Circadian clock | EPFL Graph Search

A circadian clock, or circadian oscillator, is a biochemical oscillator that cycles with a stable phase and is synchronized with solar time. Such a clock's in vivo period is necessarily almost exactly 24 hours (the earth's current solar day). In most living things, internally synchronized circadian clocks make it possible for the organism to anticipate daily environmental changes corresponding with the day–night cycle and adjust its biology and behavior accordingly. The term circadian derives from the Latin circa (about) dies (a day), since when taken away from external cues (such as environmental light), they do not run to exactly 24 hours. Clocks in humans in a lab in constant low light, for example, will average about 24.2 hours per day, rather than 24 hours exactly. The normal body clock oscillates with an endogenous period of exactly 24 hours, it entrains, when it receives sufficient daily corrective signals from the environment, primarily daylight and darkness. Circadian cloc

On the Relationship Between Protein-DNA Interactions and Circadian Gene Expression in Mouse Liver

Guillaume Rey

Virtually every cell in our body tracks daily time using an endogenous mechanism called the circadian clock, which has a period of about 24 hours. This timing system offers a selective advantage by enabling organisms to anticipate periodic changes in environmental conditions, such as light-dark cycles. As a consequence, most behavioral and physiological processes including sleep/wake patterns, body temperature and blood pressure are influenced and thus occur at the appropriate time of the day. Such molecular oscillators have been identified almost over the full tree of life, ranging from bacteria to human, as well as insects, fungi and plants. In the mouse liver, the circadian system has been involved in several processes such as glucose homeostasis, cholesterol biosynthesis and gating of the cell cycle. At the molecular level, the circadian clock uses interlocked feedback loops in which the heterodimeric transcription factor BMAL1/CLOCK in mammals or CLK/CYC in flies, plays a central role. In this transcriptional regulatory network, BMAL1/CLOCK activates via E-box motifs transcription of several repressors: the PER and CRY proteins, which repress their own activation by BMAL1/CLOCK, and the nuclear receptor REV-ERB??, which negatively regulates the transcription of the Bmal1 gene. BMAL1/CLOCK also drives rhythmic transcription of PAR-bZIP transcription factors such as DBP/HLF/TEF, which allow for phase-specific expression of many metabolic genes in the liver. While there is prominent control of physiological and behavioral functions by the circadian clock, the detailed links between circadian regulators and downstream targets are poorly known. Thus the main goal of this research was to apply combined experimental and computational methods to further elucidate these interactions, both in flies and in mammals. Identifying the targets of the heterodimeric CLK/CYC (or BMAL1/CLOCK in mammals) basic-helix-loop-helix (bHLH) transcription factor poses challenges and it has been difficult to decipher its specific sequence affinity beyond a canonical E-box motif. We used a comparative genomics approach to identify and model CLK/CYC bound enhancers. The presence of two highly conserved tandem E-box motifs (E1-E2) was detected among CLK/CYC targets genes in flies. A probabilistic model was derived from these sequences and validated with functional genomics datasets. A phylogenetic analysis showed that this motif is evolutionarily conserved among mammals, fishes and insects. Subsequently, we developed computational and experimental methods aimed at defining the target genes of BMAL1/CLOCK in a complex tissue such as the mouse liver. To this end, we mapped all DNA-binding sites of BMAL1 during one circadian cycle using chromatin immunoprecipitation combined with deep sequencing (ChIP-Seq). As a control experiment, we performed a ChIP-Seq experiment for the heterodimeric partner, CLOCK. We developed a novel deconvolution-based method for optimal detection of binding sites from ChIP-Seq data and implemented a bioinformatics pipeline to establish the relationship between transcription factor binding and mRNA expression. Our computational analysis revealed widespread daily rhythms in DNA binding, with maximum levels peaking at midday. In the list of candidate targets, genes belonging to the core circadian clockwork stood out as the most strongly bound, often showing multiple binding sites. In addition, BMAL1 targets were highly enriched in genes involved in carbohydrate and lipid metabolism, cancer pathways, and also in transcription factors, in particular nuclear receptors. Thus, our results show that BMAL1 directly controls precise processes such as glucose homeostasis and, at the same time, uses other transcriptional regulators to spread the circadian signal to a large number of indirect targets. Additionally, we characterized the DNA-binding specificity of BMAL1 using a combination of biochemical and bioinformatics methods. Genomic sequence analysis of the sites using Hidden Markov Models (HMMs) identified E-boxes and tandem E-box (E1-E2) consensus elements. Interestingly, we found that strongly bound BMAL1 sites exhibit high phylogenetic conservation among placental mammals, which was even more pronounced in core circadian genes. Electromobility shift assays showed that E1-E2 sites are bound by a dimer of BMAL1/CLOCK heterodimers with a spacing-dependent cooperative interaction, a finding that was further validated in transactivation assays. A highly significant fraction of BMAL1 target genes showed cyclic mRNA expression profiles with a phase distribution delayed by about 4 hours compared to the binding. Importantly, sites with E1-E2 elements showed tighter phase distribution in mRNA accumulation. These analyses and experiments revealed the importance of tandem E1-E2 elements, which may favor strong binding and precise timing of daily gene expression. Finally, analyzing the temporal profiles of BMAL1 binding, precursor mRNA and mature mRNA levels showed how transcriptional and posttranscriptional regulation contribute differentially to circadian expression phase. Taken together, our interdisciplinary approach clearly established BMAL1/CLOCK's primary function as a master regulator of the core circadian oscillator, while demonstrating also its direct contribution to a variety of output programs. Thus our results may help to better understand metabolic diseases linked to dysfunctions of the circadian clockwork. Moreover, a more precise description of the clock-controlled liver functions might be also useful in chronotherapy in order to improve effectiveness and tolerance of drugs by treating patients at the right moment of the day.

