Regulation of BMAL1 Protein Stability and Circadian Function by GSK3β-Mediated Phosphorylation

Circadian rhythms govern a large array of physiological and metabolic functions. To achieve plasticity in circadian regulation, proteins constituting the molecular clock machinery undergo various post-translational modifications (PTMs), which influence their activity and intracellular localization. The core clock protein BMAL1 undergoes several PTMs. Here we report that the Akt-GSK3beta signaling pathway regulates BMAL1 protein stability and activity.GSK3beta phosphorylates BMAL1 specifically on Ser 17 and Thr 21 and primes it for ubiquitylation. In the absence of GSK3beta-mediated phosphorylation, BMAL1 becomes stabilized and BMAL1 dependent circadian gene expression is dampened. Dopamine D2 receptor mediated signaling, known to control the Akt-GSK3beta pathway, influences BMAL1 stability and in vivo circadian gene expression in striatal neurons.These findings uncover a previously unknown mechanism of circadian clock control. The GSK3beta kinase phosphorylates BMAL1, an event that controls the stability of the protein and the amplitude of circadian oscillation. BMAL1 phosphorylation appears to be an important regulatory step in maintaining the robustness of the circadian clock

Dynamic circadian protein-protein interaction networks predict temporal organization of cellular functions.

Essentially all biological processes depend on protein-protein interactions (PPIs). Timing of such interactions is crucial for regulatory function. Although circadian (~24-hour) clocks constitute fundamental cellular timing mechanisms regulating important physiological processes, PPI dynamics on this timescale are largely unknown. Here, we identified 109 novel PPIs among circadian clock proteins via a yeast-two-hybrid approach. Among them, the interaction of protein phosphatase 1 and CLOCK/BMAL1 was found to result in BMAL1 destabilization. We constructed a dynamic circadian PPI network predicting the PPI timing using circadian expression data. Systematic circadian phenotyping (RNAi and overexpression) suggests a crucial role for components involved in dynamic interactions. Systems analysis of a global dynamic network in liver revealed that interacting proteins are expressed at similar times likely to restrict regulatory interactions to specific phases. Moreover, we predict that circadian PPIs dynamically connect many important cellular processes (signal transduction, cell cycle, etc.) contributing to temporal organization of cellular physiology in an unprecedented manner

A Large Scale shRNA Barcode Screen Identifies the Circadian Clock Component ARNTL as Putative Regulator of the p53 Tumor Suppressor Pathway

BACKGROUND: The p53 tumor suppressor gene is mutated in about half of human cancers, but the p53 pathway is thought to be functionally inactivated in the vast majority of cancer. Understanding how tumor cells can become insensitive to p53 activation is therefore of major importance. Using an RNAi-based genetic screen, we have identified three novel genes that regulate p53 function. RESULTS: We have screened the NKI shRNA library targeting 8,000 human genes to identify modulators of p53 function. Using the shRNA barcode technique we were able to quickly identify active shRNA vectors from a complex mixture. Validation of the screening results indicates that the shRNA barcode technique can reliable identify active shRNA vectors from a complex pool. Using this approach we have identified three genes, ARNTL, RBCK1 and TNIP1, previously unknown to regulate p53 function. Importantly, ARNTL (BMAL1) is an established component of the circadian regulatory network. The latter finding adds to recent observations that link circadian rhythm to the cell cycle and cancer. We show that cells having suppressed ARNTL are unable to arrest upon p53 activation associated with an inability to activate the p53 target gene p21(CIP1). CONCLUSIONS: We identified three new regulators of the p53 pathway through a functional genetic screen. The identification of the circadian core component ARNTL strengthens the link between circadian rhythm and cancer

Circadian Clock Genes Contribute to the Regulation of Hair Follicle Cycling

Hair follicles undergo recurrent cycling of controlled growth (anagen), regression (catagen), and relative quiescence (telogen) with a defined periodicity. Taking a genomics approach to study gene expression during synchronized mouse hair follicle cycling, we discovered that, in addition to circadian fluctuation, CLOCK–regulated genes are also modulated in phase with the hair growth cycle. During telogen and early anagen, circadian clock genes are prominently expressed in the secondary hair germ, which contains precursor cells for the growing follicle. Analysis of Clock and Bmal1 mutant mice reveals a delay in anagen progression, and the secondary hair germ cells show decreased levels of phosphorylated Rb and lack mitotic cells, suggesting that circadian clock genes regulate anagen progression via their effect on the cell cycle. Consistent with a block at the G1 phase of the cell cycle, we show a significant upregulation of p21 in Bmal1 mutant skin. While circadian clock mechanisms have been implicated in a variety of diurnal biological processes, our findings indicate that circadian clock genes may be utilized to modulate the progression of non-diurnal cyclic processes

