Snf1-Dependent Transcription Confers Glucose-Induced Decay upon the mRNA Product.

In the yeast Saccharomyces cerevisiae, the switch from respiratory metabolism to fermentation causes rapid decay of transcripts encoding proteins uniquely required for aerobic metabolism. Snf1, the yeast ortholog of AMP-activated protein kinase, has been implicated in this process because inhibiting Snf1 mimics the addition of glucose. In this study, we show that the SNF1-dependent ADH2 promoter, or just the major transcription factor binding site, is sufficient to confer glucose-induced mRNA decay upon heterologous transcripts.

Yeast 14-3-3 protein functions as a comodulator of transcription by inhibiting coactivator functions.

In eukaryotes combinatorial activation of transcription is an important component of gene regulation. In the budding yeast Saccharomyces cerevisiae, Adr1-Cat8 and Adr1-Oaf1/Pip2 are pairs of activators that act together to regulate two diverse sets of genes. Transcription activation of both sets is regulated positively by the yeast AMP-activated protein kinase homolog, Snf1, in response to low glucose or the presence of a non-fermentable carbon source and negatively by two redundant 14-3-3 isoforms, Bmh1 and Bmh2.

Coupling mRNA synthesis and decay.

What has been will be again, what has been done will be done again; there is nothing new under the sun. -Ecclesiastes 1:9 (New International Version) Posttranscriptional regulation of gene expression has an important role in defining the phenotypic characteristics of an organism. Well-defined steps in mRNA metabolism that occur in the nucleus-capping, splicing, and polyadenylation-are mechanistically linked to the process of transcription.

Phosphoproteomic analysis identifies proteins involved in transcription-coupled mRNA decay as targets of Snf1 signaling.

Stresses, such as glucose depletion, activate Snf1, the Saccharomyces cerevisiae ortholog of adenosine monophosphate-activated protein kinase (AMPK), enabling adaptive cellular responses. In addition to affecting transcription, Snf1 may also promote mRNA stability in a gene-specific manner. To understand Snf1-mediated signaling, we used quantitative mass spectrometry to identify proteins that were phosphorylated in a Snf1-dependent manner.

Binding and transcriptional regulation by 14-3-3 (Bmh) proteins requires residues outside of the canonical motif.

Evolutionarily conserved 14-3-3 proteins have important functions as dimers in numerous cellular signaling processes, including regulation of transcription. Yeast 14-3-3 proteins, known as Bmh, inhibit a post-DNA binding step in transcription activation by Adr1, a glucose-regulated transcription factor, by binding to its regulatory domain, residues 226 to 240. The domain was originally defined by regulatory mutations, ADR1(c) alleles that alter activator-dependent gene expression.

14-3-3 (Bmh) proteins regulate combinatorial transcription following RNA polymerase II recruitment by binding at Adr1-dependent promoters in Saccharomyces cerevisiae.

Adr1 and Cat8 are nutrient-regulated transcription factors in Saccharomyces cerevisiae that coactivate genes necessary for growth in the absence of a fermentable carbon source. Transcriptional activation by Adr1 is dependent on the AMP-activated protein kinase Snf1 and is inhibited by binding of Bmh, yeast 14-3-3 proteins, to the phosphorylated Adr1 regulatory domain. We show here that Bmh inhibits transcription by binding to Adr1 at promoters that contain a preinitiation complex, demonstrating that Bmh-mediated inhibition is not due to nuclear exclusion, inhibition of DNA binding, or RNA polymerase II (Pol II) recruitment.

The AMP-activated protein kinase Snf1 regulates transcription factor binding, RNA polymerase II activity, and mRNA stability of glucose-repressed genes in Saccharomyces cerevisiae.

AMP-activated protein kinase, the "energy sensor of the cell," responds to low cellular energy stores by regulating enzymes and transcription factors that allow the cell to adapt to limiting nutritional conditions. Snf1, the yeast ortholog of AMP-activated protein kinase, has an essential role in respiratory metabolism in Saccharomyces cerevisiae that includes activating the transcription factor Adr1. How Snf1 regulates Adr1 activity is poorly understood.

Pichia pastoris 14-3-3 regulates transcriptional activity of the methanol inducible transcription factor Mxr1 by direct interaction.

The zinc-finger transcription factor, Mxr1 activates methanol utilization and peroxisome biogenesis genes in the methylotrophic yeast, Pichia pastoris. Expression of Mxr1-dependent genes is regulated in response to various carbon sources by an unknown mechanism. We show here that this mechanism involves the highly conserved 14-3-3 proteins.

