GSE114693
An RpaA-dependent sigma factor cascade sets the timing of circadian transcriptional rhythms in Synechococcus elongatus
The circadian clock of the cyanobacterium Synechococcus elongatus PCC 7942 drives oscillations in global mRNA abundances with 24 hour periodicity under constant light conditions. The transcription factor RpaA, a circadian clock-regulated transcription factor, controls the timing of circadian gene expression, but the mechanisms underlying this control are not well understood. Here we show that four RpaA-dependent sigma factors – RpoD2, RpoD6, RpoD5, and SigF2 – are sequentially activated downstream of active RpaA and are required for proper expression of circadian mRNAs. We find that RpoD6, RpoD5, and SigF2 exhibit circadian oscillations with different timing relative to each other at the level of mRNA expression and protein abundance. By measuring global gene expression in strains modified to individually lack rpoD2, rpoD6, rpoD5, and sigF2 we identify how expression of circadian mRNAs – including sigma factor mRNAs – is altered in the absence of each sigma factor. Broadly, our findings suggest that a single transcription factor, RpaA, is sufficient to generate complex circadian expression patterns in part by regulating an interdependent sigma factor cascade. Overall design: Mapping of RNA polymerase locked at initiation sites over circadian time was determined by time-series ChIP-seq: immunoprecipitation of RNA polymerase bound to DNA in cells treated with rifampicin to lock RNA polymerase at initiation sites genome-wide.Transcriptome profiling by RNA-seq was performed over time in wild-type cells and in sigF2? cells in constant light conditions. Transcriptome profiling by RNA-seq was performed over time in OX-D53E cells (kaiBC?, rpaA?, Ptrc::rpaA(D53E), hereafter OX-D53E strain), in OX-D53E rpoD2? cells, in OX-D53E rpoD6? cells, and in OX-D53E rpoD5? cells grown in light following induction of active RpaA(D53E) by addition of IPTG after T=0h (dawn following two-consecutive 12h light-12h dark entrainment cycles).