The Circadian Clock Coordinates Ribosome Biogenesis

Biological rhythms play a fundamental role in the physiology and behavior of most living organisms. Rhythmic circadian expression of clock-controlled genes is orchestrated by a molecular clock that relies on interconnected negative feedback loops of transcription regulators. Here we show that the circadian clock exerts its function also through the regulation of mRNA translation. Namely, the circadian clock influences the temporal translation of a subset of mRNAs involved in ribosome biogenesis by controlling the transcription of translation initiation factors as well as the clock-dependent rhythmic activation of signaling pathways involved in their regulation. Moreover, the circadian oscillator directly regulates the transcription of ribosomal protein mRNAs and ribosomal RNAs. Thus the circadian clock exerts a major role in coordinating transcription and translation steps underlying ribosome biogenesis.

Rhythmic and clock-dependent chromatin loops regulate circadian gene expression

Jérôme Mermet

In mammals the circadian clock drives daily behavioural and physiological changes that resonate with environmental cues, which can be observed, for example, in the intricate timing of rest during the night and activity during the day in humans. The circadian clock consists of a network of hierarchical clocks in which the central pacemaker is located in the suprachiasmatic nucleus, which is sensitive to external light and propagates time to the peripheral organ-based clocks. Within organs, clocks tick in each individual cell and are molecularly encoded in the genome as a network of dynamic feedback loops. By mediating the expression of the appropriate gene products at the correct moment of the day, the molecular clocks op-timize physiological functions of virtually every cell in our body, allowing us to synchronize with daily fluc-tuations in the environment. In mammalian cells the chromatin organization in the nucleus consists of a topological network, in which chromatin interactions between distal regulatory elements such as enhancers and target genes are essen-tial to regulate gene expression. Important questions remain: is the complex chromatin folding dynamic and if so on which genomic and temporal scales? In fact, dynamic rearrangements of the chromatin have been reported on multiple genomic and time scales, from the entire genome to enhancer-promoter con-tacts, across developmental transitions, cellular differentiation, and transcription cycles. With this per-spective in mind, the circadian oscillator provides a unique model to study the dynamics of genomic inter-actions in the context of fluctuating transcription. Here, we explored chromatin contacts of core-clock and clock-controlled gene promoters in the mouse. We aimed at evaluating the putative dynamics of chromatin contacts, at estimating their conservation across tissues, and at exploring the contribution of the circadian clock to this interaction network. To achieve this goal, we performed 4C-sequencing assays at two circadian time points in the liver and kidney of WT and clock-deficient animals. Overall, we observe that chromatin contacts are largely conserved across circadian time points and genetic backgrounds, and are tissue-specific. Our experiments reveal that the promoters of core-clock and clock-controlled genes contact distal genomic regions containing en-hancer chromatin signatures, suggesting a functional role of these distal sites in target gene regulation. Our data also suggest a clock-driven module of rhythmic genes that are colocalized in the nucleus. However, we also observe dynamic contacts between oscillating gene promoter and enhancer-like ge-nomic regions. In this thesis, we discuss in detail two examples of dynamic chromatin looping that are ob-served at the Cry1 and Gys2 loci. Our data reveal not only a dynamic coupling between the chromatin loop and the transcriptional state but also that the dynamic of chromatin looping depends on a functional clock. Furthermore, genome editing experiments using CRISPR-Cas9 tools reveal that the Cry1 distal site is essential for the transcriptional regulation of Cry1 and contributes to the circadian clock robustness both in-vivo and in cultured cells. These results demonstrate how local chromatin rearrangements take place in a 24-hour time-scale and set chromatin looping between gene promoters and distal regions as a new reg-ulatory layer for circadian gene expression.

EPFL2017

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

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