Circadian rhythm and cell cycle

Circadian rhythms are biological processes found in most living organisms, displaying a roughly 24-hour period, responding primarily to light darkness cycles in an organism's environment. At the cellular level, the circa-24h rhythmicity is generated by a molecular clock based on a transcription-translation feedback network and consists of a cell-autonomous and self-sustained oscillator. In conditions where cells proliferate, the cell division cycle can be also considered as an oscillator. Since both processes run with similar periods in several mammalian cells, it is reasonable to expect that interactions between these two cycles may cause synchronization. Many studies reported evidences of interactions between the circadian and the cell cycles in different organisms. In particular, it appeared that in several systems, specific cell cycle phases occur in distinct temporal windows rather than being randomly distributed in time. These findings led to the concept of circadian gating of the cell cycle, through which the circadian clock can favor or forbid cell cycle transitions at specific circadian phases. However, it was also reported the converse, namely an effect of cell division on the circadian oscillator. Even though interactions between the circadian clock and the cell cycle have been identified in both directions, the dynamical consequences and the directionality of the coupling at the single-cell level were not extensively investigated. In order to better characterize the potential synchronization in mammalian cells, we estimated the mutual interactions between circadian clock and cell cycle in NIH3T3 mouse fibroblasts by the use of time-lapse fluorescent microscopy in combination with statistical analysis and mathematical modeling. NIH3T3 cells, harboring a fluorescent reporter under the control of the circadian RevErb-a gene promoter, were imaged for several days allowing the simultaneous detection of circadian oscillations and time of divisions. The analysis of thousands of circadian cycles in dividing cells indicated that both oscillators are synchronized, with cell divisions occurring about 5 h before the peak of the circadian RevErb-a reporter. We tested several perturbations such as different serum concentrations, different temperatures, treatment with pharmacological compounds and shRNA-mediated knockdown of circadian regulators. Surprisingly, this showed that circadian rhythm and cell cycle remain synchronized over the wide range of conditions probed. Our data showed that this synchronization state reflects an unexpected predominant influence of the cell cycle on the circadian oscillator, and did not support the leading hypothesis about a circadian gating of the cell cycle. The stochastic modeling of two interacting phase oscillators allowed us to identify the parameters of the coupling functions, revealing an acceleration of circadian phase after the division. The work presented in this thesis sheds light on the interaction between two fundamentally recurrent cellular processes in mammalian cells and provides a deeper understanding of the role of the circadian clock in proliferating cells and tissues. These findings might have significant implications for chronobiology and chronotherapeutics.

Spatio-temporal synchronization of biological oscillators

Jonathan Bieler

The circadian clock is a cell-autonomous and self-sustained oscillator with a period of about 24 hours that controls many aspects of cellular physiology. The cell cycle is also a fundamental periodic process, with a period in the range of one day in mammals. A large number of studies showed that these two cycles interact at the molecular level. Mathematical theory of coupled oscillators predicts that in the presence of coupling, two cycles with similar periods can synchronize. Consequently, the molecular interactions between the cell cycle and the circadian clock could lead to their synchronization. Such synchrony is consistent with a number of studies showing that cells divide more at specific circadian times. Moreover, a few works also report effects of cell divisions on the circadian oscillator. More detailed mathematical models of the molecular networks involved also predict that the two cycles can synchronize. To better characterize this potential synchronization in mammalian cells, we monitored the mutual interactions between the two oscillators by time-lapse imaging of single NIH3T3 fibroblasts during several days. To do so, we used a fluorescent reporter under the control of a circadian promoter and monitored the cell cycle progression by detecting the time of mitosis. The analysis of thousands of circadian cycles in dividing cells clearly indicated that both oscillators tick in a 1:1 mode-locked state, with cell divisions occurring tightly 5 hours before the peak in circadian Rev-Erbα -YFP reporter expression. In principle, such synchrony may be caused by coupling from one oscillator onto the other, or in both directions. While gating of cell division by the circadian cycle has been most studied, our data combined with stochastic modeling strongly suggest that reverse coupling is predominant in NIH3T3 cells. In order to further test the direction of the coupling, we performed several perturbation experiments; we changed the cell cycle period by acquiring recordings at different temperatures, we analyzed cell lines containing genetic knock-down of circadian genes and used drugs that target specific proteins of the cell cycle and the circadian clock. These experiments showed that the two interacting cellular oscillators adopt a synchronized state that is highly robust over a wide range of parameters. Moreover, the set of perturbations on the periods of either oscillator induced shifts in the relative phases of circadian and cell cycles that were consistent with a scenario in which the circadian cycle resonates to the cell cycle. These findings have implications for circadian function in proliferative tissues, including epidermis, immune cells, and cancer.