EPFL2011

Circadian dynamics of RNA localisation in the mammalian liver

Clémence Yumie Syloun Hurni

Gene expression in eukaryotes is a complex multi-step process. It starts in the nucleus with transcription, the synthesis of a mRNA copy from a DNA template. While in the nucleus, the RNA transcript is subject to multiple co- and post-transcriptional modifications, including splicing, capping, polyadenylation, and assembly into ribonucleoprotein complexes. Correctly processed mRNA is exported to the cytoplasm and translated by ribosomes to form a chain of amino acids: the protein. The mRNA lifetime in the cytoplasm is determined by the activity of a distinct set of RNA Binding Proteins, enzymes, and functional RNAs, which promote stability or degradation. Each step of the RNA life cycle is tightly regulated to ensure proper cellular function. The kinetic rates governing these steps are dynamic and notably adapt to the daily fluctuations of the environment related to the alternance of day and night. Indeed, most organisms possess an internal timing system called the circadian clock. The clock is a genetically encoded self-sustained transcriptional-translational feedback loop, which operates in almost every cell and tissue of the body. It controls the temporal gene expression program to synchronise cellular and physiological functions to the external world. In this thesis, I explore the transcriptome of the mouse liver by combining RNA-sequencing, mathematical modelling, and single-molecule RNA-FISH (smFISH), with an emphasis on the spatio-temporal organisation of RNA expression at the subcellular and tissue scales. I first investigate how RNA are differentially localised at the scale of the liver tissue. Hepatocytes are arranged in structural and functional units called lobules, and carry out different physiological functions depending on their spatial position within the lobule. In this collaborative work, we characterised spatio-temporal gene expression profiles, and showed that that while the expression of hundreds of genes is dually orchestrated by time and space, the circadian core clock is expressed uniformly within the liver lobule, and is therefore robust to the heterogeneous microenvironment. Second, I explore the RNA localisation at the scale of a hepatocyte. The subcellular distributions of RNA in different compartments (here, the nucleus and the cytoplasm), are dictated by the balance of a synthesis term and a decay term. To quantify the kinetic parameters driving nuclear and cytoplasmic mRNA accumulation, I sequenced RNA from both cellular fractions from mouse livers sampled at different times of the day. Using a mathematical model describing rhythmic pre-mRNA and mRNA profiles, I could estimate the nuclear export rates and cytoplasmic degradation rates of

