The circadian oscillator analysed at the single-transcript level.

The circadian clock is an endogenous and self-sustained oscillator that anticipates daily environmental cycles. While rhythmic gene expression of circadian genes is well-described in populations of cells, the single-cell mRNA dynamics of multiple core clock genes remain largely unknown. Here we use single-molecule fluorescence in situ hybridisation (smFISH) at multiple time points to measure pairs of core clock transcripts, Rev-erbα (Nr1d1), Cry1 and Bmal1, in mouse fibroblasts. The mean mRNA level oscillates over 24 h for all three genes, but mRNA numbers show considerable spread between cells. We develop a probabilistic model for multivariate mRNA counts using mixtures of negative binomials, which accounts for transcriptional bursting, circadian time and cell-to-cell heterogeneity, notably in cell size. Decomposing the mRNA variability into distinct noise sources shows that clock time contributes a small fraction of the total variability in mRNA number between cells. Thus, our results highlight the intrinsic biological challenges in estimating circadian phase from single-cell mRNA counts and suggest that circadian phase in single cells is encoded post-transcriptionally.

Functional and Computational Genomics Reveal Unprecedented Flexibility in Stage-Specific Toxoplasma Metabolism.

To survive and proliferate in diverse host environments with varying nutrient availability, the obligate intracellular parasite Toxoplasma gondii reprograms its metabolism. We have generated and curated a genome-scale metabolic model (iTgo) for the fast-replicating tachyzoite stage, harmonized with experimentally observed phenotypes. To validate the importance of four metabolic pathways predicted by the model, we have performed in-depth in vitro and in vivo phenotyping of mutant parasites including targeted metabolomics and CRISPR-Cas9 fitness screening of all known metabolic genes. This led to unexpected insights into the remarkable flexibility of the parasite, addressing the dependency on biosynthesis or salvage of fatty acids (FAs), purine nucleotides (AMP and GMP), a vitamin (pyridoxal-5P), and a cofactor (heme) in both the acute and latent stages of infection. Taken together, our experimentally validated metabolic network leads to a deeper understanding of the parasite's biology, opening avenues for the development of therapeutic intervention against apicomplexans.

Modulation of transcriptional burst frequency by histone acetylation.

Many mammalian genes are transcribed during short bursts of variable frequencies and sizes that substantially contribute to cell-to-cell variability. However, which molecular mechanisms determine bursting properties remains unclear. To probe putative mechanisms, we combined temporal analysis of transcription along the circadian cycle with multiple genomic reporter integrations, using both short-lived luciferase live microscopy and single-molecule RNA-FISH. Using the Bmal1 circadian promoter as our model, we observed that rhythmic transcription resulted predominantly from variations in burst frequency, while the genomic position changed the burst size. Thus, burst frequency and size independently modulated Bmal1 transcription. We then found that promoter histone-acetylation level covaried with burst frequency, being greatest at peak expression and lowest at trough expression, while remaining unaffected by the genomic location. In addition, specific deletions of ROR-responsive elements led to constitutively elevated histone acetylation and burst frequency. We then investigated the suggested link between histone acetylation and burst frequency by dCas9p300-targeted modulation of histone acetylation, revealing that acetylation levels influence burst frequency more than burst size. The correlation between acetylation levels at the promoter and burst frequency was also observed in endogenous circadian genes and in embryonic stem cell fate genes. Thus, our data suggest that histone acetylation-mediated control of transcription burst frequency is a common mechanism to control mammalian gene expression.

Clock-dependent chromatin topology modulates circadian transcription and behavior.

The circadian clock in animals orchestrates widespread oscillatory gene expression programs, which underlie 24-h rhythms in behavior and physiology. Several studies have shown the possible roles of transcription factors and chromatin marks in controlling cyclic gene expression. However, how daily active enhancers modulate rhythmic gene transcription in mammalian tissues is not known. Using circular chromosome conformation capture (4C) combined with sequencing (4C-seq), we discovered oscillatory promoter-enhancer interactions along the 24-h cycle in the mouse liver and kidney. Rhythms in chromatin interactions were abolished in arrhythmic Bmal1 knockout mice. Deleting a contacted intronic enhancer element in the Cryptochrome 1 (Cry1) gene was sufficient to compromise the rhythmic chromatin contacts in tissues. Moreover, the deletion reduced the daily dynamics of Cry1 transcriptional burst frequency and, remarkably, shortened the circadian period of locomotor activity rhythms. Our results establish oscillating and clock-controlled promoter-enhancer looping as a regulatory layer underlying circadian transcription and behavior.

