Quantification of protein abundance and interaction defines a mechanism for operation of the circadian clock
- University of Manchester, United Kingdom
- Medical Research Council, United Kingdom
- University of California, Santa Cruz, United States
Abstract
The mammalian circadian clock exerts control of daily gene expression through cycles of DNA binding. Here we develop a quantitative model of how a finite pool of BMAL1 protein can regulate thousands of target sites over daily time scales. We used quantitative imaging to track dynamic changes in endogenous labelled proteins across peripheral tissues and the SCN. We determine the contribution of multiple rhythmic processes coordinating BMAL1 DNA binding, including cycling molecular abundance, binding affinities and repression. We find nuclear BMAL1 concentration determines corresponding CLOCK through heterodimerization and define a DNA residence time of this complex. Repression of CLOCK:BMAL1 is achieved through rhythmic changes to BMAL1:CRY1 association and high affinity interactions between PER2:CRY1 which mediates CLOCK:BMAL1 displacement from DNA. Finally, stochastic modelling reveals a dual role for PER:CRY complexes in which increasing concentrations of PER2:CRY1 promotes removal of BMAL1:CLOCK from genes consequently enhancing ability to move to new target sites.
Data availability
Modelling and analtyical code has been made publicly available via GitHub. The FCS analysis software is at https://github.com/LoudonLab/FcsAnalysisPipeline and the modeling link is https://github.com/LoudonLab/CLOCK-BMAL1-DNA-Binding.Source Data files have been provided for all FCS measurements and FRAP measurements in Figures 1, 2, 3 ,4, and 6.
Article and author information
Author details
Funding
Biotechnology and Biological Sciences Research Council (BB/P017347/1)
- James S Bagnall
- Nicola Begley
- Andrew SI Loudon
Biotechnology and Biological Sciences Research Council (BB/P017355/1)
- Nicola J Smyllie
- Michael H Hastings
Medical Research Council (MC_U105170643)
- Michael H Hastings
National Institutes of Health (GM107069)
- Carrie L Partch
National Institutes of Health (GM141849)
- Carrie L Partch
Wellcome Trust (107851/Z/15/Z)
- Andrew SI Loudon
Wellcome Trust (216416/Z/19/Z)
- Alex Ashton Koch
University of California
- Jennifer L Fribourgh
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Ethics
Animal experimentation: All experimental procedures were carried out in accordance with the Animals (Scientific Procedures) Act of 1986, UK (License number PP7901495).
Copyright
© 2022, Koch et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
Metrics
-
- 1,990
- views
-
- 327
- downloads
-
- 28
- citations
Views, downloads and citations are aggregated across all versions of this paper published by eLife.
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Quantification of protein abundance and interaction defines a mechanism for operation of the circadian clock
eLife 11:e73976.
https://doi.org/10.7554/eLife.73976
Further reading
-
- Cell Biology
G protein-coupled receptors (GPCRs) are integral membrane proteins which closely interact with their plasma membrane lipid microenvironment. Cholesterol is a lipid enriched at the plasma membrane with pivotal roles in the control of membrane fluidity and maintenance of membrane microarchitecture, directly impacting on GPCR stability, dynamics, and function. Cholesterol extraction from pancreatic beta cells has previously been shown to disrupt the internalisation, clustering, and cAMP responses of the glucagon-like peptide-1 receptor (GLP-1R), a class B1 GPCR with key roles in the control of blood glucose levels via the potentiation of insulin secretion in beta cells and weight reduction via the modulation of brain appetite control centres. Here, we unveil the detrimental effect of a high cholesterol diet on GLP-1R-dependent glucoregulation in vivo, and the improvement in GLP-1R function that a reduction in cholesterol synthesis using simvastatin exerts in pancreatic islets. We next identify and map sites of cholesterol high occupancy and residence time on active vs inactive GLP-1Rs using coarse-grained molecular dynamics (cgMD) simulations, followed by a screen of key residues selected from these sites and detailed analyses of the effects of mutating one of these, Val229, to alanine on GLP-1R-cholesterol interactions, plasma membrane behaviours, clustering, trafficking and signalling in INS-1 832/3 rat pancreatic beta cells and primary mouse islets, unveiling an improved insulin secretion profile for the V229A mutant receptor. This study (1) highlights the role of cholesterol in regulating GLP-1R responses in vivo; (2) provides a detailed map of GLP-1R - cholesterol binding sites in model membranes; (3) validates their functional relevance in beta cells; and (4) highlights their potential as locations for the rational design of novel allosteric modulators with the capacity to fine-tune GLP-1R responses.
-
- Cell Biology
- Immunology and Inflammation
Macrophages are crucial in the body’s inflammatory response, with tightly regulated functions for optimal immune system performance. Our study reveals that the RAS–p110α signalling pathway, known for its involvement in various biological processes and tumourigenesis, regulates two vital aspects of the inflammatory response in macrophages: the initial monocyte movement and later-stage lysosomal function. Disrupting this pathway, either in a mouse model or through drug intervention, hampers the inflammatory response, leading to delayed resolution and the development of more severe acute inflammatory reactions in live models. This discovery uncovers a previously unknown role of the p110α isoform in immune regulation within macrophages, offering insight into the complex mechanisms governing their function during inflammation and opening new avenues for modulating inflammatory responses.