\sim

1400 genes. Nuclear export occurs on a much shorter time-scale than cytoplasmic degradation, and nuclear lifetime has only a minor contribution to the total RNA lifetime. However, a subset of metabolic genes remain in the nucleus for more than one hour (up to four hours), which accounts for the long phase delay between the peak times of transcription and of cytoplasmic accumulation. Furthermore, nuclear export contributes to the modulation and generation of rhythmic profiles of ~10% of the cycling nuclear mRNA. This study provides a comprehensive estimation of the nuclear and cytoplasmic life times in the liver and contributes to a better understanding of the dynamic regulation of the transcriptome during the feeding-fasting cycle.

EPFL2021

Phase specific transcriptional regulation of circadian clock and metabolism in mouse liver

Jonathan Aryeh Sobel

The molecular clock has been conserved from cyanobacteria to mammals and is believed to align behavioral and biochemical processes with the diurnal cycle. This cellular mechanism has been an advantage to increase the fitness of organisms through the ability to anticipate food availability or predator presence. Accumulating evidence has revealed that the circadian clock is intimately interconnected with the metabolic cycle. But, the nature of these interconnections is yet not clear at the transcriptional level, and the contribution of cis-regulatory modules has not been elucidated. Accessible chromatin regions of the genome are implicated in diverse processes, such as gene regulation through enhancers and promoters, insulation of genomic domains or alternative splicing. They allow cell-type specific programs that are controlled by tissue-specific transcription factors and chromatin modifiers, although tissue-specific regulation of the circadian clock remains unclear. Therefore, we compared genome-wide BMAL1 binding and chromatin accessibility in the liver and in NIH3T3 fibroblasts. Moreover, for the first time, our study explores the dynamics of accessible regions, histone 3 lysine 27 acetylation (H3K27ac) and RNA polymerase II (Pol II) every 4 hours over one day in mouse liver. In this study, we show that a substantial fraction of these accessible sites are oscillating during a diurnal cycle with a circadian period. We observed that these accessible regions are enriched in the proximity of actively transcribed genes, and that they are dynamically affected by the binding of transcription factors such as BMAL1. We investigated wild-type (WT) and Bmal1-/- genotypes, in night restricted feeding regimen and in Light-Dark (LD) cycle, to study the circadian clock regulatory network underlying diurnal transcription. Therefore, we applied a penalized generalized linear model to infer the activity of transcription factor binding motifs in oscillating accessible sites using Pol II loadings at the transcription start sites of nearby genes. We were able to recapitulate the known regulatory elements of the circadian clock, notably E-box, D-Box, and ROR-responsive elements (RRE) in the WT genotype. On the other hand, we found that Forkhead box (FOX), glucocorticoids responsive elements (GRE), and C-AMP Response Element (CRE) were the main contributors of the regulation of oscillating genes in Bmal1-/-. Finally, using a mixture model to detect footprints with a base pair resolution, we studied the dynamics of the accessibility overlapping E-box. Our last analysis suggested that BMAL1/CLOCK is binding on double E-boxes with a spacer of 6 or 7 bp in a hetero-tetramer configuration. A 3D structure model further supported this binding mode. In Summary, we used DNase I-seq and ChIP-seq of Pol II and H3K27ac to study the circadian chromatin landscape in a 4h time-resolved experimental design. We uncovered the underlying circadian transcriptional regulatory network and, we dissected the chromatin accessibility around BMAL1 binding sites at a base pair resolution, which led to an unappreciated mode of binding of BMAL1/CLOCK in a hetero-tetramer conformation on double E-boxes. Lastly, we found tissue-specific factors that might contribute to tissue-specific binding of BMAL1/CLOCK.

EPFL2016