Macromolecular Assemblies of the Mammalian Circadian Clock.
ABSTRACT: The mammalian circadian clock is built on a feedback loop in which PER and CRY proteins repress their own transcription. We found that in mouse liver nuclei all three PERs, both CRYs, and Casein Kinase-1? (CK1?) are present together in an ?1.9-MDa repressor assembly that quantitatively incorporates its CLOCK-BMAL1 transcription factor target. Prior to incorporation, CLOCK-BMAL1 exists in an ?750-kDa complex. Single-particle electron microscopy (EM) revealed nuclear PER complexes purified from mouse liver to be quasi-spherical ?40-nm structures. In the cytoplasm, PERs, CRYs, and CK1? were distributed into several complexes of ?0.9-1.1 MDa that appear to constitute an assembly pathway regulated by GAPVD1, a cytoplasmic trafficking factor. Single-particle EM of two purified cytoplasmic PER complexes revealed ?20-nm and ?25-nm structures, respectively, characterized by flexibly tethered globular domains. Our results define the macromolecular assemblies comprising the circadian feedback loop and provide an initial structural view of endogenous eukaryotic clock machinery.
Project description:Mammalian circadian clocks are driven by transcription/translation feedback loops composed of positive transcriptional activators (BMAL1 and CLOCK) and negative repressors (CRYPTOCHROMEs (CRYs) and PERIODs (PERs)). CRYs, in complex with PERs, bind to the BMAL1/CLOCK complex and repress E-box-driven transcription of clock-associated genes. There are two individual CRYs, with CRY1 exhibiting higher affinity to the BMAL1/CLOCK complex than CRY2. It is known that this differential binding is regulated by a dynamic serine-rich loop adjacent to the secondary pocket of both CRYs, but the underlying features controlling loop dynamics are not known. Here we report that allosteric regulation of the serine-rich loop is mediated by Arg-293 of CRY1, identified as a rare <i>CRY1</i> SNP in the Ensembl and 1000 Genomes databases. The p.Arg293His CRY1 variant caused a shortened circadian period in a <i>Cry1</i> <sup>-/-</sup> <i>Cry2</i> <sup>-/-</sup> double knockout mouse embryonic fibroblast cell line. Moreover, the variant displayed reduced repressor activity on BMAL1/CLOCK driven transcription, which is explained by reduced affinity to BMAL1/CLOCK in the absence of PER2 compared with CRY1. Molecular dynamics simulations revealed that the p.Arg293His CRY1 variant altered a communication pathway between Arg-293 and the serine loop by reducing its dynamicity. Collectively, this study provides direct evidence that allosterism in CRY1 is critical for the regulation of circadian rhythm.
Project description:The mammalian circadian clock is a molecular oscillator composed of a feedback loop that involves transcriptional activators CLOCK and BMAL1, and repressors Cryptochrome (CRY) and Period (PER). Here we show that a direct CLOCK-BMAL1 target gene, Gm129, is a novel regulator of the feedback loop. ChIP analysis revealed that the CLOCK:BMAL1:CRY1 complex strongly occupies the promoter region of Gm129. Both mRNA and protein levels of GM129 exhibit high amplitude circadian oscillations in mouse liver, and Gm129 gene encodes a nuclear-localized protein that directly interacts with BMAL1 and represses CLOCK:BMAL1 activity. In vitro and in vivo protein-DNA interaction results demonstrate that, like CRY1, GM129 functions as a repressor by binding to the CLOCK:BMAL1 complex on DNA. Although Gm129-/- or Cry1-/- Gm129-/- mice retain a robust circadian rhythm, the peaks of Nr1d1 and Dbp mRNAs in liver exhibit significant phase delay compared to control. Our results suggest that, in addition to CRYs and PERs, GM129 protein contributes to the transcriptional feedback loop by modulating CLOCK:BMAL1 activity as a transcriptional repressor. Examination of 3 transcriptional regulators in mouse liver
Project description:Non-alcoholic fatty liver disease (NAFLD) is a common disease, which is characterized by the accumulation of triglycerides in the hepatocytes without excess alcohol intake. Circadian rhythms can participate in lipid, glucose, and cholesterol metabolism and are closely related to metabolism seen in this disease. Circadian clock genes can modulate liver lipid metabolism. Desynchrony of circadian rhythms and the influences imparted by external environmental stimuli can increase morbidity. By contrast, synchronizing circadian rhythms can help to alleviate the metabolic disturbance seen in NAFLD. In this review, we have discussed the current research connections that exist between the circadian clock and the metabolism of NAFLD, and we have specifically focused on the key circadian clock genes, Bmal1, Clock, Rev-Erbs, Rors, Pers, Crys, Nocturnin, and DECs.