What shapes eukaryotic transcriptional bursting?

Isogenic cells in a common environment present a large degree of heterogeneity in gene expression. Part of this variability is attributed to transcriptional bursting: the stochastic activation and inactivation of promoters that leads to the discontinuous production of mRNA. The diversity in bursting patterns displayed by different genes suggests the existence of a connection between bursting and gene regulation. Experimental strategies such as single-molecule RNA FISH, MS2-GFP or short-lived protein reporters allow the quantification of transcriptional bursting and the comparison of bursting kinetics between conditions, allowing therefore the identification of molecular mechanisms modulating transcriptional bursting. In this review we recapitulate the impact on transcriptional bursting of different molecular aspects of transcription such as the chromatin environment, nucleosome occupancy, histone modifications, the number and affinity of regulatory elements, DNA looping and transcription factor availability. More specifically, we examine their role in tuning the burst size or the burst frequency. While some molecular mechanisms involved in transcription such as histone marks can affect every aspect of bursting, others predominantly influence the burst size (e.g. the number and affinity of cis-regulatory elements) or frequency (e.g. transcription factor availability).

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.

CAST: An automated segmentation and tracking tool for the analysis of transcriptional kinetics from single-cell time-lapse recordings.

Fluorescence and bioluminescence time-lapse imaging allows to investigate a vast range of cellular processes at single-cell or even subcellular resolution. In particular, time-lapse imaging can provide uniquely detailed information on the fine kinetics of transcription, as well as on biological oscillations such as the circadian and cell cycles. However, we face a paucity of automated methods to quantify time-lapse imaging data with single-cell precision, notably throughout multiple cell cycles. We developed CAST (Cell Automated Segmentation and Tracking platform) to automatically and robustly detect the position and size of cells or nuclei, quantify the corresponding light signals, while taking into account both cell divisions (lineage tracking) and migration events. We present here how CAST analyzes bioluminescence data from a short-lived transcriptional luciferase reporter. However, our flexible and modular implementation makes it easily adaptable to a wide variety of time-lapse recordings. We exemplify how CAST efficiently quantifies single-cell gene expression over multiple cell cycles using mouse NIH3T3 culture cells with a luminescence expression driven by the Bmal1 promoter, a central gene of the circadian oscillator. We further illustrate how such data can be used to quantify transcriptional bursting in conditions of lengthened circadian period, revealing thereby remarkably similar bursting signature compared to the endogenous circadian condition despite marked period lengthening. In summary, we establish CAST as novel tool for the efficient segmentation, signal quantification, and tracking of time-lapse images from mammalian cell culture.

Comparative genome analysis of Pseudomonas knackmussii†B13, the first bacterium known to degrade chloroaromatic compounds.

Pseudomonas knackmussii B13 was the first strain to be isolated in 1974 that could degrade chlorinated aromatic hydrocarbons. This discovery was the prologue for subsequent characterization of numerous bacterial metabolic pathways, for genetic and biochemical studies, and which spurred ideas for pollutant bioremediation. In this study, we determined the complete genome sequence of B13 using next generation sequencing technologies and optical mapping. Genome annotation indicated that B13 has a variety of metabolic pathways for degrading monoaromatic hydrocarbons including chlorobenzoate, aminophenol, anthranilate and hydroxyquinol, but not polyaromatic compounds. Comparative genome analysis revealed that B13 is closest to Pseudomonas denitrificans and Pseudomonas aeruginosa. The B13 genome contains at least eight genomic islands [prophages and integrative conjugative elements (ICEs)], which were absent in closely related pseudomonads. We confirm that two ICEs are identical copies of the 103 kb self-transmissible element ICEclc that carries the genes for chlorocatechol metabolism. Comparison of ICEclc showed that it is composed of a variable and a 'core' region, which is very conserved among proteobacterial genomes, suggesting a widely distributed family of so far uncharacterized ICE. Resequencing of two spontaneous B13 mutants revealed a number of single nucleotide substitutions, as well as excision of a large 220 kb region and a prophage that drastically change the host metabolic capacity and survivability.