Measuring mRNA Levels Over Time During the Yeast <em>S. cerevisiae</em> Hypoxic Response

Complex changes in gene expression typically mediate a large portion of a cellular response. Each gene may change expression with unique kinetics as the ...

Rowan University

Until now, it has been difficult to establish exactly how a specific DNA lesion signals apoptosis because each DNA damaging agent produces a collection of distinct DNA lesions and produces damage in RNA, protein, and lipids. We have developed a system in human cells that focuses on the response to a single type of DNA lesion, namely O(6)-methylguanine (O(6)MeG). We dissect the signaling pathways involved in O(6)MeG-induced apoptosis, a response dependent on the MutSalpha heterodimer that is normally involved in DNA mismatch repair. O(6)MeG triggers robust activation of caspases associated with both death receptor- and mitochondrial-mediated apoptosis. Despite this, O(6)MeG/MutSalpha-triggered apoptosis is only partly dependent on caspase activation; moreover, it is mediated solely by mitochondrial signaling and not at all by death receptor signaling. Finally, while Bcl-2 and Bcl-x(L), negative regulators of mitochondrial-regulated apoptosis, could effectively block O(6)MeG/MutSalpha-dependent apoptosis, they were unable to prevent the cells from ultimately dying.

Apoptotic signaling in response to a single type of DNA lesion, O(6)-methylguanine.

Changes in oxygen levels cause widespread changes in gene expression in organisms ranging from bacteria to humans. In Saccharomyces cerevisiae, this response is mediated in part by Hap1, originally identified as a heme-dependent transcriptional activator that functions during aerobic growth. We show here that Hap1 also plays a significant and direct role under hypoxic conditions, not as an activator, but as a repressor. The repressive activity of Hap1 controls several genes, including three ERG genes required for ergosterol biosynthesis. Chromatin immunoprecipitation experiments showed that Hap1 binds to the ERG gene promoters, while additional experiments showed that the corepressor Tup1/Ssn6 is recruited by Hap1 and is also required for repression. Furthermore, mutational analysis demonstrated that conserved Hap1 binding sites in the ERG5 5' regulatory region are required for repression. The switch of Hap1 from acting as a hypoxic repressor to an aerobic activator is determined by heme, which is synthesized only in the presence of oxygen. The ability of Hap1 to function as a ligand-dependent repressor and activator is a property shared with mammalian nuclear hormone receptors and likely allows greater transcriptional control by Hap1 in response to changing oxygen levels.

Heme levels switch the function of Hap1 of Saccharomyces cerevisiae between transcriptional activator and transcriptional repressor.

We have studied hypoxic induction of transcription by studying the seripauperin (PAU) genes of Saccharomyces cerevisiae. Previous studies showed that PAU induction requires the depletion of heme and is dependent upon the transcription factor Upc2. We have now identified additional factors required for PAU induction during hypoxia, including Hog1, a mitogen-activated protein kinase (MAPK) whose signaling pathway originates at the membrane. Our results have led to a model in which heme and ergosterol depletion alters membrane fluidity, thereby activating Hog1 for hypoxic induction. Hypoxic activation of Hog1 is distinct from its previously characterized response to osmotic stress, as the two conditions cause different transcriptional consequences. Furthermore, Hog1-dependent hypoxic activation is independent of the S. cerevisiae general stress response. In addition to Hog1, specific components of the SAGA coactivator complex, including Spt20 and Sgf73, are also required for PAU induction. Interestingly, the mammalian ortholog of Spt20, p38IP, has been previously shown to interact with the mammalian ortholog of Hog1, p38. Taken together, our results have uncovered a previously unknown hypoxic-response pathway that may be conserved throughout eukaryotes.

The Hog1 mitogen-activated protein kinase mediates a hypoxic response in Saccharomyces cerevisiae.