Project description:To study the alterations of sleep quality and circadian rhythm genes expressions upon elderly thyroid nodule patients, the risk factors associated with thyroid malignancies, and the potential relationship involved. The elderly people enrolled in our study were divided into three groups according to the thyroid histopathology: malignant nodule group, benign nodule group, and normal group, and the clinical data and sleep quality were collected. Among the patients of surgery, 56 fresh thyroid tissues were collected for real-time PCR, immunohistochemistry and western blotting analysis of CLOCK, BMAL1, CRYs and PERs. Poor sleep quality, sleep latency and daytime dysfunction were the independent risk factors of malignant nodule after adjusted by other impacts. The expression levels of CLOCK, BMAL1 and PER2 in thyroid malignant group were significantly higher than benign and normal groups, while CRY2 was decreased, p < 0.05. In addition, CLOCK and BMAL1 protein levels were positively correlated with PSQI of corresponding patients and CRY2 was negatively correlated. Circadian rhythm genes mainly altered in malignant nodules, and sleep disorders may be involved in the occurrence of elderly thyroid malignancy through the high expressions of CLOCK and BMAL1, and low expression of CRY2.
Project description:The mammalian molecular clock is based on a transcription-translation feedback loop (TTFL) comprising the Period1, 2 (Per1, 2), Cryptochrome1, 2 (Cry1, 2), and Brain and Muscle ARNT-Like 1 (Bmal1) genes. The robustness of the TTFL is attributed to genetic redundancy among some essential clock genes, deterring genetic studies on molecular clocks using genome editing targeting single genes. To manipulate multiple clock genes in a streamlined and efficient manner, we developed a CRISPR-Cas9-based single adeno-associated viral (AAV) system targeting the circadian clock (CSAC) for essential clock genes including Pers, Crys, or Bmal1. First, we tested several single guide RNAs (sgRNAs) targeting individual clock genes in silico and validated their efficiency in Neuro2a cells. To target multiple genes, multiplex sgRNA plasmids were constructed using Golden Gate assembly and packaged into AAVs. CSAC efficiency was evident through protein downregulation in vitro and ablated molecular oscillation ex vivo. We also measured the efficiency of CSAC in vivo by assessing circadian rhythms after injecting CSAC into the suprachiasmatic nuclei of Cas9-expressing knock-in mice. Circadian locomotor activity and body temperature rhythms were severely disrupted in these mice, indicating that our CSAC is a simple yet powerful tool for investigating the molecular clock in vivo.
Project description:Numerous physiological processes of mammals, including bone metabolism, are regulated by the circadian clock system, which consists of a central regulator, the suprachiasmatic nucleus (SCN), and the peripheral oscillators of the BMAL1/CLOCK-PERs/CRYs system. Various bone turnover markers and bone metabolism-regulating hormones such as melatonin and parathyroid hormone (PTH) display diurnal rhythmicity. According to previous research, disruption of the circadian clock due to shift work, sleep restriction, or clock gene knockout is associated with osteoporosis or other abnormal bone metabolism, showing the importance of the circadian clock system for maintaining homeostasis of bone metabolism. Moreover, common causes of osteoporosis, including postmenopausal status and aging, are associated with changes in the circadian clock. In our previous research, we found that agonism of the circadian regulators REV-ERBs inhibits osteoclast differentiation and ameliorates ovariectomy-induced bone loss in mice, suggesting that clock genes may be promising intervention targets for abnormal bone metabolism. Moreover, osteoporosis interventions at different time points can provide varying degrees of bone protection, showing the importance of accounting for circadian rhythms for optimal curative effects in clinical treatment of osteoporosis. In this review, we summarize current knowledge about circadian rhythms and bone metabolism.