Snf1/AMPK regulates Gcn5 occupancy, H3 acetylation and chromatin remodelling at S. cerevisiae ADY2 promoter.

The ability of cells to respond to changes in their environment is mediated by transcription factors that remodel chromatin and reprogram expression of specific subsets of genes. In Saccharomyces cerevisiae, changes in carbon source lead to gene induction by Adr1 and Cat8 that are known to require the upstream function of the Snf1 protein kinase, the central regulator of carbon metabolism, to exert their activating effect. How Snf1 facilitates transcription activation by Adr1 and Cat8 is not known.

Analysis of nucleosome positioning using a nucleosome-scanning assay.

The nucleosome-scanning assay (NuSA) couples isolation of mononucleosomal DNA after micrococcal nuclease (MNase) digestion with quantitative real-time PCR (qPCR) to map nucleosome positions in chromatin. It is a relatively simple, rapid procedure that can produce a high-resolution map of nucleosome location and occupancy and thus is suitable for analyzing individual promoters in great detail. The analysis can also quantify the protection of DNA sequences due to interaction with proteins other than nucleosomes and show how this protection varies when conditions change.

Transcriptional regulation in Saccharomyces cerevisiae: transcription factor regulation and function, mechanisms of initiation, and roles of activators and coactivators.

Here we review recent advances in understanding the regulation of mRNA synthesis in Saccharomyces cerevisiae. Many fundamental gene regulatory mechanisms have been conserved in all eukaryotes, and budding yeast has been at the forefront in the discovery and dissection of these conserved mechanisms. Topics covered include upstream activation sequence and promoter structure, transcription factor classification, and examples of regulated transcription factor activity.

Toward a global analysis of metabolites in regulatory mutants of yeast.

The AMP-activated protein kinase in yeast, Snf1, coordinates expression and activity of numerous intracellular signaling and developmental pathways, including those regulating cellular differentiation, response to stress, meiosis, autophagy, and the diauxic transition. Snf1 phosphorylates metabolic enzymes and transcription factors to change cellular physiology and metabolism. Adr1 and Cat8, transcription factors that activate gene expression after the diauxic transition, are regulated by Snf1; Cat8 through direct phosphorylation and Adr1 by dephosphorylation in a Snf1-dependent manner.

Activator-independent transcription of Snf1-dependent genes in mutants lacking histone tails.

Transcriptional regulation of Snf1-dependent genes occurs in part by histone-acetylation-dependent binding of the transcription factor Adr1. Analysis of previously published microarray data indicated unscheduled transcription of a large number of Snf1- and Adr1-dependent genes when either the histone H3 or H4 tail was deleted. Quantitative real-time PCR confirmed that the tails were important to preserve stringent transcriptional repression of Snf1-dependent genes when glucose was present.

Snf1 dependence of peroxisomal gene expression is mediated by Adr1.

Eukaryotes utilize fatty acids by beta-oxidation, which occurs in the mitochondria and peroxisomes in higher organisms and in the peroxisomes in yeast. The AMP-activated protein kinase regulates this process in mammalian cells, and its homolog Snf1, together with the transcription factors Adr1, Oaf1, and Pip2, regulates peroxisome proliferation and beta-oxidation in yeast. A constitutive allele of Adr1 (Adr1(c)) lacking the glucose- and Snf1-regulated phosphorylation substrate Ser-230 was found to be Snf1-independent for regulation of peroxisomal genes.

Snf1-independent, glucose-resistant transcription of Adr1-dependent genes in a mediator mutant of Saccharomyces cerevisiae.

Glucose represses transcription of a network of co-regulated genes in Saccharomyces cerevisiae, ensuring that it is utilized before poorer carbon sources are metabolized. Adr1 is a glucose-regulated transcription factor whose promoter binding and activity require Snf1, the yeast homologue of the AMP-activated protein kinase in higher eukaryotes. In this study we found that a temperature-sensitive allele of MED14, a Mediator middle subunit that tethers the tail to the body, allowed a low level of Adr1-independent ADH2 expression that can be enhanced by Adr1 in a dose-dependent manner.

Snf1 controls the activity of adr1 through dephosphorylation of Ser230.

The transcription factors Adr1 and Cat8 act in concert to regulate the expression of numerous yeast genes after the diauxic shift. Their activities are regulated by Snf1, the yeast homolog of the AMP-activated protein kinase of higher eukaryotes. Cat8 is regulated directly by Snf1, but how Snf1 regulates Adr1 is unknown.