EPFL2014

Kinetic Analysis of Transcriptional and Post-Transcriptional Processes During Circadian Cycles

Laura Symul

Every morning the sun rises, flooding the earth with its light and heat and every night it disappears leaving the Earth in the darkness of the night. Organisms living on this planet have adapted to this solar rhythm and have spread their activities throughout the day. Early in the development of life, an internal molecular clock appeared in primitive organisms and the evolutionary advantage of being able to anticipate, rather than simply passively face, environmental changes led virtually all organisms to have an internal timing system called "circadian clock." In most species, this clock is constituted of a genetic network ticking in every cell of the body. This genetic network organized in interlocked feedback loops generates oscillations with a 24-hour period. A variable number of proteins and RNAs is thus accumulating in a rhythmic fashion. For example, in mouse liver, which was the main biological model I used during my thesis, it is estimated that thousands of genes are expressed periodically. The molecular processes leading to rhythmic gene expression and protein accumulation are highly regulated. Dynamical control is needed to sustain the oscillations and to maintain their timing. During my PhD, I was particularly interested in the regulation of rhythmic gene activation via transcription factor binding in their promoter region. I also focused on gene transcription itself and its dynamics as well as on transcription associated changes in epigenetic marks. Another interest was given to the regulation of transcripts (messenger RNA) after transcription, especially to understand whether their degradation in the cytoplasm was regulated rhythmically. Finally, I contributed to a project aiming at the detection of transcripts whose translation into protein was specifically regulated in a circadian manner. The four projects on which I worked were done in collaborations with experimental biology labs. Our partners performed the experiments and provided the biological material while I was in charge of the data processing and their statistical analysis. For some projects, I also developed different mathematical models for a more accurate and complete understanding of the phenomena studied and to take into account the inherent variability of biological data. Notably in the project on the post-transcriptional regulation of transcripts, the model-based approach we developed allowed estimating the role of this regulation in a way that is less prone to arbitrary choice of criteria or thresholds compared to previous studies addressing this question. To summarize, the results I obtained show that, in human cell lines, the major transcription factor of the clock (the heterodimer CLOCK/BMAL1) binds to DNA at specific binding sites and their number is comparable to the number of binding sites observed in the mouse liver. However the fraction of rhythmic transcript among the genes with CLOCK/BMAL1 binding sites is about 10 times lower than the corresponding fraction observed in mouse liver. The results of the second project showed that, in mouse liver, rhythmic transcription is followed by dynamical changes in epigenetic marks with delays specific to the marks (tri-methylation of lysines 4 and 36 of histone 3 of nucleosomes) and we highlighted the role of post-transcriptional regulation for the rhythmic expression of a subset of mRNAs. In the third project, we further investigated the regulation at the post-transcriptional level and combining measurements of pre-mRNA and mRNA with a mathematical model, we showed that the post-transcriptional regulation plays a limited role in the rhythmic accumulation of transcripts and is involved in the regulation of only 20 % of rhythmic transcripts. Laboratories of Michael Brunner (University of Heidelberg, Germany), Frederic Gachon, Nouria Hernandez (University of Lausanne, Switzerland) and Ueli Schibler (University of Geneva, Switzerland) 3 Finally, helped by the analysis I performed on micro array data from an experiment aiming at comparing the fraction of translated mRNA to the total accumulation of these mRNAs, the team of Frederic Gachon showed that several transcripts implied in the composition of ribosomes are translated specifically during the night, when rodents are active. All of these results and their links with other published studies on circadian rhythms, show that biological tissues have largely incorporated the clock for the regulation of many other functional pathways in order to optimally synchronize their physiological actions with environmental changes due to the alternation of days and nights. In mouse liver, links with the metabolism are particularly strong and disruptions of the clock induce phenotypes reflecting a disturbed metabolism.

EPFL2013

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