Genome-Wide RNA Polymerase II Profiles and RNA Accumulation Reveal Kinetics of Transcription and Associated Epigenetic Changes During Diurnal Cycles

Interactions of cell-autonomous circadian oscillators with diurnal cycles govern the temporal compartmentalization of cell physiology in mammals. To understand the transcriptional and epigenetic basis of diurnal rhythms in mouse liver genome-wide, we generated temporal DNA occupancy profiles by RNA polymerase II (Pol II) as well as profiles of the histone modifications H3K4me3 and H3K36me3. We used these data to quantify the relationships of phases and amplitudes between different marks. We found that rhythmic Pol II recruitment at promoters rather than rhythmic transition from paused to productive elongation underlies diurnal gene transcription, a conclusion further supported by modeling. Moreover, Pol II occupancy preceded mRNA accumulation by 3 hours, consistent with mRNA half-lives. Both methylation marks showed that the epigenetic landscape is highly dynamic and globally remodeled during the 24-hour cycle. While promoters of transcribed genes had tri-methylated H3K4 even at their trough activity times, tri-methylation levels reached their peak, on average, 1 hour after Pol II. Meanwhile, rhythms in tri-methylation of H3K36 lagged transcription by 3 hours. Finally, modeling profiles of Pol II occupancy and mRNA accumulation identified three classes of genes: one showing rhythmicity both in transcriptional and mRNA accumulation, a second class with rhythmic transcription but flat mRNA levels, and a third with constant transcription but rhythmic mRNAs. The latter class emphasizes widespread temporally gated posttranscriptional regulation in the mouse liver.

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

Transcriptional regulatory logic of the diurnal cycle in the mouse liver

Matteo Dal Peraro, Bart Deplancke, Frédéric Bruno Martin Gachon, Nouria Hernandez, Alexandra Styliani Kalantzi, Irina Krier, Felix Naef, Sunil Kumar Raghav, Guillaume Rey, Jonathan Aryeh Sobel, Benjamin Dieter Weger

Many organisms exhibit temporal rhythms in gene expression that propel diurnal cycles in physiology. In the liver of mammals, these rhythms are controlled by transcription-translation feedback loops of the core circadian clock and by feeding-fasting cycles. To better understand the regulatory interplay between the circadian clock and feeding rhythms, we mapped DNase I hypersensitive sites (DHSs) in the mouse liver during a diurnal cycle. The intensity of DNase I cleavages cycled at a substantial fraction of all DHSs, suggesting that DHSs harbor regulatory elements that control rhythmic transcription. Using chromatin immunoprecipitation followed by DNA sequencing (ChIP-seq), we found that hypersensitivity cycled in phase with RNA polymerase II (Pol II) loading and H3K27ac histone marks. We then combined the DHSs with temporal Pol II profiles in wild-type (WT) and Bmal1(-/-) livers to computationally identify transcription factors through which the core clock and feeding-fasting cycles control diurnal rhythms in transcription. While a similar number of mRNAs accumulated rhythmically in Bmal1(-/-) compared to WT livers, the amplitudes in Bmal1(-/-) were generally lower. The residual rhythms in Bmal1(-/-) reflected transcriptional regulators mediating feeding-fasting responses as well as responses to rhythmic systemic signals. Finally, the analysis of DNase I cuts at nucleotide resolution showed dynamically changing footprints consistent with dynamic binding of CLOCK: BMAL1 complexes. Structural modeling suggested that these footprints are driven by a transient heterotetramer binding configuration at peak activity. Together, our temporal DNase I mappings allowed us to decipher the global regulation of diurnal transcription rhythms in the mouse liver.

Public Library of Science2017

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