Abstract
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 (∼1 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.
Highlights
Genes are often transcribed in random bursts followed by long periods of inactivity
We show that activation of the white collar complex (WCC) by a single short light pulse (LP) triggers a synchronized wave of transcription at a large number of promoters
A number of features suggested that the light-activatable transcription factor WCC of Neurospora and the frequency promoter are suitable for studying biochemical processes associated with transcriptional bursting in a synchronized population approach (Supplementary Fig. 1a,b). (i) In darkness frq is rhythmically transcribed at low levels under control of WCC that binds to the clock-box located B1.2 kb upstream of the transcription start site[28]
Summary
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. Recent evidence suggests that processes associated with transcription itself appear to induce extended time periods during which genes are refractory towards activation[12,22,23]. Molecular mechanisms underlying such refractoriness of promoters are not known. By analysing burst statistics under distinct experimental regimes, single-cell-based studies provide valuable insight into the dynamics of transcription They are generally not suited for unravelling underlying biochemical processes, as conventional biochemical measurements are usually performed on an ensemble of cells and cannot readily be applied to single cells. Genome-wide analysis indicates that many light-inducible promoters become refractory after activation and suggests that the extent of refractoriness is a promoter-specific feature
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