Elongating under Stress

In response to extracellular stimuli, mitogen-activated protein kinases (MAPKs) modulate gene expression to maximize cell survival. Exposure of yeast to high osmolarity results in activation of the p38-related MAPK Hog1, which plays a key role in reprogramming the gene expression pattern required for cell survival upon osmostress. Hog1 not only regulates initiation but also modulates other steps of the transcription process. Recent work indicates that other yeast signalling MAPKs such as Mpk1 modulate transcriptional elongation in response to cell wall stress. Similarly, mammalian MAPKs have also been found associated to coding regions of stress-responsive genes. In this paper, significant progress in MAPK-regulated events that occur during the transcriptional elongation step is summarized, and future directions are discussed. We expect that the principles learned from these studies will provide a new understanding of the regulation of gene expression by signalling kinases

Biochemical characterization of recombinant yeast PPZ1, a protein phosphatase involved in salt tolerance

AbstractThe Saccharomyces cerevisiae gene PPZ1 codes for a 692-residues protein that shows in its carboxyl-terminal half about 60% identity with the catalytic subunit of mammalian and yeast protein phosphatase-1 and that is involved in salt homeostasis. T The complete PPZ1 protein has been succesfully expressed as a soluble glutathione-S-transferase fusion protein. The recombinant protein, after purification by a single affinity chromatography step, displayed phosphatase activity towards a number of substrates, including myelin basic protein, histone 2A and casein, but was ineffective in dephosphorylating glycogen phosphorylase. It was also active towards p-nitrophenylphosphate. The activity was severalfold increased by the presence of Mn2+ ions and by limited trypsinolysis. The enzyme was inhibited by okadaic acid and microcystin-LR at concentrations comparable to what is found for type 1 protein phosphatase although it was much less sensitive to inhibitor-2. The recombinant protein was phosphorylated in vitro by cAMP-dependent protein kinase, protein kinase C and casein kinase-2. Phosphorylation affected preferentially sites located in the amino-terminal half of the protein and did not alter the activity of the phosphatase

Shaping the Transcriptional Landscape through MAPK Signaling

A change in the transcriptional landscape is an equilibrium-breaking event important for many biological processes. Mitogen-activated protein kinase (MAPK) signaling pathways are dedicated to sensing extracellular cues and are highly conserved across eukaryotes. Modulation of gene expression in response to the extracellular environment is one of the main mechanisms by which MAPK regulates proteome homeostasis to orchestrate adaptive responses that determine cell fate. A massive body of knowledge generated from population and single-cell analyses has led to an understanding of how MAPK pathways operate. MAPKs have thus emerged as fundamental transcriptome regulators that function through a multi-layered control of gene expression, a process often deregulated in disease, which therefore provides an attractive target for therapeutic strategies. Here, we summarize the current understanding of the mechanisms underlying MAPK-mediated gene expression in organisms ranging from yeast to mammals

Biophysical properties of Saccharomyces cerevisiae and their relationship with HOG pathway activation

Parameterized models of biophysical and mechanical cell properties are important for predictive mathematical modeling of cellular processes. The concepts of turgor, cell wall elasticity, osmotically active volume, and intracellular osmolarity have been investigated for decades, but a consistent rigorous parameterization of these concepts is lacking. Here, we subjected several data sets of minimum volume measurements in yeast obtained after hyper-osmotic shock to a thermodynamic modeling framework. We estimated parameters for several relevant biophysical cell properties and tested alternative hypotheses about these concepts using a model discrimination approach. In accordance with previous reports, we estimated an average initial turgor of 0.6 ± 0.2 MPa and found that turgor becomes negligible at a relative volume of 93.3 ± 6.3% corresponding to an osmotic shock of 0.4 ± 0.2 Osm/l. At high stress levels (4 Osm/l), plasmolysis may occur. We found that the volumetric elastic modulus, a measure of cell wall elasticity, is 14.3 ± 10.4 MPa. Our model discrimination analysis suggests that other thermodynamic quantities affecting the intracellular water potential, for example the matrix potential, can be neglected under physiological conditions. The parameterized turgor models showed that activation of the osmosensing high osmolarity glycerol (HOG) signaling pathway correlates with turgor loss in a 1:1 relationship. This finding suggests that mechanical properties of the membrane trigger HOG pathway activation, which can be represented and quantitatively modeled by turgor

