RESUMEN
Cell cycle progression relies on coordinated changes in the composition and subcellular localization of the proteome. By applying two distinct convolutional neural networks on images of millions of live yeast cells, we resolved proteome-level dynamics in both concentration and localization during the cell cycle, with resolution of â¼20 subcellular localization classes. We show that a quarter of the proteome displays cell cycle periodicity, with proteins tending to be controlled either at the level of localization or concentration, but not both. Distinct levels of protein regulation are preferentially utilized for different aspects of the cell cycle, with changes in protein concentration being mostly involved in cell cycle control and changes in protein localization in the biophysical implementation of the cell cycle program. We present a resource for exploring global proteome dynamics during the cell cycle, which will aid in understanding a fundamental biological process at a systems level.
Asunto(s)
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Células Eucariotas/metabolismo , Redes Neurales de la Computación , Proteoma/metabolismo , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Ribosome assembly requires precise coordination between the production and assembly of ribosomal components. Mutations in ribosomal proteins that inhibit the assembly process or ribosome function are often associated with ribosomopathies, some of which are linked to defects in proteostasis. In this study, we examine the interplay between several yeast proteostasis enzymes, including deubiquitylases (DUBs) Ubp2 and Ubp14, and E3 ligases Ufd4 and Hul5, and we explore their roles in the regulation of the cellular levels of K29-linked unanchored polyubiquitin (polyUb) chains. Accumulating K29-linked unanchored polyUb chains associate with maturing ribosomes to disrupt their assembly, activate the ribosome assembly stress response (RASTR), and lead to the sequestration of ribosomal proteins at the intranuclear quality control compartment (INQ). These findings reveal the physiological relevance of INQ and provide insights into mechanisms of cellular toxicity associated with ribosomopathies.
Asunto(s)
Poliubiquitina , Proteínas Ribosómicas , Ribosomas , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Proteínas Ribosómicas/metabolismo , Proteínas Ribosómicas/genética , Ribosomas/metabolismo , Ribosomas/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Poliubiquitina/metabolismo , Poliubiquitina/genética , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitinación , Proteostasis , Núcleo Celular/metabolismoRESUMEN
Proteomics has proved invaluable in generating large-scale quantitative data; however, the development of systems approaches for examining the proteome in vivo has lagged behind. To evaluate protein abundance and localization on a proteome scale, we exploited the yeast GFP-fusion collection in a pipeline combining automated genetics, high-throughput microscopy, and computational feature analysis. We developed an ensemble of binary classifiers to generate localization data from single-cell measurements and constructed maps of â¼3,000 proteins connected to 16 localization classes. To survey proteome dynamics in response to different chemical and genetic stimuli, we measure proteome-wide abundance and localization and identified changes over time. We analyzed >20 million cells to identify dynamic proteins that redistribute among multiple localizations in hydroxyurea, rapamycin, and in an rpd3Δ background. Because our localization and abundance data are quantitative, they provide the opportunity for many types of comparative studies, single cell analyses, modeling, and prediction. VIDEO ABSTRACT.
Asunto(s)
Proteoma/análisis , Proteínas de Saccharomyces cerevisiae/análisis , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/citología , Máquina de Vectores de Soporte , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Análisis de la Célula IndividualRESUMEN
Selective protein degradation by the ubiquitin-proteasome system (UPS) is involved in all cellular processes. However, the substrates and specificity of most UPS components are not well understood. Here we systematically characterized the UPS in Saccharomyces cerevisiae. Using fluorescent timers, we determined how loss of individual UPS components affects yeast proteome turnover, detecting phenotypes for 76% of E2, E3, and deubiquitinating enzymes. We exploit this dataset to gain insights into N-degron pathways, which target proteins carrying N-terminal degradation signals. We implicate Ubr1, an E3 of the Arg/N-degron pathway, in targeting mitochondrial proteins processed by the mitochondrial inner membrane protease. Moreover, we identify Ylr149c/Gid11 as a substrate receptor of the glucose-induced degradation-deficient (GID) complex, an E3 of the Pro/N-degron pathway. Our results suggest that Gid11 recognizes proteins with N-terminal threonines, expanding the specificity of the GID complex. This resource of potential substrates and relationships between UPS components enables exploring functions of selective protein degradation.
