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ChIP-Seq analysis of RpaA binding dynamics

ABSTRACT: The response regulator RpaA is required for control of genome-wide gene expression by the cyanobacterial circadian clock. RpaA is predicted to be a DNA binding protein based on sequence homology, but prior studies have been unable to detect binding in vitro or in vivo to a small panel of promoters. We used ChIP-Seq to determine whether RpaA associates with DNA in vivo, and if so, with what dynamics. We find that RpaA binds to over 100 location in the genome in a circadian manner, with strongest binding occuring around subjective dusk. Analysis of these binding sites shows that RpaA directly regulates the expression of clock components to generate feedback on the core oscillator, and also regulates expression of a small set of circadian effectors that in turn orchestrate global expression rhythms.

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

PROVIDER: GSE51093 | GEO | 2013/12/04

SECONDARY ACCESSION(S): PRJNA221155

REPOSITORIES: GEO

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ChIP-Seq analysis of RpaA binding dynamics

Project description:The response regulator RpaA is required for control of genome-wide gene expression by the cyanobacterial circadian clock. RpaA is predicted to be a DNA binding protein based on sequence homology, but prior studies have been unable to detect binding in vitro or in vivo to a small panel of promoters. We used ChIP-Seq to determine whether RpaA associates with DNA in vivo, and if so, with what dynamics. We find that RpaA binds to over 100 location in the genome in a circadian manner, with strongest binding occuring around subjective dusk. Analysis of these binding sites shows that RpaA directly regulates the expression of clock components to generate feedback on the core oscillator, and also regulates expression of a small set of circadian effectors that in turn orchestrate global expression rhythms. Crosslinked samples were acquired every four hours from a turbidostatic wild-type (AMC408) cultures in constant light (LL) following entrainment with two light-dark (LD) cycles. As a negative control, we acquired samples similarly from an M-NM-^TrpaA culture. Chromatin immunoprecipitation was performed on each sample from wht wild-type and from a pool of all samples from the M-NM-^TrpaA culture. Libraries were prepared from the ChIP samples and sequenced with Illumina technology. For the wild-type, biological replicate samples were acquired at times of maximum and minimum RpaA binding.

2013-12-04 | E-GEOD-51093 | biostudies-arrayexpress

The role of the Synechocystis sp. PCC 6803 homolog of the circadian clock output regulator RpaA in day-night transitions

Project description:Like many other organisms, cyanobacteria exhibit rhythmic gene expression with a period length of 24 hours to adapt to daily environmental changes. In the model organism Synechococcus elongatus PCC 7942 the central oscillator consists of three proteins: KaiA, KaiB and KaiC and utilizes the histidine kinase SasA and its response regulator RpaA as output-signaling pathway. Synechocystis sp. PCC 6803 contains two additional homologs of the kaiB and kaiC genes. Here we demonstrate that RpaA interacts with the core oscillator KaiAB1C1 of Synechocystis sp. PCC 6803 via SasA, similar to Synechococcus elongatus PCC 7942. However, interaction with the additional Kai homologs was not detected, suggesting different signal transduction components for the clock homologs. Inactivation of rpaA in Synechocystis sp. PCC 6803, lead to reduced viability of the mutant in light-dark cycles that aggravated under mixotrophic growth conditions. Chemoheterotrophic growth in the dark was abolished completely. In accordance, transcriptomic data revealed that RpaA is involved in the regulation of genes related to CO2‑acclimation and carbon metabolism under diurnal light conditions. Further, our results indicate that RpaA functions in the posttranslational regulation of glycogen metabolism as well, and a potential link between the circadian clock and motility was identified.

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

Project description: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.

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Project description:The transcription factor RpaA is the master regulator of circadian transcription in cyanobacteria, driving genome-wide oscillations in mRNA abundance. Deletion of rpaA has no effect on viability in constant light conditions, but renders cells inviable in cycling conditions when light and dark periods alternate. We investigated the mechanisms underlying this viability defect. We performed RNA-seq experiment using the rpaA- “clock rescue” and “clock rescue” strains and we show that the rpaA- “clock rescue” strain is defective in transcription of genes crucial for utilization of carbohydrate stores at night.

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Global Mapping at the Core of the Arabidopsis Circadian Clock Defines the Network Structure of the Oscillator

2012-03-08 | E-GEOD-35952 | biostudies-arrayexpress

Circadian and diurnal expression data of Synechococcus

Project description:In the unicellular cyanobacterium, Synechococcus elongatus PCC 7942, essentially all promoter activities are under the control of the circadian clock in continuous light (LL) conditions. Here, we employed high-density oligonucleotide arrays to explore comprehensive profiles of genome-wide Synechococcus gene expression in wild type, kaiABC-null and kaiC-overexpressor strains under LL and continuous dark (DD) conditions. In the wild type strain more than 30% of transcripts significantly oscillated in a circadian fashion, peaking at subjective dawn and dusk. Such circadian control was nullified in kaiABC-null strains. Although KaiC has been proposed to globally repress gene expression, our analysis revealed that dawn expressing genes were upregulated by kaiC-overexpression, such that the clock was arrested at subjective dawn. Transfer of cells to continuous dark (DD) conditions from LL immediately suppressed expression of most of genes, while the clock keeps time even in the absence of transcriptional feedback. Thus, the Synechococcus genome seems primarily regulated by the light/dark cycles and dramatically modified by the protein-based circadian oscillator. Keywords: timecourse data (~48 hours under continuous light or darkness) from Synechococcus elongatus PCC 7942 (wild type, kaiABC-null, and inducible kaiC-overexpressor) strains

2009-08-01 | GSE14225 | GEO

Circadian expression data of Synechococcus in WT, rpoD6-null, sasA-null and rpaA-null strains

Project description:In the unicellular cyanobacterium, Synechococcus elongatus PCC 7942, most genes show rhythmic expression controlled by the Kai-based clock under continuous light conditions (LL). We found that rpoD6-null mutants impaired expression of clock-controlled genes peaking at hours 8-10 in LL, while sasA-null or rpaA-null mutants each arrested the expression profiles at subjective dawn. Time-course data (0-24 h) of wild type (WT), rpoD6-null, sasA-null and rpaA-null S. elongatus PCC 7942 strains analyzed under continuous light (LL) conditions after two 12h:12h light:dark (LD) cycles using Affymetrix high-density oligonucleotide microarrays (GeneChip CustomExpress Arrays) representing 2,515 predicted protein-coding genes on the genome of Synechococcus elongatus PCC 6301, which can be used also for the almost homologous strain, S. elongatus PCC 7942.

2012-03-01 | E-GEOD-28430 | biostudies-arrayexpress

Analysis of the regulation of circadian gene expression in cyanobacteria with RNA sequencing

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.

2013-12-04 | GSE51112 | GEO

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