RESUMEN
Upstream events that trigger initiation of cell division, at a point called START in yeast, determine the overall rates of cell proliferation. The identity and complete sequence of those events remain unknown. Previous studies relied mainly on cell size changes to identify systematically genes required for the timely completion of START. Here, we evaluated panels of non-essential single gene deletion strains for altered DNA content by flow cytometry. This analysis revealed that most gene deletions that altered cell cycle progression did not change cell size. Our results highlight a strong requirement for ribosomal biogenesis and protein synthesis for initiation of cell division. We also identified numerous factors that have not been previously implicated in cell cycle control mechanisms. We found that CBS, which catalyzes the synthesis of cystathionine from serine and homocysteine, advances START in two ways: by promoting cell growth, which requires CBS's catalytic activity, and by a separate function, which does not require CBS's catalytic activity. CBS defects cause disease in humans, and in animals CBS has vital, non-catalytic, unknown roles. Hence, our results may be relevant for human biology. Taken together, these findings significantly expand the range of factors required for the timely initiation of cell division. The systematic identification of non-essential regulators of cell division we describe will be a valuable resource for analysis of cell cycle progression in yeast and other organisms.
Asunto(s)
División Celular/genética , Puntos de Control de la Fase G1 del Ciclo Celular/genética , Ribosomas , Saccharomyces cerevisiae , Proliferación Celular , Tamaño de la Célula , ADN/análisis , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Eliminación de Gen , Regulación Fúngica de la Expresión Génica , Redes Reguladoras de Genes , Homocigoto , Ribosomas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrolloRESUMEN
Transcriptional activation is often associated with chromatin remodeling. However, little is known about the dynamics of remodeling of nucleosome arrays in vivo. Upon induction of Saccharomyces cerevisiae PHO5, a novel kinetic assay of DNA methyltransferase accessibility showed that nucleosomes adjacent to the histone-free upstream activating sequence (UASp1) are disrupted earlier and at higher frequency in the cell population than are those more distal. Individually cloned molecules, each representing the chromatin state of a full promoter from a single cell, revealed multiple promoter classes with either no remodeling or variable numbers of disrupted nucleosomes. Individual promoters in the remodeled fraction were highly enriched for contiguous blocks of disrupted nucleosomes, the majority of which overlapped the UAS region. These results support a probabilistic model in which chromatin remodeling at PHO5 spreads from sites of transactivator association with DNA and attenuates with distance.
Asunto(s)
Nucleosomas/genética , Nucleosomas/metabolismo , Regiones Promotoras Genéticas/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Fosfatasa Ácida , Ensamble y Desensamble de Cromatina , Regulación Fúngica de la Expresión Génica , Cinética , Metiltransferasas/metabolismo , Secuencias Reguladoras de Ácidos Nucleicos/genéticaRESUMEN
Non-invasive methods for mapping chromatin structure are necessary for creating an accurate view of genome function and dynamics in vivo. Ectopic induction of cytosine-5 DNA methyltransferases (C5 MTases) in Saccharomyces cerevisiae is a powerful technique for probing chromatin structure with minimal disruption to yeast physiology. Accessibility of MTases to their cognate sites is impaired based on the strength and span of the protein-DNA interaction to be probed. Methylated cytosines that resist chemical deamination are detected positively by the PCR-based technique of bisulfite genomic sequencing. PCR amplicons can be sequenced directly yielding an average m(5)C frequency or accessibility of each target site within the population, a technique termed methyltransferase accessibility protocol (MAP). More recently, the sequencing of cloned molecules in MAP for individual templates (MAPit) enables assignment of the methylation status of each target site along a continuous DNA strand from a single cell. The unique capability to score methylation at multiple sites in single molecules permits detection of inherent structural variability in chromatin. Here, MAPit analysis of the repressed and induced PHO5 promoter of budding yeast, using a C5 MTase with dinucleotide recognition specificity, reveals considerable cell-to-cell heterogeneity in chromatin structure. Substantial variation is observed in the extent to which the MTase gains entry to each of the nucleosomes positioned at PHO5, suggesting differences in their intrinsic thermodynamic stability in vivo. MAPit should be readily adaptable to the analysis of chromatin structure and non-histone protein-DNA interactions in a variety of model systems.
Asunto(s)
Cromatina/química , Cromatina/metabolismo , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Biología Molecular/métodos , Secuencia de Bases , Cromatina/genética , Metilación de ADN , ADN de Hongos/metabolismo , Datos de Secuencia Molecular , Regiones Promotoras Genéticas/genética , Saccharomyces cerevisiae/enzimología , Proteínas de Saccharomyces cerevisiae/metabolismo , Análisis de Secuencia de ADNRESUMEN
Effective methods of probing chromatin structure without disrupting DNA-protein interactions and associations are necessary for creating an accurate picture of chromatin and its processes in vivo. Expression of cytidine-5 DNA methyltransferases (C5 DMTases) in Saccharomyces cerevisiae provides a powerful noninvasive method of assaying relative DNA accessibility in chromatin. DNA MTases are occluded from protein-associated DNA based on the strength and span of the DNA-protein interaction. Ectopic regulation of C5 DMTase expression systems allows for minimal disruption of yeast physiology. Methylated sites are detected by bisulfite genomic sequencing, which leads to a positive signal corresponding to modified cytidine residues. High-resolution C5 DMTases with dinucleotide recognition specificity are shown to provide sufficient coverage to map interactions spanning a relatively short distance.
