Regulation of Mammalian Physiology by interconnected Circadian and Feeding Rhythms

Circadian clocks are endogenous timekeeping systems that adapt in an anticipatory fashion the physiology and behavior of most living organisms. In mammals, the master pacemaker resides in the suprachiasmatic nucleus and entrains peripheral clocks using a wide range of signals that differentially schedule physiology and gene expression in a tissue-specific manner. The peripheral clocks, such as those found in the liver, are particularly sensitive to rhythmic external cues like feeding behavior, which modulate the phase and amplitude of rhythmic gene expression. Consequently, the liver clock temporally tunes the expression of many genes involved in metabolism and physiology. However, the circadian modulation of cellular functions also relies on multiple layers of posttranscriptional and posttranslational regulation. Strikingly, these additional regulatory events may happen independently of any transcriptional oscillations, showing that complex regulatory networks ultimately drive circadian output functions. These rhythmic events also integrate feeding-related cues and adapt various metabolic processes to food availability schedules. The importance of such temporal regulation of metabolism is illustrated by metabolic dysfunctions and diseases resulting from circadian clock disruption or inappropriate feeding patterns. Therefore, the study of circadian clocks and rhythmic feeding behavior should be of interest to further advance our understanding of the prevention and therapy of metabolic diseases.

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

Circadian clock- and feeding-dependent regulation of translation initiation and elongation in the liver

Cédric Gobet

Circadian rhythms allow organisms to anticipate and adapt to periodic environmental changes related to the day-night cycle. These rhythms originate from a genetic network ticking in almost every cell of our body and the coordination between different organs, thus allowing to generate physiological outputs at the right time of the day. In the liver, which is one of the major metabolic organ of the body, gene expression has to be highly regulated by the circadian clock and feeding rhythms to generate such a timely behavior. Indeed, at every step from transcription to post-translational modifications, the circadian clock and metabolism interplay. In this context, we quantified transcription, mRNA accumulation and translation in liver from wild-type and clock-deficient mice fed ad libitum or only during the night period. RNA-Sequencing and ribosome profiling combined with mathematical modeling were used to assess rhythmic regulation along the gene expression process. Transcription was found to be the main driver of temporal mRNA accumulation and its subsequent translation. We observed a diurnal translation efficiency in genes with 5'-Terminal Oligo Pyrimidine tract (5'-TOP) sequences or with Translation Initiator of Short 5'-UTR (TISU) motifs. Remarkably, the rhythmic translation efficiency was mainly driven by feeding cues although clock-deficiency slightly affected the amplitude and phase of the oscillations. In a subsequent work, we emphasized the above analysis of translation regulation with a focus on the ribosome elongation process. A statistical model and bioinformatics pipeline were developed to infer codon dwell times from ribosome profiling data. The method has the particularity of integrating pairs of codons between the sites of the ribosome and to fit every sequencing read on an individual basis. We validated our approach on a yeast dataset and then studied extensively the mouse liver translation elongation landscape. A large dynamic range between the fastest and longest codons was uncovered in the ribosome A and P sites with strong synergic interaction of pairs of codons clustering by amino acids. In order to challenge our system, we performed drug free ribosome profiling, modified to reduce library biases, in liver of mice fed ad libitum or starved. Unexpectedly, codon dwell times were conserved between the two conditions despite a gene expression landscape reflecting low energy state of the mice and subsequent amino acid shortage. Finally, to understand how the ribosome elongation velocity is regulated by (aminoacyl)-tRNA levels, we adapted and modified a published DNA hybridization-based tRNA profiling method in liver of mice fed ad libitum or starved. While the quantified tRNAs abundance showed a large dynamic range and specific isoacceptors patterns, no differences in (aminoacyl)-tRNAs were found between the two feeding conditions. Nevertheless, tRNAs for isoleucine, asparagine, aspartate and arginine exhibited a relative low loading state compared to other tRNA in both conditions. Altogether, correlation between codon usage, codon dwell times and tRNA abundances provided insights of how translation elongation is regulated in mammals. Together, this thesis aimed to dissect the mechanisms regulating gene expression by the interplay of the circadian clock and metabolism, as well as understand how translation elongation is regulated in mammals.

EPFL2018

Cross-regulatory circuits linking inflammation, high-fat diet, and the circadian clock

Frédéric Bruno Martin Gachon, Felix Naef, Jake Yeung

Mammalian physiology resonates with the daily changes in the external environment, allowing processes such as rest-activity cycles, metabolism, and body temperature to synchronize with daily changes in the surroundings. Studies have identified the molecular underpinnings of robust oscillations in gene expression occurring over the 24-h day, but how acute or chronic perturbations modulate gene expression rhythms, physiology, and behavior is still relatively unknown. In this issue of Genes & Development, Hong and colleagues (pp. 1367-1379) studied how acute and chronic inflammation interacts with the circadian clock. They found that NF-kappa B signaling can modify chromatin states and modulate expression of genes in the core clock network as well as circadian locomotor behavior. Interestingly, a high-fat diet (HFD) fed to mice also triggers this inflammation pathway, suggesting that cross-regulatory circuits link inflammation, HFD, and the circadian clock.