A yeast strain lacking Met4p, the primary transcriptional regulator of the sulfur assimilation pathway, cannot synthesize methionine. This apparently simple auxotroph did not grow well in rich media containing excess methionine, forming small colonies on yeast extract/peptone/dextrose plates. Faster-growing large colonies were abundant when overnight cultures were plated, suggesting that spontaneous suppressors of the growth defect arise with high frequency. To identify the suppressor mutations, we used genome-wide single-nucleotide polymorphism and standard genetic analyses. The most common suppressors were loss-of-function mutations in OPI1, encoding a transcriptional repressor of phospholipid metabolism. Using a new system that allows rapid and specific degradation of Met4p, we could study the dynamic expression of all genes following loss of Met4p. Experiments using this system with and without Opi1p showed that Met4 activates and Opi1p represses genes that maintain levels of S-adenosylmethionine (SAM), the substrate for most methyltransferase reactions. Cells lacking Met4p grow normally when either SAM is added to the media or one of the SAM synthetase genes is overexpressed. SAM is used as a methyl donor in three Opi1p-regulated reactions to create the abundant membrane phospholipid, phosphatidylcholine. Our results show that rapidly growing cells require significant methylation, likely for the biosynthesis of phospholipids.

Coordinated regulation of sulfur and phospholipid metabolism reflects the importance of methylation in the growth of yeast.

Une souche de levure sans Met4p, le principal r&eacute;gulateur transcriptionnel de la voie d&#39;assimilation de soufre, ne peut pas synth&eacute;tiser la m&eacute;thionine. Cette auxotrophe apparemment simple ne pousse pas bien dans un milieu riche contenant des exc&egrave;s m&eacute;thionine, formant de petites colonies sur les plaques de dextrose/peptone/Extrait de levure. Croissance rapide de grandes colonies &eacute;taient abondants lorsque les cultures une nuit ou Pluss ont &eacute;t&eacute; ensemenc&eacute;s, sugg&eacute;rant que les suppresseurs spontan&eacute;s de la d&eacute;fectuosit&eacute; de la croissance se posent avec haute fr&eacute;quence. Pour identifier les mutations de l&#39;&eacute;liminateur, nous avons utilis&eacute; le genome-wide single-nucleotide polymorphism et analyses g&eacute;n&eacute;tiques standard. Les suppresseurs plus communs &eacute;taient des mutations perte de fonction OPI1, codant un r&eacute;presseur transcriptionnel du m&eacute;tabolisme des phospholipides. &Agrave; l&#39;aide d&#39;un nouveau syst&egrave;me qui permet une d&eacute;gradation rapide et pr&eacute;cise de Met4p, nous pourrions &eacute;tudier l&#39;expression dynamique de tous les g&egrave;nes suite &agrave; perte de Met4p. Exp&eacute;riences &agrave; l&#39;aide de ce syst&egrave;me avec et sans Opi1p a montr&eacute; que Met4 active et Opi1p r&eacute;prime les g&egrave;nes qui maintiennent les niveaux de la S-ad&eacute;nosylm&eacute;thionine (SAM), le substrat pour la plupart des r&eacute;actions m&eacute;thyltransf&eacute;rase. Cellules d&eacute;pourvues Met4p poussent normalement quand une SAM est ajout&eacute; aux m&eacute;dias ou un des g&egrave;nes SAM synth&eacute;tase est surexprim&eacute;. SAM est utilis&eacute;e comme un donneur de m&eacute;thyl dans trois r&eacute;actions Opi1p r&eacute;glement&eacute;s pour cr&eacute;er le phospholipide de la membrane abondante, la phosphatidylcholine. Nos r&eacute;sultats montrent que les cellules en pleine croissance exigent importante m&eacute;thylation, probablement pour la biosynth&egrave;se des phospholipides.

R&eacute;gulation coordonn&eacute;e du m&eacute;tabolisme du soufre et phospholipides refl&egrave;te l&#39;importance de la m&eacute;thylation dans la croissance des levures.

