Rhythmic modulation of transcriptional burst frequency in circadian gene promoters

Isogenic cells sharing a common environment present a large degree of heterogeneity in gene expression, and stochasticity inherent to transcription substantially participates in this cell-to-cell variability. Notably, a majority of mammalian genes are transcribed during short periods termed transcription bursts followed by longer periods of transcriptional inactivity. Interestingly, genes display variability in the frequencies and sizes of their bursts, implying that different regulatory mechanisms actively participate in shaping gene-specific bursting signatures. However, the nature of these molecular mechanisms and their precise contribution to transcriptional bursting remains elusive. In this work, we used a short-lived luciferase reporter under the control of a Bmal1 circadian promoter stably inserted into the genome of NIH-3T3 cultured fibroblasts to quantify its transcriptional bursting along the circadian cycle and at three different reporter integration sites. By recording dynamic variations of the luminescence signal at the single-cell level and counting individual transcripts using smRNA-FISH at specific time-points, we could infer the transcriptional bursting parameters characteristic of these conditions using a telegraph (on-off) model of gene expression. We observed that while the integration site-specific differences in expression levels mainly arose from burst size dissimilarities, the burst frequency predominantly modulated the temporal variations in expression of Bmal1 over the circadian cycle. Thus, both parameters are uncoupled and can be independently modulated to regulate expression levels. By focusing on the molecular origins of bursting, we found that the rhythmic circadian modulation of burst frequencies depended on the presence of ROR responsive elements (ROREs) on Bmal1 promoter. These DNA motifs recruit the REV-ERBs repressors involved in the rhythmic regulation of the histone acetylation state at target promoters. Indeed, the H3K27ac profile in the Bmal1 promoter corresponded to that of its burst frequency. More generally, higher histone acetylation levels were observed during Bmal1 circadian peaks of expression, while H3K27ac signal did not vary between clones harboring different reporter integration sites. Similar properties were observed on other rhythmically expressed genes: despite variability in promoter motifs and expression phases, endogenous circadian genes displaying rhythmic variations in their promoter acetylation state also modulated their burst frequencies over the circadian period. By inferring the transcriptional bursting parameters of non-circadian genes using smRNA-FISH datasets, we also observed significant correlations between histone acetylation signal around promoters and the burst frequency. In conclusion, this study identified an association between the burst frequency and the histone acetylation state of promoters. While the molecular mechanisms behind this association remain elusive, it could be related to the facilitated binding of transcription regulators upon histone acetylation-mediated chromatin loosening. In this thesis we clarified how transcription of circadian genes is rhythmically modulated, and we further elucidated the link between molecular events and transcriptional bursting, with particular focus on histone acetylation.

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

Quantitative analysis of gene expression and transcriptional memory in mouse embryonic stem cells

Aleksandra Mandic

Quantitative studies of gene expression in have shown widespread variability in transcript and protein product abundance at the single-cell level. Such fluctuations in gene expression have been shown to lead to heterogeneous cellular responses and phenotypes, acting in a range of systems, from immunity, cancer to development. To better understand heterogeneity in cellular populations, we developed a novel method for the precise quantification of gene expression in single living cells. Furthermore, we focused on whether and to which extent transcriptional expression profiles are transmitted during cell division. In the first section, we described a new approach for quantitative measurements of gene expression, based on internal tagging of endogenous proteins with a reporter allowing luminescence and fluorescence time-lapse imaging. Using the currently brightest luminescent reporter, fluctuations of protein expression levels could be monitored in single living cells with high sensitivity and temporal resolution over extended time periods. Moreover, our system allowed measurement of single-cell protein degradation rates in the absence of protein synthesis inhibitors, as well as calculation of absolute synthesis rates over the cell cycle. Finally, the internal tag could be excised by inducible expression of Cre recombinase, which enabled estimation of endogenous mRNA half-lives by monitoring the decay of luminescent signal. Second, to monitor transcriptional activity and quantify memory across multiple endogenous genes in mouse embryonic stem cells, we generated cell lines in which a short-lived luciferase reporter allowed sensitive monitoring of transcriptional kinetics by luminescence imaging at high time resolution over long periods of time. By coupling live single-cell monitoring of transcriptional activity of 9 different promoters with mathematical modelling, we found that different levels of expression fluctuations shape transcriptional memory. Long fluctuations (1-10 generations) and short fluctuations (1-3 h), as well as their amplitudes, vary widely between across different genes and together shape transcriptional memory. Surprisingly, we have shown that there was a large level of gene-specific cell-to-cell synchronicity in transcriptional profiles. Overall, we uncovered that noise and timescales of slow and fast fluctuations, as well as their synchronisation over the cell cycle, defined how transcriptional dynamics were correlated within a lineage of related cells. In conclusion, this thesis described an innovative method that allows measurement of absolute protein expression levels, protein turnover, as well as mRNA half-lives for endogenously expressed genes. Our approach opens new avenues in quantitative studies of gene expression in single living cells. Furthermore, we revealed different levels of transcriptional fluctuations that shape gene specific transcriptional memory and its timescales in mouse embryonic stem cells.

EPFL2018