Whole genome analysis of p38 SAPK-mediated gene expression upon stress

<p>Abstract</p> <p>Background</p> <p>Cells have the ability to respond and adapt to environmental changes through activation of stress-activated protein kinases (SAPKs). Although p38 SAPK signalling is known to participate in the regulation of gene expression little is known on the molecular mechanisms used by this SAPK to regulate stress-responsive genes and the overall set of genes regulated by p38 in response to different stimuli.</p> <p>Results</p> <p>Here, we report a whole genome expression analyses on mouse embryonic fibroblasts (MEFs) treated with three different p38 SAPK activating-stimuli, namely osmostress, the cytokine TNFα and the protein synthesis inhibitor anisomycin. We have found that the activation kinetics of p38α SAPK in response to these insults is different and also leads to a complex gene pattern response specific for a given stress with a restricted set of overlapping genes. In addition, we have analysed the contribution of p38α the major p38 family member present in MEFs, to the overall stress-induced transcriptional response by using both a chemical inhibitor (SB203580) and p38α deficient (p38α^-/-) MEFs. We show here that p38 SAPK dependency ranged between 60% and 88% depending on the treatments and that there is a very good overlap between the inhibitor treatment and the ko cells. Furthermore, we have found that the dependency of SAPK varies depending on the time the cells are subjected to osmostress.</p> <p>Conclusions</p> <p>Our genome-wide transcriptional analyses shows a selective response to specific stimuli and a restricted common response of up to 20% of the stress up-regulated early genes that involves an important set of transcription factors, which might be critical for either cell adaptation or preparation for continuous extra-cellular changes. Interestingly, up to 85% of the up-regulated genes are under the transcriptional control of p38 SAPK. Thus, activation of p38 SAPK is critical to elicit the early gene expression program required for cell adaptation to stress.</p

Structural disruption of BAF chromatin remodeller impairs neuroblastoma metastasis by reverting an invasiveness epigenomic program

Background Epigenetic programming during development is essential for determining cell lineages, and alterations in this programming contribute to the initiation of embryonal tumour development. In neuroblastoma, neural crest progenitors block their course of natural differentiation into sympathoadrenergic cells, leading to the development of aggressive and metastatic paediatric cancer. Research of the epigenetic regulators responsible for oncogenic epigenomic networks is crucial for developing new epigenetic-based therapies against these tumours. Mammalian switch/sucrose non-fermenting (mSWI/SNF) ATP-dependent chromatin remodelling complexes act genome-wide translating epigenetic signals into open chromatin states. The present study aimed to understand the contribution of mSWI/SNF to the oncogenic epigenomes of neuroblastoma and its potential as a therapeutic target. Methods Functional characterisation of the mSWI/SNF complexes was performed in neuroblastoma cells using proteomic approaches, loss-of-function experiments, transcriptome and chromatin accessibility analyses, and in vitro and in vivo assays. Results Neuroblastoma cells contain three main mSWI/SNF subtypes, but only BRG1-associated factor (BAF) complex disruption through silencing of its key structural subunits, ARID1A and ARID1B, impairs cell proliferation by promoting cell cycle blockade. Genome-wide chromatin remodelling and transcriptomic analyses revealed that BAF disruption results in the epigenetic repression of an extensive invasiveness-related expression program involving integrins, cadherins, and key mesenchymal regulators, thereby reducing adhesion to the extracellular matrix and the subsequent invasion in vitro and drastically inhibiting the initiation and growth of neuroblastoma metastasis in vivo. Conclusions We report a novel ATPase-independent role for the BAF complex in maintaining an epigenomic program that allows neuroblastoma invasiveness and metastasis, urging for the development of new BAF pharmacological structural disruptors for therapeutic exploitation in metastatic neuroblastoma

Control of Ubp3 ubiquitin protease activity by the Hog1 SAPK modulates transcription upon osmostress

Here, the Hog1 kinase interacts with and activates the ubiquitin protease Ubp3 in a stress-dependent manner. The phosphorylation of Ubp3 enhances RNA polymerase II occupancy on osmotic stress-responsive genes

Design, Synthesis and Characterization of a Highly Effective Inhibitor for Analog-Sensitive (as) Kinases

Highly selective, cell-permeable and fast-acting inhibitors of individual kinases are sought-after as tools for studying the cellular function of kinases in real time. A combination of small molecule synthesis and protein mutagenesis, identified a highly potent inhibitor (1-Isopropyl-3-(phenylethynyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine) of a rationally engineered Hog1 serine/threonine kinase (Hog1T100G). This inhibitor has been successfully used to study various aspects of Hog1 signaling, including a transient cell cycle arrest and gene expression changes mediated by Hog1 in response to stress. This study also underscores that the general applicability of this approach depends, in part, on the selectivity of the designed the inhibitor with respect to activity versus the engineered and wild type kinases. To explore this specificity in detail, we used a validated chemogenetic assay to assess the effect of this inhibitor on all gene products in yeast in parallel. The results from this screen emphasize the need for caution and for case-by-case assessment when using the Analog-Sensitive Kinase Allele technology to assess the physiological roles of kinases