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O'Shea, Erin

The remarkably stable circadian oscillations of single cyanobacteria enable a population of growing cells to maintain synchrony for weeks. The cyanobacterial pacemaker is a posttranslational regulation (PTR) circuit that generates circadian oscillations in the phosphorylation state of the clock protein KaiC. Layered on top of the PTR is transcriptional-translational feedback regulation (TTR), common to all circadian systems, consisting of a negative feedback loop in which KaiC regulates its own production. We found that the PTR circuit is sufficient to generate oscillations in growing cyanobacteria. However, in the absence of TTR, individual oscillators were less stable and synchrony was not maintained in a population of cells. Experimentally constrained mathematical modeling reproduced sustained oscillations in the PTR circuit alone and demonstrated the importance of TTR for oscillator synchrony.

The cyanobacterium Synechococcus elongatus PCC 7942 exhibits oscillations in mRNA transcript abundance with 24-h periodicity under continuous light conditions. The mechanism underlying these oscillations remains elusive—neither cis nor trans-factors controlling circadian gene expression phase have been identified. Here, we show that the topological status of the chromosome is highly correlated with circadian gene expression state. We also demonstrate that DNA sequence characteristics of genes that appear monotonically activated and monotonically repressed by chromosomal relaxation during the circadian cycle are similar to those of supercoiling-responsive genes in Escherichia coli. Furthermore, perturbation of superhelical status within the physiological range elicits global changes in gene expression similar to those that occur during the normal circadian cycle.

The circadian clock of the cyanobacterium Synechococcuselongatus PCC 7942 is governed by a core oscillator consisting of the proteins KaiA, KaiB, and KaiC. Remarkably, circadian oscillations in the phosphorylation state of KaiC can be reconstituted in a test tube by mixing the three Kai proteins and adenosine triphosphate. The in vitro oscillator provides a well-defined system in which experiments can be combined with mathematical analysis to understand the mechanism of a highly robust biological oscillator. In this Review, we summarize the biochemistry of the Kai proteins and examine models that have been proposed to explain how oscillations emerge from the properties of the oscillator’s constituents.

Background: Previous molecular and mechanistic studies have identified several principles of prokaryotic transcription, but less is known about the global transcriptional architecture of bacterial genomes. Here we perform a comprehensive study of a cyanobacterial transcriptome, that of Synechococcus elongatus PCC 7942, generated by combining three high-resolution data sets: RNA sequencing, tiling expression microarrays, and RNA polymerase chromatin immunoprecipitation sequencing. Results: We report absolute transcript levels, operon identification, and high-resolution mapping of 5' and 3' ends of transcripts. We identify several interesting features at promoters, within transcripts and in terminators relating to transcription initiation, elongation, and termination. Furthermore, we identify many putative non-coding transcripts. Conclusions: We provide a global analysis of a cyanobacterial transcriptome. Our results uncover insights that reinforce and extend the current views of bacterial transcription.

The cyanobacterium Synechococcus elongatus PCC 7942 exhibits global biphasic circadian oscillations in gene expression under constant-light conditions. Class I genes are maximally expressed in the subjective dusk, whereas class II genes are maximally expressed in the subjective dawn. Here, we identify sequence features that encode the phase of circadian gene expression. We find that, for multiple genes, an ∼70-nucleotide promoter fragment is sufficient to specify class I or II phase. We demonstrate that the gene expression phase can be changed by random mutagenesis and that a single-nucleotide substitution is sufficient to change the phase. Our study provides insight into how the gene expression phase is encoded in the cyanobacterial genome.

The cyanobacterial circadian pacemaker consists of a three-protein clock—KaiA, KaiB, and KaiC—that generates oscillations in the phosphorylation state of KaiC. Here we investigate how temporal information encoded in KaiC phosphorylation is transduced to RpaA, a transcription factor required for circadian gene expression. We show that phosphorylation of RpaA is regulated by two antagonistic histidine kinases, SasA and CikA, which are sequentially activated at distinct times by the Kai clock complex. SasA acts as a kinase toward RpaA, whereas CikA, previously implicated in clock input, acts as a phosphatase that dephosphorylates RpaA. CikA and SasA cooperate to generate an oscillation of RpaA activity that is distinct from that generated by either enzyme alone and offset from the rhythm of KaiC phosphorylation. Our observations reveal how circadian clocks can precisely control the timing of output pathways via the concerted action of two oppositely acting enzymes.