Deficient of a Clock Gene, Brain and Muscle Arnt-Like Protein-1 (BMAL1), Induces Dyslipidemia and Ectopic Fat Formation

A link between circadian rhythm and metabolism has long been discussed. Circadian rhythm is controlled by positive and negative transcriptional and translational feedback loops composed of several clock genes. Among clock genes, the brain and muscle Arnt-like protein-1 (BMAL1) and circadian locomotor output cycles kaput (CLOCK) play important roles in the regulation of the positive rhythmic transcription. In addition to control of circadian rhythm, we have previously shown that BMAL1 regulates adipogenesis. In metabolic syndrome patients, the function of BMAL1 is dysregulated in visceral adipose tissue. In addition, analysis of SNPs has revealed that BMAL1 is associated with susceptibility to hypertension and type II diabetes. Furthermore, the significant roles of BMAL1 in pancreatic β cells proliferation and maturation were recently reported. These results suggest that BMAL1 regulates energy homeostasis. Therefore, in this study, we examined whether loss of BMAL1 function is capable of inducing metabolic syndrome. Deficient of the Bmal1 gene in mice resulted in elevation of the respiratory quotient value, indicating that BMAL1 is involved in the utilization of fat as an energy source. Indeed, lack of Bmal1 reduced the capacity of fat storage in adipose tissue, resulting in an increase in the levels of circulating fatty acids, including triglycerides, free fatty acids, and cholesterol. Elevation of the circulating fatty acids level induced the formation of ectopic fat in the liver and skeletal muscle in Bmal1 -/- mice. Interestingly, ectopic fat formation was not observed in tissue-specific (liver or skeletal muscle) Bmal1 -/- mice even under high fat diet feeding condition. Therefore, we were led to conclude that BMAL1 is a crucial factor in the regulation of energy homeostasis, and disorders of the functions of BMAL1 lead to the development of metabolic syndrome

Timeless Links Replication Termination to Mitotic Kinase Activation

The mechanisms that coordinate the termination of DNA replication with progression through mitosis are not completely understood. The human Timeless protein (Tim) associates with S phase replication checkpoint proteins Claspin and Tipin, and plays an important role in maintaining replication fork stability at physical barriers, like centromeres, telomeres and ribosomal DNA repeats, as well as at termination sites. We show here that human Tim can be isolated in a complex with mitotic entry kinases CDK1, Auroras A and B, and Polo-like kinase (Plk1). Plk1 bound Tim directly and colocalized with Tim at a subset of mitotic structures in M phase. Tim depletion caused multiple mitotic defects, including the loss of sister-chromatid cohesion, loss of mitotic spindle architecture, and a failure to exit mitosis. Tim depletion caused a delay in mitotic kinase activity in vivo and in vitro, as well as a reduction in global histone H3 S10 phosphorylation during G2/M phase. Tim was also required for the recruitment of Plk1 to centromeric DNA and formation of catenated DNA structures at human centromere alpha satellite repeats. Taken together, these findings suggest that Tim coordinates mitotic kinase activation with termination of DNA replication

Body weight, metabolism and clock genes

Biological rhythms are present in the lives of almost all organisms ranging from plants to more evolved creatures. These oscillations allow the anticipation of many physiological and behavioral mechanisms thus enabling coordination of rhythms in a timely manner, adaption to environmental changes and more efficient organization of the cellular processes responsible for survival of both the individual and the species. Many components of energy homeostasis exhibit circadian rhythms, which are regulated by central (suprachiasmatic nucleus) and peripheral (located in other tissues) circadian clocks. Adipocyte plays an important role in the regulation of energy homeostasis, the signaling of satiety and cellular differentiation and proliferation. Also, the adipocyte circadian clock is probably involved in the control of many of these functions. Thus, circadian clocks are implicated in the control of energy balance, feeding behavior and consequently in the regulation of body weight. In this regard, alterations in clock genes and rhythms can interfere with the complex mechanism of metabolic and hormonal anticipation, contributing to multifactorial diseases such as obesity and diabetes. The aim of this review was to define circadian clocks by describing their functioning and role in the whole body and in adipocyte metabolism, as well as their influence on body weight control and the development of obesity