Un ceppo di lievito privo di Met4p, il principale regolatore di transcriptional del percorso di assimilazione dello zolfo, non pu&ograve; sintetizzare la metionina. Questo apparentemente semplice auxotroph non ha fatto crescere bene in multimediali contenenti metionina in eccesso, formando piccole colonie su piastre di lievito peptone/estrarre/destrosio. Pi&ugrave; veloce crescita grandi colonie erano abbondanti quando culture durante la notte sono state placcate, suggerendo che soppressori spontanee del difetto di crescita si presentano con alta frequenza. Per identificare le mutazioni soppressore, abbiamo usato il polimorfismo a singolo nucleotide genome-wide e standard analisi genetiche. I soppressori pi&ugrave; comuni erano mutazioni con perdita di funzione nel OPI1, codifica un repressore trascrizionale del metabolismo di fosfolipidi. Utilizzando un nuovo sistema che permette la degradazione rapida e specifica di Met4p, noi potremmo studiare l&#39;espressione dinamica di tutti i geni in seguito perdita di Met4p. Esperimenti utilizzando questo sistema con e senza Opi1p ha mostrato che Met4 attiva e Opi1p reprime geni che mantengono livelli di S-adenosilmetionina (SAM), il substrato per la maggior parte delle reazioni di metiltransferasi. Cellule manca Met4p crescono normalmente quando SAM viene aggiunto ai media o uno dei geni sintetasi SAM &egrave; overexpressed. SAM viene utilizzata come un donatore di metile in tre reazioni Opi1p regolamentati per creare i fosfolipidi di membrana abbondanti, fosfatidilcolina. I nostri risultati dimostrano che cellule in rapida crescite richiedano significativa metilazione, probabilmente per la biosintesi dei fosfolipidi.

Regolazione coordinata del metabolismo dello zolfo e fosfolipidi riflette l&#39;importanza della metilazione nella crescita del lievito.

We describe the development and characterization of a system that allows the rapid and specific induction of individual genes in the yeast Saccharomyces cerevisiae without changes in nutrients or temperature. The system is based on the chimeric transcriptional activator Gal4dbd.ER.VP16 (GEV). Upon addition of the hormone Î²-estradiol, cytoplasmic GEV localizes to the nucleus and binds to promoters containing Gal4p consensus binding sequences to activate transcription. With galactokinase Gal1p and transcriptional activator Gal4p absent, the system is fast-acting, resulting in readily detectable transcription within 5 min after addition of the inducer. Î²-Estradiol is nearly a gratuitous inducer, as indicated by genome-wide profiling that shows unintended induction (by GEV) of only a few dozen genes. Response to inducer is graded: intermediate concentrations of inducer result in production of intermediate levels of product protein in all cells. We present data illustrating several applications of this system, including a modification of the regulated degron method, which allows rapid and specific degradation of a specific protein upon addition of Î²-estradiol. These gene induction and protein degradation systems provide important tools for studying the dynamics and functional relationships of genes and their respective regulatory networks.

Fast-acting and nearly gratuitous induction of gene expression and protein depletion in Saccharomyces cerevisiae.

Nous d&eacute;crivons le d&eacute;veloppement et la caract&eacute;risation d&#39;un syst&egrave;me qui permet l&#39;induction rapide et sp&eacute;cifique des g&egrave;nes individuels dans la levure Saccharomyces cerevisiae sans changements d&#39;&eacute;l&eacute;ments nutritifs ou de temp&eacute;rature. Le syst&egrave;me est bas&eacute; sur l&#39;activateur de la transcription chim&eacute;rique Gal4dbd.ER.VP16 (GEV). Lors de l&#39;addition de l&#39;hormone &beta;-estradiol, GEV cytoplasmique se localise au noyau et se lie aux promoteurs contenant des s&eacute;quences de liaison Gal4p consensus d&#39;activer la transcription. Avec galactokinase Gal1p et activateur de la transcription Gal4p absent, le syst&egrave;me est &agrave; action rapide, ayant pour r&eacute;sultat une transcription facilement d&eacute;tectable dans les 5 min apr&egrave;s addition de l&#39;inducteur. &Beta;-Estradiol est presque un inducteur gratuit, comme indiqu&eacute; par g&eacute;nome profilage qui montre une induction involontaire (par GEV) de seulement quelques dizaines de g&egrave;nes. R&eacute;ponse &agrave; inducteur est class&eacute; : interm&eacute;diaire des concentrations de r&eacute;sultat d&#39;inducteur dans la production des niveaux interm&eacute;diaires de prot&eacute;ine produit dans toutes les cellules. Nous pr&eacute;sentons des donn&eacute;es illustrant plusieurs applications de ce syst&egrave;me, y compris une modification de la m&eacute;thode de degron r&eacute;glement&eacute;, ce qui permet une d&eacute;gradation rapide et sp&eacute;cifique d&#39;une prot&eacute;ine sp&eacute;cifique lors de l&#39;addition de &beta;-estradiol. Ces syst&egrave;mes de d&eacute;gradation de l&#39;induction et la prot&eacute;ine g&egrave;ne fournissent des outils importants pour l&#39;&eacute;tude de la dynamique et les relations fonctionnelles des g&egrave;nes et de leurs r&eacute;seaux respectifs de r&eacute;gulation.

