Genome-Wide and Phase-Specific DNA-Binding Rhythms of BMAL1 Control Circadian Output Functions in Mouse Liver

The mammalian circadian clock uses interlocked negative feedback loops in which the heterodimeric basic helix-loop-helix transcription factor BMAL1/CLOCK is a master regulator. While there is prominent control of liver functions by the circadian clock, the detailed links between circadian regulators and downstream targets are poorly known. Using chromatin immunoprecipitation combined with deep sequencing we obtained a time-resolved and genome-wide map of BMAL1 binding in mouse liver, which allowed us to identify over 2,000 binding sites, with peak binding narrowly centered around Zeitgeber time 6. Annotation of BMAL1 targets confirms carbohydrate and lipid metabolism as the major output of the circadian clock in mouse liver. Moreover, transcription regulators are largely overrepresented, several of which also exhibit circadian activity. Genes of the core circadian oscillator stand out as strongly bound, often at promoter and distal sites. Genomic sequence analysis of the sites identified E-boxes and tandem E1-E2 consensus elements. Electromobility shift assays showed that E1-E2 sites are bound by a dimer of BMAL1/CLOCK heterodimers with a spacing-dependent cooperative interaction, a finding that was further validated in transactivation assays. BMAL1 target genes showed cyclic mRNA expression profiles with a phase distribution centered at Zeitgeber time 10. Importantly, sites with E1-E2 elements showed tighter phases both in binding and mRNA accumulation. Finally, analyzing the temporal profiles of BMAL1 binding, precursor mRNA and mature mRNA levels showed how transcriptional and post-transcriptional regulation contribute differentially to circadian expression phase. Together, our analysis of a dynamic protein-DNA interactome uncovered how genes of the core circadian oscillator crosstalk and drive phase-specific circadian output programs in a complex tissue.

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

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