Disrupting Circadian Homeostasis of Sympathetic Signaling Promotes Tumor Development in Mice

and why disruption of circadian rhythm may lead to tumorigenesis. oncogenic potential, leading to tumor development in the same organ systems in wild-type and circadian gene-mutant mice. is a clock-controlled physiological function. The central circadian clock paces extracellular mitogenic signals that drive peripheral clock-controlled expression of key cell cycle and tumor suppressor genes to generate a circadian rhythm in cell proliferation. Frequent disruption of circadian rhythm is an important tumor promoting factor

Lithium and GSK3-β promoter gene variants influence white matter microstructure in bipolar disorder

Lithium is the mainstay for the treatment of bipolar disorder (BD) and inhibits glycogen synthase kinase 3-β (GSK3-β). The less active GSK3-β promoter gene variants have been associated with less detrimental clinical features of BD. GSK3-β gene variants and lithium can influence brain gray matter structure in psychiatric conditions. Diffusion tensor imaging (DTI) measures of white matter (WM) integrity showed widespred disruption of WM structure in BD. In a sample of 70 patients affected by a major depressive episode in course of BD, we investigated the effect of ongoing long-term lithium treatment and GSK3-β promoter rs334558 polymorphism on WM microstructure, using DTI and tract-based spatial statistics with threshold-free cluster enhancement. We report that the less active GSK3-β rs334558*C gene-promoter variants, and the long-term administration of the GSK3-β inhibitor lithium, were associated with increases of DTI measures of axial diffusivity (AD) in several WM fiber tracts, including corpus callosum, forceps major, anterior and posterior cingulum bundle (bilaterally including its hippocampal part), left superior and inferior longitudinal fasciculus, left inferior fronto-occipital fasciculus, left posterior thalamic radiation, bilateral superior and posterior corona radiata, and bilateral corticospinal tract. AD reflects the integrity of axons and myelin sheaths. We suggest that GSK3-β inhibition and lithium could counteract the detrimental influences of BD on WM structure, with specific benefits resulting from effects on specific WM tracts contributing to the functional integrity of the brain and involving interhemispheric, limbic, and large frontal, parietal, and fronto-occipital connections

Global loss of Bmal1 expression alters adipose tissue hormones, gene expression and glucose metabolism

Extent: 11p.The close relationship between circadian rhythm disruption and poor metabolic status is becoming increasingly evident, but role of adipokines is poorly understood. Here we investigated adipocyte function and the metabolic status of mice with a global loss of the core clock gene Bmal1 fed either a normal or a high fat diet (22% by weight). Bmal1 null mice aged 2 months were killed across 24 hours and plasma adiponectin and leptin, and adipose tissue expression of Adipoq, Lep, Retn and Nampt mRNA measured. Glucose, insulin and pyruvate tolerance tests were conducted and the expression of liver glycolytic and gluconeogenic enzyme mRNA determined. Bmal1 null mice displayed a pattern of increased plasma adiponectin and plasma leptin concentrations on both control and high fat diets. Bmal1 null male and female mice displayed increased adiposity (1.8 fold and 2.3 fold respectively) on the normal diet, but the high fat diet did not exaggerate these differences. Despite normal glucose and insulin tolerance, Bmal1 null mice had increased production of glucose from pyruvate, implying increased liver gluconeogenesis. The Bmal1 null mice had arrhythmic clock gene expression in epigonadal fat and liver, and loss of rhythmic transcription of a range of metabolic genes. Furthermore, the expression of epigonadal fat Adipoq, Retn, Nampt, AdipoR1 and AdipoR2 and liver Pfkfb3 mRNA were down-regulated. These results show for the first time that global loss of Bmal1, and the consequent arrhythmicity, results in compensatory changes in adipokines involved in the cellular control of glucose metabolism.David John Kennaway, Tamara Jayne Varcoe, Athena Voultsios, Michael James Bode