Project description:The mammalian circadian clock is a molecular oscillator composed of a feedback loop that involves transcriptional activators CLOCK and BMAL1, and repressors Cryptochrome (CRY) and Period (PER). Here we show that a direct CLOCK·BMAL1 target gene, Gm129, is a novel regulator of the feedback loop. ChIP analysis revealed that the CLOCK·BMAL1·CRY1 complex strongly occupies the promoter region of Gm129. Both mRNA and protein levels of GM129 exhibit high amplitude circadian oscillations in mouse liver, and Gm129 gene encodes a nuclear-localized protein that directly interacts with BMAL1 and represses CLOCK·BMAL1 activity. In vitro and in vivo protein-DNA interaction results demonstrate that, like CRY1, GM129 functions as a repressor by binding to the CLOCK·BMAL1 complex on DNA. Although Gm129(-/-) or Cry1(-/-) Gm129(-/-) mice retain a robust circadian rhythm, the peaks of Nr1d1 and Dbp mRNAs in liver exhibit a significant phase delay compared with control. Our results suggest that, in addition to CRYs and PERs, the GM129 protein contributes to the transcriptional feedback loop by modulating CLOCK·BMAL1 activity as a transcriptional repressor.
Project description:In the negative feedback loop driving fungal and animal circadian oscillators, negative elements (FREQUENCY [FRQ], PERIODS [PERs], and CRYPTOCHROMES [CRYs]) are understood to inhibit their own expression, in part by promoting the phosphorylation of their heterodimeric transcriptional activators (e.g., White Collar-1 [WC-1]-WC-2 [White Collar complex; WCC] and BMAL1/Circadian Locomotor Output Cycles Kaput [CLOCK]). However, correlations between heterodimer activity and phosphorylation are weak, contradictions exist, and mechanistic details are almost wholly lacking. We report mapping of 80 phosphosites on WC-1 and 15 on WC-2 and elucidation of the time-of-day-specific code, requiring both a group of phosphoevents on WC-1 and two distinct clusters on WC-2, that governs circadian repression, leading to feedback loop closure. Combinatorial control via phosphorylation also governs rhythmic WCC binding to the promoters of clock-controlled genes mediating the essential first step in circadian output, a group encoding both transcription factors and signaling proteins. These data provide a basic mechanistic understanding for fundamental events underlying circadian negative feedback and output, key aspects of circadian biology.
Project description:The circadian clock controls the expression of nearly 50% of protein coding genes in mice and most likely in humans as well. Therefore, disruption of the circadian clock is presumed to have serious pathological effects including cancer. However, epidemiological studies on individuals with circadian disruption because of night shift or rotating shift work have produced contradictory data not conducive to scientific consensus as to whether circadian disruption increases the incidence of breast, ovarian, prostate, or colorectal cancers. Similarly, genetically engineered mice with clock disruption do not exhibit spontaneous or radiation-induced cancers at higher incidence than wild-type controls. Because many cellular functions including the cell cycle and cell division are, at least in part, controlled by the molecular clock components (CLOCK, BMAL1, CRYs, PERs), it has also been expected that appropriate timing of chemotherapy may increase the efficacy of chemotherapeutic drugs and ameliorate their side effect. However, empirical attempts at chronochemotherapy have not produced beneficial outcomes. Using mice without and with human tumor xenografts, sites of DNA damage and repair following treatment with the anticancer drug cisplatin have been mapped genome-wide at single nucleotide resolution and as a function of circadian time. The data indicate that mechanism-based studies such as these may provide information necessary for devising rational chronochemotherapy regimens.
Project description:Robust rhythms of abundances and phosphorylation profiles of PERIOD proteins were thought be the master rhythms that drive mammalian circadian clock functions. PER stability was proposed to be a major determinant of period length. In mammals, CK1 forms stable complexes with PER. Here we identify the PER residues essential for PER-CK1 interaction. In cells and in mice, their mutation abolishes PER phosphorylation and CLOCK hyperphosphorylation, resulting in PER stabilization, arrhythmic PER abundance and impaired negative feedback process, indicating that PER acts as the CK1 scaffold in circadian feedback mechanism. Surprisingly, the mutant mice exhibit robust short period locomotor activity and other physiological rhythms but low amplitude molecular rhythms. PER-CK1 interaction has two opposing roles in regulating CLOCK-BMAL1 activity. These results indicate that the circadian clock can function independently of PER phosphorylation and abundance rhythms due to another PER-CRY-dependent feedback mechanism and that period length can be uncoupled from PER stability.