Mechanisms of organelle biogenesis govern stochastic fluctuations in organelle abundance

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.02678.00

Two Antagonistic Clock-Regulated Histidine Kinases Time the Activation of Circadian Gene Expression

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.Chemistry and Chemical BiologyMolecular and Cellular Biolog

Hog1 Controls Global Reallocation of RNA Pol II upon Osmotic Shock in Saccharomyces cerevisiae

When challenged with osmotic shock, Saccharomyces cerevisiae induces hundreds of genes, despite a concurrent reduction in overall transcriptional capacity. The stress-responsive MAP kinase Hog1 activates expression of specific genes through interactions with chromatin remodeling enzymes, transcription factors, and RNA polymerase II. However, it is not clear whether Hog1 is involved more globally in modulating the cell’s transcriptional program during stress, in addition to activating specific genes. Here we show that large-scale redistribution of RNA Pol II from housekeeping to stress genes requires Hog1. We demonstrate that decreased RNA Pol II occupancy is the default outcome for highly expressed genes upon stress and that Hog1 is partially required for this effect. We find that Hog1 and RNA Pol II colocalize to open reading frames that bypass global transcriptional repression. These activation targets are specified by promoter binding of two osmotic stress-responsive transcription factors. The combination of reduced global transcription with a gene-specific override mechanism allows cells to rapidly switch their transcriptional program in response to stress.Chemistry and Chemical Biolog

Sequence Determinants of Circadian Gene Expression Phase in Cyanobacteria

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.Chemistry and Chemical BiologyMolecular and Cellular Biolog

The Molecular Clockwork of a Protein-Based Circadian Oscillator

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.Molecular and Cellular Biolog

Oscillations in Supercoiling Drive Circadian Gene Expression in Cyanobacteria

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.Molecular and Cellular Biolog

A high resolution map of a cyanobacterial transcriptome

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.Molecular and Cellular Biolog

Genome-Wide Characterization of the Phosphate Starvation Response in Schizosaccharomyces pombe

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.Chemistry and Chemical BiologyMolecular and Cellular Biolog

The Innate Immune Protein Nod2 Binds Directly to MDP, a Bacterial Cell Wall Fragment

Mammalian Nod2 is an intracellular protein that is implicated in the innate immune response to the bacterial cell wall and is associated with the development of Crohn’s disease, Blau syndrome, and gastrointestinal cancers. Nod2 is required for an immune response to muramyl dipeptide (MDP), an immunostimulatory fragment of bacterial cell wall, but it is not known whether MDP binds directly to Nod2. We report the expression and purification of human Nod2 from insect cells. Using novel MDP self-assembled monolayers (SAMs), we provide the first biochemical evidence for a direct, high-affinity interaction between Nod2 and MDP.Molecular and Cellular Biolog

Regulation of Chromatin Remodeling by Inositol Polyphosphates

Chromatin remodeling is required for efficient transcription of eukaryotic genes. In a genetic selection for budding yeast mutants that were defective in induction of the phosphate-responsive PHO5 gene, we identified mutations inARG82/IPK2, which encodes a nuclear inositol polyphosphate kinase. In arg82 mutant strains, remodeling ofPHO5 promoter chromatin is impaired, and the adenosine triphosphate–dependent chromatin-remodeling complexes SWI/SNF and INO80 are not efficiently recruited to phosphate-responsive promoters. These results suggest a role for the small molecule inositol polyphosphate in the regulation of chromatin remodeling and transcription