Structure of silent transcription intervals and noise characteristics of mammalian genes

Mammalian transcription occurs stochastically in short bursts interspersed by silent intervals showing a refractory period. However, the underlying processes and consequences on fluctuations in gene products are poorly understood. Here, we use single allele time-lapse recordings in mouse cells to identify minimal models of promoter cycles, which inform on the number and durations of rate-limiting steps responsible for refractory periods. The structure of promoter cycles is gene specific and independent of genomic location. Typically, five rate-limiting steps underlie the silent periods of endogenous promoters, while minimal synthetic promoters exhibit only one. Strikingly, endogenous or synthetic promoters with TATA boxes show simplified two-state promoter cycles. Since transcriptional bursting constrains intrinsic noise depending on the number of promoter steps, this explains why TATA box genes display increased intrinsic noise genome-wide in mammals, as revealed by single-cell RNA-seq. These findings have implications for basic transcription biology and shed light on interpreting single-cell RNA-counting experiments.

Modeling of transcription mechanisms in mammals from kinetic measurements in single cells

Benjamin Zoller

In mammals, transcription was observed to occur predominantly in bursts, resulting from short and intense periods of gene transcription interspersed by longer silent periods. In order to elucidate the causes and the consequences of discontinuous transcription in mammals, we further extended a probabilistic and computational framework to model single-cell time-lapse recording of luminescent reporter based on stochastic gene expression models that describe the basic processes of gene activation, transcription, translation, and degradation of mRNA and proteins. Motivated by the recent finding of a refractory period in gene reactivation, we performed inference in promoter cycle models, where the gene activity is modeled by one active state and multiple sequential inactive states describing the promoter progression toward activation, and we characterized the rate limiting processes causing the refractory period. Our analysis demonstrated that the kinetic structure of the cycle is gene-specific. Several rate limiting steps (5 on average) are required to model the silent period of endogenous promoters while minimal synthetic promoters overall exhibit a simplified promoter cycle structure characterized by a reduced number of steps (3 on average) and longer silent period. Nevertheless, the promoter architecture might not solely be responsible for such an observed difference, as the synthetic promoters were all sharing similar chromatin context (same insertion locus). Based on the inferred kinetics, we investigate the implications on the transcriptional noise. It followed from the inferred bursting kinetics, that intrinsic noise is dominated by the fluctuations in the cycle, and globally maintained over the wide range of expression covered by the clones (100-fold). With minor modifications of the developed probabilistic framework, we investigated how the bursting kinetics of a mammalian gene (Ctgf) are modulated in response to two different induction pathways. Both stimuli resulted in a fast and acute increase in burst sizes. Whereas TGF-b1 induced a sustained increase of transcription rate and prolonged transcriptional activation, serum stimulation led to a large and temporally tight first transcriptional burst, followed by a refractory period in the range of hours. Those observations illustrated how different stimuli can result in kinetically distinct transcriptional responses of the same gene.

EPFL2014

Extensive Regulation of Diurnal Transcription and Metabolism by Glucocorticoids

Frédéric Bruno Martin Gachon, Cédric Gobet, Benjamin Dieter Weger, Meltem Weger

Altered daily patterns of hormone action are suspected to contribute to metabolic disease. It is poorly understood how the adrenal glucocorticoid hormones contribute to the coordination of daily global patterns of transcription and metabolism. Here, we examined diurnal metabolite and transcriptome patterns in a zebrafish glucocorticoid deficiency model by RNA-Seq, NMR spectroscopy and liquid chromatography-based methods. We observed dysregulation of metabolic pathways including glutaminolysis, the citrate and urea cycles and glyoxylate detoxification. Constant, non-rhythmic glucocorticoid treatment rescued many of these changes, with some notable exceptions among the amino acid related pathways. Surprisingly, the non-rhythmic glucocorticoid treatment rescued almost half of the entire dysregulated diurnal transcriptome patterns. A combination of E-box and glucocorticoid response elements is enriched in the rescued genes. This simple enhancer element combination is sufficient to drive rhythmic circadian reporter gene expression under non-rhythmic glucocorticoid exposure, revealing a permissive function for the hormones in glucocorticoid-dependent circadian transcription. Our work highlights metabolic pathways potentially contributing to morbidity in patients with glucocorticoid deficiency, even under glucocorticoid replacement therapy. Moreover, we provide mechanistic insight into the interaction between the circadian clock and glucocorticoids in the transcriptional regulation of metabolism.

Public Library Science2016

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.