Asunto(s)
ADN (Citosina-5-)-Metiltransferasas , ADN/metabolismo , Técnicas de Sonda Molecular , Proteínas Nucleares/metabolismo , Secuencia de Bases , Cromatina/genética , Cromatina/metabolismo , ADN/genética , ADN (Citosina-5-)-Metiltransferasas/genética , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Cartilla de ADN/genética , ADN de Hongos/genética , ADN de Hongos/metabolismo , Escherichia coli/genética , Reacción en Cadena de la Polimerasa , Unión Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Transformación GenéticaRESUMEN
To identify Saccharomyces cerevisiae genes required for the proper timing of cell cycle transitions, we previously reported a systematic examination of the DNA content of homozygous diploid deletion strains. However, deletion strains with complex DNA content profiles were not examined in that study. Here, we report S. cerevisiae genes that when deleted give rise to DNA content profiles consistent with roles of the corresponding gene products during DNA replication. We also identified a set of genes whose deletion leads to increased DNA content, consistent with defects in mitosis, cytokinesis, or cell separation. Finally, we examined known interactions between the gene products of each group, placing these gene products in functional networks. Taken together, the data we present further validate the roles of the corresponding gene products in these processes, facilitating efforts to delineate gene function critical for genome replication, maintenance, and segregation.
Asunto(s)
ADN de Hongos/genética , Eliminación de Gen , Saccharomyces cerevisiae/genética , Ciclo Celular , Replicación del ADN , ADN de Hongos/metabolismo , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Screening chemical libraries to identify compounds that affect overall cell proliferation is common. However, in most cases, it is not known whether the compounds tested alter the timing of particular cell cycle transitions. Here, we evaluated an FDA-approved drug library to identify pharmaceuticals that alter cell cycle progression in yeast, using DNA content measurements by flow cytometry. This approach revealed strong cell cycle effects of several commonly used pharmaceuticals. We show that the antilipemic gemfibrozil delays initiation of DNA replication, while cells treated with the antidepressant fluoxetine severely delay progression through mitosis. Based on their effects on cell cycle progression, we also examined cell proliferation in the presence of both compounds. We discovered a strong suppressive interaction between gemfibrozil and fluoxetine. Combinations of interest among diverse pharmaceuticals are difficult to identify, due to the daunting number of possible combinations that must be evaluated. The novel interaction between gemfibrozil and fluoxetine suggests that identifying and combining drugs that show cell cycle effects might streamline identification of drug combinations with a pronounced impact on cell proliferation.
Asunto(s)
Ciclo Celular/efectos de los fármacos , Fluoxetina/farmacología , Gemfibrozilo/farmacología , Proliferación Celular/efectos de los fármacos , Interacciones Farmacológicas , Citometría de Flujo , Mitosis/efectos de los fármacosRESUMEN
BACKGROUND: The unfolded protein response (UPR) is a eukaryotic signaling pathway, from the endoplasmic reticulum (ER) to the nucleus. Protein misfolding in the ER triggers the UPR. Accumulating evidence links the UPR in diverse aspects of cellular homeostasis. The UPR responds to the overall protein synthesis capacity and metabolic fluxes of the cell. Because the coupling of metabolism with cell division governs when cells start dividing, here we examined the role of UPR signaling in the timing of initiation of cell division and cell cycle progression, in the yeast Saccharomyces cerevisiae. METHODOLOGY/PRINCIPAL FINDINGS: We report that cells lacking the ER-resident stress sensor Ire1p, which cannot trigger the UPR, nonetheless completed the G1/S transition on time. Furthermore, loss of UPR signaling neither affected the nutrient and growth rate dependence of the G1/S transition, nor the metabolic oscillations that yeast cells display in defined steady-state conditions. Remarkably, however, loss of UPR signaling led to hypersensitivity to genotoxic stress and a ten-fold increase in chromosome loss. CONCLUSIONS/SIGNIFICANCE: Taken together, our results strongly suggest that UPR signaling is not necessary for the normal coupling of metabolism with cell division, but it has a role in genome maintenance. These results add to previous work that linked the UPR with cytokinesis in yeast. UPR signaling is conserved in all eukaryotes, and it malfunctions in a variety of diseases, including cancer. Therefore, our findings may be relevant to other systems, including humans.