Action rapide et presque gratuite induction d&#39;&eacute;puisement expression et prot&eacute;ine de g&egrave;ne chez Saccharomyces cerevisiae.

Descriviamo lo sviluppo e la caratterizzazione di un sistema che permette l&#39;induzione rapido e specifico dei singoli geni nel lievito saccaromices cerevisiae senza cambiamenti nella temperatura o sostanze nutritive. Il sistema si basa sull&#39;attivatore trascrizionale chimerico Gal4dbd.ER.VP16 (GEV). Con l&#39;aggiunta di ormone &beta;-estradiolo, GEV citoplasmatica si localizza nel nucleo e si lega ai promotori contenenti sequenze Gal4p consenso vincolante per attivare la trascrizione. Con galattochinasi Gal1p e attivatore trascrizionale Gal4p assente, il sistema &egrave; fast-acting, con conseguente trascrizione facilmente rilevabili entro 5 min dopo l&#39;aggiunta dell&#39;induttore. &Beta;-estradiolo &egrave; quasi un induttore gratuito, come indicato dal genome-wide profilatura che mostra ad induzione non intenzionale (da GEV) di solo qualche decina di geni. Risposta a induttore &egrave; classificato: intermedio concentrazioni del risultato dell&#39;induttore nella produzione dei livelli intermedi di proteina prodotto in tutte le cellule. Vi presentiamo i dati che illustrano diverse applicazioni di questo sistema, tra cui una modifica del metodo degron regolamentati, che consente la degradazione rapida e specifica di una proteina specifica con l&#39;aggiunta di &beta;-estradiolo. Questi sistemi di degradazione ad induzione e proteina del gene forniscono strumenti importanti per lo studio delle dinamiche e rapporti funzionali di geni e le rispettive reti regolamentari.

Fast-acting e quasi gratuito ad induzione di esaurimento della proteina e di espressione di gene in Saccharomyces cerevisiae.

The genome of budding yeast (Saccharomyces cerevisiae) contains approximately 5800 protein-encoding genes, the majority of which are associated with some known biological function. Yet the extent of amino acid sequence conservation of these genes over all phyla has only been partially examined. Here we provide a more comprehensive overview and visualization of the conservation of yeast genes and a means for browsing and exploring the data in detail, down to the individual yeast gene, at http://yeast-phylogroups.princeton.edu. We used data from the OrthoMCL database, which has defined orthologs from approximately 150 completely sequenced genomes, including diverse representatives of the archeal, bacterial, and eukaryotic domains. By clustering genes based on similar patterns of conservation, we organized and visualized all the protein-encoding genes in yeast as a single heat map. Most genes fall into one of eight major clusters, called "phylogroups." Gene ontology analysis of the phylogroups revealed that they were associated with specific, distinct trends in gene function, generalizations likely to be of interest to a wide range of biologists.

Phylogenetic portrait of the Saccharomyces cerevisiae functional genome.

