Circadian dynamics of RNA localisation in the mammalian liver

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.

Comprehensive analysis of the circadian nuclear and cytoplasmic transcriptome in mouse liver

Cédric Gobet, Clémence Yumie Syloun Hurni, Felix Naef, Benjamin Dieter Weger

In eukaryotes, RNA is synthesised in the nucleus, spliced, and exported to the cytoplasm where it is translated and finally degraded. Any of these steps could be subject to temporal regulation during the circadian cycle, resulting in daily fluctuations of RNA accumulation and affecting the distribution of transcripts in different subcellular compartments. Our study analysed the nuclear and cytoplasmic, poly(A) and total transcriptomes of mouse livers collected over the course of a day. These data provide a genome-wide temporal inventory of enrichment in subcellular RNA, and revealed specific signatures of splicing, nuclear export and cytoplasmic mRNA stability related to transcript and gene lengths. Combined with a mathematical model describing rhythmic RNA profiles, we could test the rhythmicity of export rates and cytoplasmic degradation rates of approximately 1400 genes. With nuclear export times usually much shorter than cytoplasmic half-lives, we found that nuclear export contributes to the modulation and generation of rhythmic profiles of 10% of the cycling nuclear mRNAs. This study contributes to a better understanding of the dynamic regulation of the transcriptome during the day-night cycle. Author summary Why was this study done? The circadian clock coordinates temporal gene expression programs to synchronise cellular and physiological functions with environmental time.The circadian clock impinges on many layers of gene regulation, from transcription to mRNA degradation in the cytoplasm.Specifically, whether temporal modulation of nuclear export could influence rhythmic mRNA expression was not known. What did the researchers do and find? We sequenced RNA from nuclear and cytoplasmic fractions from mouse livers sampled at different times of the day.Using a mathematical model describing temporal accumulation profiles of pre-mRNA, nuclear mRNA and cytoplasmic mRNA, we could estimate the nuclear export rates and cytoplasmic degradation rates of approximately 1400 genes.We observed rhythmic patterns in the nucleus that most likely originate from a rhythmic regulation of the nuclear export rate, affecting approximately 10% of the oscillations of the nuclear transcriptome in the liver. What do these findings mean? Our fractionated RNA analysis contributes to a better understanding of the dynamic regulation of the transcriptome during the daily cycle.The majority of the RNA transcripts are exported to the cytoplasm in less than 30 minutes, however, some are retained in the nucleus for several hours. Thus, cellular fractionation allows a finer resolution of temporal profiles in the different subcellular compartment, which is otherwise masked at bulk-level.

PUBLIC LIBRARY SCIENCE2022

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

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