Asunto(s)
Proteínas Mitocondriales/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Ubiquitina-Proteína Ligasas/genética , Perfilación de la Expresión Génica , Regulación Fúngica de la Expresión Génica , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Proteínas Mitocondriales/clasificación , Proteínas Mitocondriales/metabolismo , Transporte de Proteínas , Proteolisis , Proteómica/métodos , Saccharomyces cerevisiae/enzimología , Proteínas de Saccharomyces cerevisiae/metabolismo , Treonina/metabolismo , Ubiquitina/genética , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligasas/clasificación , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación , Proteína Fluorescente RojaRESUMEN
A network of transcription factors (TFs) coordinates transcription with cell cycle events in eukaryotes. Most TFs in the network are phosphorylated by cyclin-dependent kinase (CDK), which limits their activities during the cell cycle. Here, we investigate the physiological consequences of disrupting CDK regulation of the paralogous repressors Yhp1 and Yox1 in yeast. Blocking Yhp1/Yox1 phosphorylation increases their levels and decreases expression of essential cell cycle regulatory genes which, unexpectedly, increases cellular fitness in optimal growth conditions. Using synthetic genetic interaction screens, we find that Yhp1/Yox1 mutations improve the fitness of mutants with mitotic defects, including condensin mutants. Blocking Yhp1/Yox1 phosphorylation simultaneously accelerates the G1/S transition and delays mitotic exit, without decreasing proliferation rate. This mitotic delay partially reverses the chromosome segregation defect of condensin mutants, potentially explaining their increased fitness when combined with Yhp1/Yox1 phosphomutants. These findings reveal how altering expression of cell cycle genes leads to a redistribution of cell cycle timing and confers a fitness advantage to cells.
Asunto(s)
Genes cdc , Proteínas de Saccharomyces cerevisiae , Ciclo Celular/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Quinasas Ciclina-Dependientes/genética , Quinasas Ciclina-Dependientes/metabolismo , Mitosis/genética , Fosforilación , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
We present FLEX (Functional evaluation of experimental perturbations), a pipeline that leverages several functional annotation resources to establish reference standards for benchmarking human genome-wide CRISPR screen data and methods for analyzing them. FLEX provides a quantitative measurement of the functional information captured by a given gene-pair dataset and a means to explore the diversity of functions captured by the input dataset. We apply FLEX to analyze data from the diverse cell line screens generated by the DepMap project. We identify a predominant mitochondria-associated signal within co-essentiality networks derived from these data and explore the basis of this signal. Our analysis and time-resolved CRISPR screens in a single cell line suggest that the variable phenotypes associated with mitochondria genes across cells may reflect screen dynamics and protein stability effects rather than genetic dependencies. We characterize this functional bias and demonstrate its relevance for interpreting differential hits in any CRISPR screening context. More generally, we demonstrate the utility of the FLEX pipeline for performing robust comparative evaluations of CRISPR screens or methods for processing them.
Asunto(s)
Redes Reguladoras de Genes , Pruebas Genéticas/métodos , Mitocondrias/genética , Biología de Sistemas/métodos , Algoritmos , Benchmarking , Sesgo , Sistemas CRISPR-Cas , Línea Celular , Células HEK293 , HumanosRESUMEN
The ability to switch a gene from off to on and monitor dynamic changes provides a powerful approach for probing gene function and elucidating causal regulatory relationships. Here, we developed and characterized YETI (Yeast Estradiol strains with Titratable Induction), a collection in which > 5,600 yeast genes are engineered for transcriptional inducibility with single-gene precision at their native loci and without plasmids. Each strain contains SGA screening markers and a unique barcode, enabling high-throughput genetics. We characterized YETI using growth phenotyping and BAR-seq screens, and we used a YETI allele to identify the regulon of Rof1, showing that it acts to repress transcription. We observed that strains with inducible essential genes that have low native expression can often grow without inducer. Analysis of data from eukaryotic and prokaryotic systems shows that native expression is a variable that can bias promoter-perturbing screens, including CRISPRi. We engineered a second expression system, Z3 EB42, that gives lower expression than Z3 EV, a feature enabling conditional activation and repression of lowly expressed essential genes that grow without inducer in the YETI library.
Asunto(s)
Genes Esenciales , Saccharomyces cerevisiae , Biblioteca de Genes , Plásmidos , Regiones Promotoras Genéticas , Saccharomyces cerevisiae/genéticaRESUMEN
The phenotypic consequence of a given mutation can be influenced by the genetic background. For example, conditional gene essentiality occurs when the loss of function of a gene causes lethality in one genetic background but not another. Between two individual Saccharomyces cerevisiae strains, S288c and Σ1278b, â¼1% of yeast genes were previously identified as "conditional essential." Here, in addition to confirming that some conditional essential genes are modified by a nonchromosomal element, we show that most cases involve a complex set of genomic modifiers. From tetrad analysis of S288C/Σ1278b hybrid strains and whole-genome sequencing of viable hybrid spore progeny, we identified complex sets of multiple genomic regions underlying conditional essentiality. For a smaller subset of genes, including CYS3 and CYS4, each of which encodes components of the cysteine biosynthesis pathway, we observed a segregation pattern consistent with a single modifier associated with conditional essentiality. In natural yeast isolates, we found that the CYS3/CYS4 conditional essentiality can be caused by variation in two independent modifiers, MET1 and OPT1, each with roles associated with cellular cysteine physiology. Interestingly, the OPT1 allelic variation appears to have arisen independently from separate lineages, with rare allele frequencies below 0.5%. Thus, while conditional gene essentiality is usually driven by genetic interactions associated with complex modifier architectures, our analysis also highlights the role of functionally related, genetically independent, and rare variants.
