Temporal mapping during a circadian day of binding sites for the BMAL1 transcription factor in mouse liver reveals genome-wide daily rhythms in DNA binding and uncovers output functions that are controlled by the circadian oscillator.

Gene regulation is a complex non-equilibrium process. Here, we show that quantitating the temporal regulation of key gene states (transcriptionally inactive, active, and refractory) provides a parsimonious framework for analyzing gene regulation. Our theory makes two non-intuitive predictions. First, for transcription factors (TFs) that regulate transcription burst frequency, as opposed to amplitude or duration, weak TF binding is sufficient to elicit strong transcriptional responses. Second, refractoriness of a gene after a transcription burst enables rapid responses to stimuli. We validate both predictions experimentally by exploiting the natural, optogenetic-like responsiveness of the Neurospora GATA-type TF White Collar Complex (WCC) to blue light. Further, we demonstrate that differential regulation of WCC target genes is caused by different gene activation rates, not different TF occupancy, and that these rates are tuned by both the core promoter and the distance between TF-binding site and core promoter. In total, our work demonstrates the relevance of a kinetic, non-equilibrium framework for understanding transcriptional regulation.

BackgroundCircadian clocks control rhythmic expression of a large number of genes in coordination with the 24 hour day-night cycle. The mechanisms generating circadian rhythms, their amplitude and circadian phase are dependent on a transcriptional network of immense complexity. Moreover, the contribution of post-transcriptional mechanisms in generating rhythms in RNA abundance is not known.ResultsHere, we analyzed the clock-controlled transcriptome of Neurospora crassa together with temporal profiles of elongating RNA polymerase II. Our data indicate that transcription contributes to the rhythmic expression of the vast majority of clock-controlled genes (ccgs) in Neurospora. The ccgs accumulate in two main clusters with peak transcription and expression levels either at dawn or dusk. Dawn-phased genes are predominantly involved in catabolic and dusk-phased genes in anabolic processes, indicating a clock-controlled temporal separation of the physiology of Neurospora. Genes whose expression is strongly dependent on the core circadian activator WCC fall mainly into the dawn-phased cluster while rhythmic genes regulated by the glucose-dependent repressor CSP1 fall predominantly into the dusk-phased cluster. Surprisingly, the number of rhythmic transcripts increases about twofold in the absence of CSP1, indicating that rhythmic expression of many genes is attenuated by the activity of CSP1.ConclusionsThe data indicate that the vast majority of transcript rhythms in Neurospora are generated by dawn and dusk specific transcription. Our observations suggest a substantial plasticity of the circadian transcriptome with respect to the number of rhythmic genes as well as amplitude and phase of the expression rhythms and emphasize a major role of the circadian clock in the temporal organization of metabolism and physiology.Electronic supplementary materialThe online version of this article (doi:10.1186/s12915-015-0126-4) contains supplementary material, which is available to authorized users.

Genes are often transcribed in random bursts followed by long periods of inactivity. Here we employ the light-activatable white collar complex (WCC) of Neurospora to study the transcriptional bursting with a population approach. Activation of WCC by a light pulse triggers a synchronized wave of transcription from the frequency promoter followed by an extended period (B1 h) during which the promoter is refractory towards restimulation. When challenged by a second light pulse, the newly activated WCC binds to refractory promoters and has the potential to recruit RNA polymerase II (Pol II). However, accumulation of Pol II and phosphorylation of its C-terminal domain repeats at serine 5 are impaired. Our results suggest that refractory promoters carry a physical memory of their recent transcription history. Genome-wide analysis of light-induced transcription suggests that refractoriness is rather widespread and a property of promoter architecture.

We show that firefly luciferase is a stable protein when expressed at 25°C in Neurospora, which limits its use as transcription reporter. We created a short-lived luciferase by fusing a PEST signal to its C-terminus (LUC-PEST) and applied the LUC-PEST reporter system to record in vivo transcription dynamics associated with the Neurospora circadian clock and its blue-light photosensory system over the course of several days. We show that the tool is suitable to faithfully monitor rapid, but also subtle changes in transcription in a medium to high throughput format.