF4A is rhythmically regulated in the mouse liver. ChIP-seq analyses revealed cooccupancy of HNF4A and CLOCK: BMAL1 at a wide array of metabolic genes involved in lipid, glucose, and amino acid homeostasis. Taken together, we establish that HNF4A defines a feedback loop in tissue-specific mammalian oscillators and demonstrate its recruitment in the circadian regulation of metabolic pathways.

Mammalian circadian rhythms are generated by a transcription-based feedback loop in which CLOCK:BMAL1 drives transcription of its repressors (PER1/2, CRY1/2), which ultimately interact with CLOCK:BMAL1 to close the feedback loop with ~24 hr periodicity. Here we pinpoint a key difference between CRY1 and CRY2 that underlies their differential strengths as transcriptional repressors. Both cryptochromes bind the BMAL1 transactivation domain similarly to sequester it from coactivators and repress CLOCK:BMAL1 activity. However, we find that CRY1 is recruited with much higher affinity to the PAS domain core of CLOCK:BMAL1, allowing it to serve as a stronger repressor that lengthens circadian period. We discovered a dynamic serine-rich loop adjacent to the secondary pocket in the photolyase homology region (PHR) domain that regulates differential binding of cryptochromes to the PAS domain core of CLOCK:BMAL1. Notably, binding of the co-repressor PER2 remodels the serine loop of CRY2, making it more CRY1-like and enhancing its affinity for CLOCK:BMAL1.

Obstructive sleep apnea (OSA) is associated with circadian rhythm dysregulation plausibly through affecting clock genes. The study’s purpose was to investigate the effect of one-night continuous positive airway pressure treatment (CPAP treatment) on circadian clock genes: BMAL1 , CLOCK , CRY1 , and PER1 at mRNA and protein levels. The study included 30 OSA patients, who underwent diagnostic polysomnography (PSG) and next a one-night effective CPAP treatment with PSG monitoring (CPAP). The blood was collected in the evening before and the morning after PSG and CPAP. Protein levels and mRNA expression were measured using ELISA and qRT-PCR, respectively. The increase in PER1 expression was observed in the morning after compared to the evening before CPAP ( p  = 0.005); additionally, PER1 protein level decreased in the morning after CPAP compared to the morning after PSG ( p  = 0.035). In CLOCK protein levels significant changes were observed: an increase in the morning after CPAP compared to the morning after PSG ( p  = 0.049), an increase in the morning after CPAP compared to the evening before ( p  = 0.006), and an increase in difference between the morning after and evening before CPAP vs. difference between morning after and evening before PSG ( p  = 0.012). Obtained results suggest that even short-term effective CPAP treatment might reverse circadian clock signaling pathway disruption in OSA.

The mammalian circadian clock consists of a transcription–translation feedback loop (TTFL) composed of CLOCK–BMAL1 transcriptional activators and CRY–PER transcriptional repressors. Previous work showed that CRY inhibits CLOCK–BMAL1-activated transcription by a “blocking”-type mechanism and that CRY–PER inhibits CLOCK–BMAL1 by a “displacement”-type mechanism. While the mechanism of CRY-mediated repression was explained by both in vitro and in vivo experiments, the CRY–PER-mediated repression in vivo seemed in conflict with the in vitro data demonstrating PER removes CRY from the CLOCK–BMAL1–E-box complex. Here, we show that CRY–PER participates in the displacement-type repression by recruiting CK1δ to the nucleus and mediating an increased local concentration of CK1δ at CLOCK–BMAL1-bound promoters/enhancers and thus promoting the phosphorylation of CLOCK and dissociation of CLOCK–BMAL1 along with CRY from the E-box. Our findings bring clarity to the role of PER in the dynamic nature of the repressive phase of the TTFL.