Asunto(s)
Genes Modificadores , Antecedentes Genéticos , Saccharomyces cerevisiae/genética , Alelos , Vías Biosintéticas , Cisteína/biosíntesis , Genes Esenciales , Genoma Fúngico , Filogenia , Saccharomyces cerevisiae/aislamiento & purificaciónRESUMEN
The phenotypic consequences of a given mutation can vary across individuals. This so-called background effect is widely observed, from mutant fitness of loss-of-function variants in model organisms to variable disease penetrance and expressivity in humans; however, the underlying genetic basis often remains unclear. Taking insights gained from recent large-scale surveys of genetic interaction and suppression analyses in yeast, we propose that the genetic network context for a given mutation may shape its propensity of exhibiting background-dependent phenotypes. We argue that further efforts in systematically mapping the genetic interaction networks beyond yeast will provide not only key insights into the functional properties of genes, but also a better understanding of the background effects and the (un)predictability of traits in a broader context.
Asunto(s)
Expresión Génica , Redes Reguladoras de Genes , Antecedentes Genéticos , Fenotipo , Animales , Epistasis Genética , Evolución Molecular , Estudios de Asociación Genética , Genética de Población , Humanos , Mutación , Sitios de Carácter Cuantitativo , Carácter Cuantitativo Heredable , Levaduras/genéticaRESUMEN
Our ability to understand the genotype-to-phenotype relationship is hindered by the lack of detailed understanding of phenotypes at a single-cell level. To systematically assess cell-to-cell phenotypic variability, we combined automated yeast genetics, high-content screening and neural network-based image analysis of single cells, focussing on genes that influence the architecture of four subcellular compartments of the endocytic pathway as a model system. Our unbiased assessment of the morphology of these compartments-endocytic patch, actin patch, late endosome and vacuole-identified 17 distinct mutant phenotypes associated with ~1,600 genes (~30% of all yeast genes). Approximately half of these mutants exhibited multiple phenotypes, highlighting the extent of morphological pleiotropy. Quantitative analysis also revealed that incomplete penetrance was prevalent, with the majority of mutants exhibiting substantial variability in phenotype at the single-cell level. Our single-cell analysis enabled exploration of factors that contribute to incomplete penetrance and cellular heterogeneity, including replicative age, organelle inheritance and response to stress.
Asunto(s)
Mutación , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Análisis de la Célula Individual/métodos , Pleiotropía Genética , Variación Genética , Microscopía Fluorescente , Redes Neurales de la Computación , Penetrancia , Fenotipo , Saccharomyces cerevisiae/genética , Biología de Sistemas , Imagen de Lapso de TiempoRESUMEN
Essential genes tend to be highly conserved across eukaryotes, but, in some cases, their critical roles can be bypassed through genetic rewiring. From a systematic analysis of 728 different essential yeast genes, we discovered that 124 (17%) were dispensable essential genes. Through whole-genome sequencing and detailed genetic analysis, we investigated the genetic interactions and genome alterations underlying bypass suppression. Dispensable essential genes often had paralogs, were enriched for genes encoding membrane-associated proteins, and were depleted for members of protein complexes. Functionally related genes frequently drove the bypass suppression interactions. These gene properties were predictive of essential gene dispensability and of specific suppressors among hundreds of genes on aneuploid chromosomes. Our findings identify yeast's core essential gene set and reveal that the properties of dispensable essential genes are conserved from yeast to human cells, correlating with human genes that display cell line-specific essentiality in the Cancer Dependency Map (DepMap) project.