Asunto(s)
Cromosomas Fúngicos/genética , Fase G1 , Fase S , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/genética , Respuesta de Proteína Desplegada , División Celular , Cromosomas Fúngicos/metabolismo , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Toll-like receptor 3 (TLR3) can signal the production of a suite of cytokines and chemokines in response to double-stranded RNA (dsRNA) ligands or the dsRNA mimic poly(I-C). Using a human embryonic kidney 293T cell line to express human TLR3, we determined that poly(I-C)-induced signal could be significantly inhibited by single-stranded DNAs (ssDNAs), but not ssRNA or dsDNA. The ssDNA molecules that down-modulated TLR3 signaling did not affect TLR4 and do not require the hypomethylated CpG motif found in TLR9 ligands. The degree of modulation can be altered by the length, base sequence, and modification state of the ssDNAs. An inhibitory ssDNA was found to colocalize with TLR3 in transfected cells and in a cell line that naturally expresses TLR3. The inhibitory ssDNAs can compete efficiently with dsRNA for binding purified TLR3 ectodomains in vitro, while noninhibitory nucleic acids do not. The ssDNAs also decrease the levels of several cytokines produced by the human bronchial epithelial cell line BEAS-2B and by human peripheral blood mononuclear cells in response to poly(I-C) stimulation of native TLR3. These activities indicate that ssDNAs could be used to regulate the inflammatory response through TLR3.
Asunto(s)
ADN de Cadena Simple/inmunología , Receptor Toll-Like 3/inmunología , Línea Celular , Citocinas/inmunología , ADN de Cadena Simple/análisis , Endosomas/química , Genes Reporteros , Humanos , Leucocitos Mononucleares , FN-kappa B/genética , Oligonucleótidos/inmunología , Poli I-C/inmunología , Receptor Toll-Like 3/análisisRESUMEN
Probing chromatin structure with DNA methyltransferases offers advantages over more commonly used nuclease-based and chromatin immunoprecipitation methods for detection of nucleosomes and non-histone protein-DNA interactions. Here, we describe two related methods in which the readout of MTase accessibility is obtained by assaying 5-methylcytosine in DNA through the PCR-based technique of bisulfite genomic sequencing. The methyltransferase accessibility protocol (MAP) determines the relative frequency at which the enzyme accesses each of its target sites over an entire population of PCR amplified product. While MAP yields much quantitative information about relative accessibility of a region of chromatin, a complementary single-molecule view of methyltransferase accessibility, termed MAP for individual templates (MAP-IT), is provided by analysis of cloned PCR products. Absolute rather than relative methylation frequencies in a region are obtained by summing the methylation status at each site over a cohort of clones. Moreover, as the integrity of individual molecules is maintained in MAP-IT, unique information about the distribution of multiple footprints along continuous regions is gleaned. In principle, the population MAP and single-molecule MAP-IT strategies can be used to analyze chromatin structure in a variety of model systems. Here, we describe the application of MAP in living Saccharomyces cerevisiae cells and MAP-IT in the analysis of a mammalian tumor suppressor gene in nuclei. This application of MAP-IT provides the first means to simultaneously determine CpG methylation of mammalian genes and their overlying chromatin structure in the same single DNA molecule.
Asunto(s)
Ensamble y Desensamble de Cromatina/fisiología , Metilación de ADN , Metilasas de Modificación del ADN/metabolismo , Biología Molecular/métodos , Nucleosomas/metabolismo , 5-Metilcitosina/metabolismo , Núcleo Celular/enzimología , Células Cultivadas , Clonación Molecular/métodos , Islas de CpG/fisiología , Cartilla de ADN , Genes Supresores de Tumor/fisiología , Genoma , Genoma Fúngico , Humanos , Nucleosomas/genética , Reacción en Cadena de la Polimerasa/métodos , Regiones Promotoras Genéticas/genética , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Sulfitos/metabolismoRESUMEN
Cytosine-5 DNA methyltransferases (C5 DMTases) are effective reagents for analyzing chromatin and footprinting DNA-bound factors in vivo. Cytosine methylation in accessible regions is assayed positively by the PCR-based technique of bisulfite sequencing. In this article, we outline two complementary uses for the DNA methyltransferase CviPI (M.CviPI, GC specificity) in probing chromatin organization. First, we describe the use of the naturally occurring, free enzyme as a diffusible probe to map changes in nucleosome structure and to footprint factor interactions at cis-regulatory sequences. In a second application, termed targeted gene methylation (TAGM), the DMTase is targeted via in-frame fusion to a DNA-binding factor. The rapid accumulation of DNA methylation enables highly sensitive detection of factor binding. Both strategies can be applied with any C5 DMTase, such as M.SssI, which also possesses a short-recognition specificity (CG). A description of methods for constructing C5 DMTase-expressing strains of Saccharomyces cerevisiae and analyzing chromatin regions is provided. We also include comprehensive protocols for the isolation and bisulfite treatment of genomic DNA as well as the subsequent bisulfite sequencing steps. Data demonstrating the efficacy of both DMTase probing techniques, theoretical considerations, and experimental analyses are presented at GAL1 and PHO5.