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An RpaA-dependent sigma factor cascade sets the timing of circadian transcriptional rhythms in Synechococcus elongatus

ABSTRACT: 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.

ORGANISM(S): Synechococcus elongatus PCC 7942 = FACHB-805

PROVIDER: GSE114693 | GEO | 2019/05/20

REPOSITORIES: GEO

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Project description:The cyanobacterium Synechococcus elongatus contains a circadian clock which coordinates circadian changes in gene expression of a large percentage of its genes. The response regulator RpaA has been implicated as an important regulator of many circadian genes, but the role of this protein in regulating changes in gene expression genome-wide is not known. We show that deletion of rpaA abrogates circadian gene expression genome-wide and arrests cells in a gene expression state highly similar to that of wildtype cells in the morning. Furthermore, we show that RpaA binds DNA in an circadian manner that is dependent on phosphorylation of the protein. To demonstrate the sufficiency of phosphorylated RpaA in driving global changes in gene expression, we used RNA sequencing to measure changes in gene expression elicited by a phosphomimetic of RpaA (RpaA D53E) and compared these changes to those that occur during a circadian cycle in wildtype cells. This analysis reveals that induction of RpaA D53E is sufficient to drive all circadian gene expression changes that happen from dawn to dusk in wildtype cells. Interestingly, the dynamics of gene expression elicited by RpaA D53E induction mirror those observed during a circadian cycle in wildtype cells, suggesting that the dynamics of circadian gene expression and hard-wired in the regulon downstream of RpaA. Enriched mRNA was prepared from synchronized wildtype S. elongatus cells every four hours over a circadian cycle and sequenced using the Illumina TruSeq Stranded mRNA Sample Prep Kit and Illumina HiSeq technology. To test the role of phosphorylated RpaA in driving circadian gene expression, we generated a strain which we refer to as OX-D53E that lacks core clock components (M-NM-^TrpaA, M-NM-^TkaiBC) with an RpaA phosphomimetic (RpaA D53E) under the control of an IPTG-inducible promoter (Ptrc::rpaAD53E). We used the same methodology to measure gene expression in OX-D53E before and after induction of RpaA D53E. As a control, we also measured gene expression in the OX-D53E strain over time in the absence of IPTG. Also, we generated a strain similar to OX-D53E in which the only difference was that no gene was inserted downstream of the IPTG inducible promoter (OX-Empty). We measured gene expressio in OX-Empty before and after IPTG addition to test for off-target effects of IPTG.

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An RpaA-dependent sigma factor cascade sets the timing of circadian transcriptional rhythms in Synechococcus elongatus

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