The dynamics of adaptation determine which mutations fix in a population, and hence how reproducible evolution will be. This is central to understanding the spectra of mutations recovered in the evolution of antibiotic resistance, the response of pathogens to immune selection, and the dynamics of cancer progression. In laboratory evolution experiments, demonstrably beneficial mutations are found repeatedly, but are often accompanied by other mutations with no obvious benefit. Here we use whole-genome whole-population sequencing to examine the dynamics of genome sequence evolution at high temporal resolution in 40 replicate Saccharomyces cerevisiae populations growing in rich medium for 1,000 generations. We find pervasive genetic hitchhiking: multiple mutations arise and move synchronously through the population as mutational 'cohorts'. Multiple clonal cohorts are often present simultaneously, competing with each other in the same population. Our results show that patterns of sequence evolution are driven by a balance between these chance effects of hitchhiking and interference, which increase stochastic variation in evolutionary outcomes, and the deterministic action of selection on individual mutations, which favours parallel evolutionary solutions in replicate populations.

Pervasive genetic hitchhiking and clonal interference in forty evolving yeast populations.

Studies of natural populations of many organisms have shown that traits are often complex, caused by contributions of mutations in multiple genes. In contrast, genetic studies in the laboratory primarily focus on studying the phenotypes caused by mutations in a single gene. However, the single mutation approach may be limited with respect to the breadth and degree of new phenotypes that can be found. We have taken the approach of isolating complex, or polygenic mutants in the lab to study the regulation of transcriptional activation distance in yeast. While most aspects of eukaryotic transcription are conserved from yeast to human, transcriptional activation distance is not. In Saccharomyces cerevisiae, the upstream activating sequence (UAS) is generally found within 450 base pairs of the transcription start site (TSS) and when the UAS is moved too far away, activation no longer occurs. In contrast, metazoan enhancers can activate from as far as several hundred kilobases from the TSS. Previously, we identified single mutations that allow transcription activation to occur at a greater-than-normal distance from the GAL1 UAS. As the single mutant phenotypes were weak, we have now isolated polygenic mutants that possess strong long-distance phenotypes. By identification of the causative mutations we have accounted for most of the heritability of the phenotype in each strain and have provided evidence that the Mediator coactivator complex plays both positive and negative roles in the regulation of transcription activation distance.

Analysis of Polygenic Mutants Suggests a Role for Mediator in Regulating Transcriptional Activation Distance in Saccharomyces cerevisiae.

Many cells experience hypoxia, or low oxygen, and respond by dramatically altering gene expression. In the yeast Saccharomyces cerevisiae, genes that respond are required for many oxygen-dependent cellular processes, such as respiration, biosynthesis, and redox regulation. To more fully characterize the global response to hypoxia, we exposed yeast to hypoxic conditions, extracted RNA at different times, and performed RNA sequencing (RNA-seq) analysis. Time-course statistical analysis revealed hundreds of genes that changed expression by up to 550-fold. The genes responded with varying kinetics suggesting that multiple regulatory pathways are involved. We identified most known oxygen-regulated genes and also uncovered new regulated genes. Reverse transcription-quantitative PCR (RT-qPCR) analysis confirmed that the lysine methyltransferase EFM6 and the recombinase DMC1, both conserved in humans, are indeed oxygen-responsive. Looking more broadly, oxygen-regulated genes participate in expected processes like respiration and lipid metabolism, but also in unexpected processes like amino acid and vitamin metabolism. Using principle component analysis, we discovered that the hypoxic response largely occurs during the first 2 hr and then a new steady-state expression state is achieved. Moreover, we show that the oxygen-dependent genes are not part of the previously described environmental stress response (ESR) consisting of genes that respond to diverse types of stress. While hypoxia appears to cause a transient stress, the hypoxic response is mostly characterized by a transition to a new state of gene expression. In summary, our results reveal that hypoxia causes widespread and complex changes in gene expression to prepare the cell to function with little or no oxygen.

Mark J. Hickman

Department of Biological Sciences

Rowan University

Measuring mRNA Levels Over Time During the Yeast S. cerevisiae Hypoxic Response

Mark J. Hickman

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Biological Sciences

Rowan University

Measuring mRNA Levels Over Time During the Yeast S. cerevisiae Hypoxic Response

Department of Biological Sciences