ABSTRACT
For many years, a connection between circadian clocks and cancer has been postulated. Here we describe an unexpected function for the circadian repressor CRY2 as a component of an FBXL3-containing E3 ligase that recruits T58-phosphorylated c-MYC for ubiquitylation. c-MYC is a critical regulator of cell proliferation; T58 is central in a phosphodegron long recognized as a hotspot for mutation in cancer. This site is also targeted by FBXW7, although the full machinery responsible for its turnover has remained obscure. CRY1 cannot substitute for CRY2 in promoting c-MYC degradation. Their unique functions may explain prior conflicting reports that have fueled uncertainty about the relationship between clocks and cancer. We demonstrate that c-MYC is a target of CRY2-dependent protein turnover, suggesting a molecular mechanism for circadian control of cell growth and a new paradigm for circadian protein degradation.
Subject(s)
Cell Transformation, Neoplastic/genetics , Circadian Clocks/genetics , Cryptochromes/genetics , F-Box Proteins/genetics , Gene Expression Regulation, Neoplastic , Lymphoma/genetics , Proto-Oncogene Proteins c-myc/genetics , Animals , Carrier Proteins/chemistry , Carrier Proteins/genetics , Carrier Proteins/metabolism , Cell Transformation, Neoplastic/metabolism , Cell Transformation, Neoplastic/pathology , Circadian Rhythm/genetics , Cryptochromes/chemistry , Cryptochromes/metabolism , Cullin Proteins/chemistry , Cullin Proteins/genetics , Cullin Proteins/metabolism , F-Box Proteins/chemistry , F-Box Proteins/metabolism , Fibroblasts , HEK293 Cells , Humans , Lymphoma/metabolism , Lymphoma/mortality , Lymphoma/pathology , Mice , Mice, Knockout , Models, Molecular , Protein Stability , Protein Structure, Secondary , Proteolysis , Proto-Oncogene Proteins c-myc/chemistry , Proto-Oncogene Proteins c-myc/metabolism , S-Phase Kinase-Associated Proteins/chemistry , S-Phase Kinase-Associated Proteins/genetics , S-Phase Kinase-Associated Proteins/metabolism , Signal Transduction , Survival AnalysisABSTRACT
Cellular metabolite balance and mitochondrial function are under circadian control, but the pathways connecting the molecular clock to these functions are unclear. Peroxisome proliferator-activated receptor delta (PPARδ) enables preferential utilization of lipids as fuel during exercise and is a major driver of exercise endurance. We show here that the circadian repressors CRY1 and CRY2 function as co-repressors for PPARδ. Cry1-/-;Cry2-/- myotubes and muscles exhibit elevated expression of PPARδ target genes, particularly in the context of exercise. Notably, CRY1/2 seem to repress a distinct subset of PPARδ target genes in muscle compared to the co-repressor NCOR1. In vivo, genetic disruption of Cry1 and Cry2 enhances sprint exercise performance in mice. Collectively, our data demonstrate that CRY1 and CRY2 modulate exercise physiology by altering the activity of several transcription factors, including CLOCK/BMAL1 and PPARδ, and thereby alter energy storage and substrate selection for energy production.
Subject(s)
Cryptochromes/metabolism , PPAR delta/metabolism , Physical Conditioning, Animal , Animals , Cells, Cultured , Cryptochromes/genetics , Gene Deletion , Gene Expression Regulation , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Muscles/physiology , Protein Interaction MapsABSTRACT
Metformin is widely used in the treatment of type 2 diabetes to lower blood glucose. Although metformin is a relatively safe and effective drug, its clinical efficacy is variable and under certain circumstances it may contribute to life-threatening lactic acidosis. Thus, additional understanding of metformin pharmacokinetics and pharmacodynamics could provide important information regarding therapeutic use of this widely prescribed drug. Here we report a significant effect of time of day on acute blood glucose reduction in response to metformin administration and on blood lactate levels in healthy mice. Furthermore, we demonstrate that while metformin transport into hepatocytes is unaltered by time of day, the kinetics of metformin-induced activation of AMP-activated protein kinase (AMPK) in the liver are remarkably altered with circadian time. Liver-specific ablation of Bmal1 expression alters metformin induction of AMPK and blood glucose response but does not completely abolish time of day differences. Together, these data demonstrate that circadian rhythms affect the biological responses to metformin in a complex manner.
Subject(s)
Circadian Clocks/drug effects , Liver/drug effects , Liver/physiology , Metformin/administration & dosage , AMP-Activated Protein Kinases , Animals , Blood Glucose/drug effects , Circadian Rhythm/drug effects , Diabetes Mellitus, Type 2/drug therapy , Hepatocytes/drug effects , Hepatocytes/physiology , Lactates/blood , Male , Mice , Protein Serine-Threonine KinasesABSTRACT
The circadian transcriptional repressors cryptochrome 1 (Cry1) and 2 (Cry2) evolved from photolyases, bacterial light-activated DNA repair enzymes. In this study, we report that while they have lost DNA repair activity, Cry1/2 adapted to protect genomic integrity by responding to DNA damage through posttranslational modification and coordinating the downstream transcriptional response. We demonstrate that genotoxic stress stimulates Cry1 phosphorylation and its deubiquitination by Herpes virus associated ubiquitin-specific protease (Hausp, a.k.a Usp7), stabilizing Cry1 and shifting circadian clock time. DNA damage also increases Cry2 interaction with Fbxl3, destabilizing Cry2. Thus, genotoxic stress increases the Cry1/Cry2 ratio, suggesting distinct functions for Cry1 and Cry2 following DNA damage. Indeed, the transcriptional response to genotoxic stress is enhanced in Cry1-/- and blunted in Cry2-/- cells. Furthermore, Cry2-/- cells accumulate damaged DNA. These results suggest that Cry1 and Cry2, which evolved from DNA repair enzymes, protect genomic integrity via coordinated transcriptional regulation.