Phenotypic screens allow the identification of small molecules with promising anticancer activity, but the difficulty in characterizing the mechanism of action of these compounds in human cells often undermines their value as drug leads. Here, we used a loss-of-function genetic screen in human haploid KBM7 cells to discover the mechanism of action of the anticancer natural product ophiobolin A (OPA). We found that genetic inactivation of de novo synthesis of phosphatidylethanolamine (PE) mitigates OPA cytotoxicity by reducing cellular PE levels. OPA reacts with the ethanolamine head group of PE in human cells to form pyrrole-containing covalent cytotoxic adducts and these adducts lead to lipid bilayer destabilization. Our characterization of this unusual cytotoxicity mechanism, made possible by unbiased genetic screening in human cells, suggests that the selective antitumor activity displayed by OPA may be due to altered membrane PE levels in cancer cells. DOI: http://dx.doi.org/10.7554/eLife.14601.001

Background: Inorganic phosphate is an essential nutrient required by organisms for growth. During phosphate starvation, Saccharomyces cerevisiae activates the phosphate signal transduction (PHO) pathway, leading to expression of the secreted acid phosphatase, PHO5. The fission yeast, Schizosaccharomyces pombe, regulates expression of the ScPHO5 homolog (pho1+) via a non-orthologous PHO pathway involving genetically identified positive (pho7+) and negative (csk1+) regulators. The genes induced by phosphate limitation and the molecular mechanism by which pho7+ and csk1+ function are unknown. Here we use a combination of molecular biology, expression microarrays, and chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-Seq) to characterize the role of pho7+ and csk1+ in the PHO response. Results: We define the set of genes that comprise the initial response to phosphate starvation in S. pombe. We identify a conserved PHO response that contains the ScPHO5 (pho1+), ScPHO84 (SPBC8E4.01c), and ScGIT1 (SPBC1271.09) orthologs. We identify members of the Pho7 regulon and characterize Pho7 binding in response to phosphate-limitation and Csk1 activity. We demonstrate that activation of pho1+ requires Pho7 binding to a UAS in the pho1+ promoter and that Csk1 repression does not regulate Pho7 enrichment. Further, we find that Pho7-dependent activation is not limited to phosphate-starvation, as additional environmental stress response pathways require pho7+ for maximal induction. Conclusions: We provide a global analysis of the transcriptional response to phosphate limitation in S. pombe. Our results elucidate the conserved core regulon induced in response to phosphate starvation in this ascomycete distantly related to S. cerevisiae and provide a better understanding of flexibility in environmental stress response networks.

Fluctuations in organelle abundance can profoundly limit the precision of cell biological processes from secretion to metabolism. We modeled the dynamics of organelle biogenesis and predicted that organelle abundance fluctuations depend strongly on the specific mechanisms that increase or decrease the number of a given organelle. Our model exactly predicts the size of experimentally measured Golgi apparatus and vacuole abundance fluctuations, suggesting that cells tolerate the maximum level of variability generated by the Golgi and vacuole biogenesis pathways. We observe large increases in peroxisome abundance fluctuations when cells are transferred from glucose-rich to fatty acid-rich environments. These increased fluctuations are significantly diminished in mutants lacking peroxisome fission factors, leading us to infer that peroxisome biogenesis switches from de novo synthesis to primarily fission. Our work provides a general framework for exploring stochastic organelle biogenesis and using fluctuations to quantitatively unravel the biophysical pathways that control the abundance of subcellular structures. DOI: http://dx.doi.org/10.7554/eLife.02678.001

Understanding chromatin function requires knowing the precise location of nucleosomes. MNase-seq methods have been widely applied to characterize nucleosome organization in vivo, but generally lack the accuracy to determine the precise nucleosome positions. Here we develop a computational approach leveraging digestion variability to determine nucleosome positions at a base-pair resolution from MNase-seq data. We generate a variability template as a simple error model for how MNase digestion affects the mapping of individual nucleosomes. Applied to both yeast and human cells, this analysis reveals that alternatively positioned nucleosomes are prevalent and create significant heterogeneity in a cell population. We show that the periodic occurrences of dinucleotide sequences relative to nucleosome dyads can be directly determined from genome-wide nucleosome positions from MNase-seq. Alternatively positioned nucleosomes near transcription start sites likely represent different states of promoter nucleosomes during transcription initiation. Our method can be applied to map nucleosome positions in diverse organisms at base-pair resolution. DOI: http://dx.doi.org/10.7554/eLife.16970.001