The disorderly study of ordered recruitment.

Activated transcription in eukaryotes requires the aid of numerous co-factors to overcome the physical barriers chromatin poses to activation, bridge the gap between activators and polymerase, and ensure appropriate regulation. S. cerevisiae has long been a model organism for studying the role of co-activators in the steps leading up to gene activation.

The transcriptional coactivators SAGA, SWI/SNF, and mediator make distinct contributions to activation of glucose-repressed genes.

The paradigm of activation via ordered recruitment has evolved into a complicated picture as the influence of coactivators and chromatin structures on gene regulation becomes understood. We present here a comprehensive study of many elements of activation of ADH2 and FBP1, two glucose-regulated genes. We identify SWI/SNF as the major chromatin-remodeling complex at these genes, whereas SAGA (Spt-Ada-Gcn5-acetyltransferase complex) is required for stable recruitment of other coactivators.

Time-dependent profiling of metabolites from Snf1 mutant and wild type yeast cells.

The effect of sampling time in the context of growth conditions on a dynamic metabolic system was investigated in order to assess to what extent a single sampling time may be sufficient for general application, as well as to determine if useful kinetic information could be obtained. A wild type yeast strain (W) was compared to a snf1Delta mutant yeast strain (S) grown in high-glucose medium (R) and in low-glucose medium containing ethanol (DR). Under these growth conditions, different metabolic pathways for utilizing the different carbon sources are expected to be active.

Promoter binding by the Adr1 transcriptional activator may be regulated by phosphorylation in the DNA-binding region.

Background: Post-translational modification regulates promoter-binding by Adr1, a Zn-finger transcriptional activator of glucose-regulated genes. Support for this model includes the activation of an Adr1-dependent gene in the absence of Adr1 protein synthesis, and a requirement for the kinase Snf1 for Adr1 DNA-binding. A fusion protein with the Adr1 DNA-binding domain and a heterologous activation domain is glucose-regulated, suggesting that the DNA binding region is the target of regulation.

Artificial recruitment of mediator by the DNA-binding domain of Adr1 overcomes glucose repression of ADH2 expression.

The transcription factor Adr1 activates numerous genes in nonfermentable carbon source metabolism. An unknown mechanism prevents Adr1 from stably binding to the promoters of these genes in glucose-grown cells. Glucose depletion leads to Snf1-dependent binding.

Adr1 and Cat8 mediate coactivator recruitment and chromatin remodeling at glucose-regulated genes.

Background: Adr1 and Cat8 co-regulate numerous glucose-repressed genes in S. cerevisiae, presenting a unique opportunity to explore their individual roles in coactivator recruitment, chromatin remodeling, and transcription.

Methodology/principal Findings: We determined the individual contributions of Cat8 and Adr1 on the expression of a cohort of glucose-repressed genes and found three broad categories: genes that need both activators for full derepression, genes that rely mostly on Cat8 and genes that require only Adr1.

Identification and evaluation of cycling yeast metabolites in two-dimensional comprehensive gas chromatography-time-of-flight-mass spectrometry data.

A yeast metabolome exhibiting oscillatory behavior was analyzed using comprehensive two-dimensional gas chromatography-time-of-flight-mass spectrometry (GC x GC-TOF-MS) and in-house developed data analysis software methodology, referred to as a signal ratio method (S(ratio) method). In this study, 44 identified unique metabolites were found to exhibit cycling, with a depth-of-modulation amplitude greater than three. After the initial locations are found using the S(ratio) software, and identified preliminarily using ChromaTOF software, the refined mass spectra and peak volumes were subsequently obtained using parallel factor analysis (PARAFAC).

A poised initiation complex is activated by SNF1.

Snf1, the yeast AMP kinase homolog, is essential for derepression of glucose-repressed genes that are activated by Adr1. Although required for Adr1 DNA binding, the precise role of Snf1 is unknown. Deletion of histone deacetylase genes allowed constitutive promoter binding of Adr1 and Cat8, another activator of glucose-repressed genes.

Cyclic changes in metabolic state during the life of a yeast cell.

Budding yeast undergo robust oscillations in oxygen consumption during continuous growth in a nutrient-limited environment. Using liquid chromatography-mass spectrometry and comprehensive 2D gas chromatography-mass spectrometry-based metabolite profiling methods, we have determined that the intracellular concentrations of many metabolites change periodically as a function of these metabolic cycles. These results reveal the logic of cellular metabolism during different phases of the life of a yeast cell.