Asunto(s)
Genes Esenciales , Genes Fúngicos , Saccharomyces cerevisiae/genética , Supresión Genética , Aneuploidia , Evolución Molecular , Eliminación de Gen , Duplicación de Gen , Redes Reguladoras de Genes , Genes Supresores , Complejos Multiproteicos/metabolismoRESUMEN
To better understand the health implications of personal genomes, we now face a largely unmet challenge to identify functional variants within disease-associated genes. Functional variants can be identified by trans-species complementation, e.g., by failure to rescue a yeast strain bearing a mutation in an orthologous human gene. Although orthologous complementation assays are powerful predictors of pathogenic variation, they are available for only a few percent of human disease genes. Here we systematically examine the question of whether complementation assays based on paralogy relationships can expand the number of human disease genes with functional variant detection assays. We tested over 1,000 paralogous human-yeast gene pairs for complementation, yielding 34 complementation relationships, of which 33 (97%) were novel. We found that paralog-based assays identified disease variants with success on par with that of orthology-based assays. Combining all homology-based assay results, we found that complementation can often identify pathogenic variants outside the homologous sequence region, presumably because of global effects on protein folding or stability. Within our search space, paralogy-based complementation more than doubled the number of human disease genes with a yeast-based complementation assay for disease variation.
Asunto(s)
Genes Fúngicos , Prueba de Complementación Genética/métodos , Enfermedades Genéticas Congénitas/genética , Genoma Humano , Homología de Secuencia , Levaduras/genética , Alelos , HumanosRESUMEN
We can now routinely identify coding variants within individual human genomes. A pressing challenge is to determine which variants disrupt the function of disease-associated genes. Both experimental and computational methods exist to predict pathogenicity of human genetic variation. However, a systematic performance comparison between them has been lacking. Therefore, we developed and exploited a panel of 26 yeast-based functional complementation assays to measure the impact of 179 variants (101 disease- and 78 non-disease-associated variants) from 22 human disease genes. Using the resulting reference standard, we show that experimental functional assays in a 1-billion-year diverged model organism can identify pathogenic alleles with significantly higher precision and specificity than current computational methods.
Asunto(s)
Prueba de Complementación Genética/métodos , Enfermedades Genéticas Congénitas , Saccharomyces cerevisiae , Transcripción Genética , Enfermedades Genéticas Congénitas/genética , Enfermedades Genéticas Congénitas/metabolismo , Humanos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMEN
In parallel with evolutionary developments, the Hsp90 molecular chaperone system shifted from a simple prokaryotic factor into an expansive network that includes a variety of cochaperones. We have taken high-throughput genomic and proteomic approaches to better understand the abundant yeast p23 cochaperone Sba1. Our work revealed an unexpected p23 network that displayed considerable independence from known Hsp90 clients. Additionally, our data uncovered a broad nuclear role for p23, contrasting with the historical dogma of restricted cytosolic activities for molecular chaperones. Validation studies demonstrated that yeast p23 was required for proper Golgi function and ribosome biogenesis, and was necessary for efficient DNA repair from a wide range of mutagens. Notably, mammalian p23 had conserved roles in these pathways as well as being necessary for proper cell mobility. Taken together, our work demonstrates that the p23 chaperone serves a broad physiological network and functions both in conjunction with and sovereign to Hsp90.
Asunto(s)
Núcleo Celular/metabolismo , Chaperonas Moleculares/genética , Secuencia de Aminoácidos , Citosol/metabolismo , Reparación del ADN/fisiología , Proteínas HSP90 de Choque Térmico/genética , Proteínas HSP90 de Choque Térmico/metabolismo , Chaperonas Moleculares/metabolismo , Datos de Secuencia Molecular , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Recent analysis of genome sequences has identified individuals that are healthy despite carrying severe disease-associated mutations. A possible explanation is that these individuals carry a second genomic perturbation that can compensate for the detrimental effects of the disease allele, a phenomenon referred to as suppression. In model organisms, suppression interactions are generally divided into two classes: genomic suppressors which are secondary mutations in the genome that bypass a mutant phenotype, and dosage suppression interactions in which overexpression of a suppressor gene rescues a mutant phenotype. Here, we describe the general properties of genomic and dosage suppression, with an emphasis on the budding yeast. We propose that suppression interactions between genetic variants are likely relevant for determining the penetrance of human traits. Consequently, an understanding of suppression mechanisms may guide the discovery of protective variants in healthy individuals that carry disease alleles, which could direct the rational design of new therapeutics.
Asunto(s)
Variación Genética/genética , Genoma/genética , Supresión Genética/genética , Alelos , Animales , Genómica/métodos , HumanosRESUMEN
The cell cycle-regulated expression of core histone genes is required for DNA replication and proper cell cycle progression in eukaryotic cells. Although some factors involved in histone gene transcription are known, the molecular mechanisms that ensure proper induction of histone gene expression during S phase remain enigmatic. Here we demonstrate that S-phase transcription of the model histone gene HTA1 in yeast is regulated by a novel attach-release mechanism involving phosphorylation of the conserved chromatin boundary protein Yta7 by both cyclin-dependent kinase 1 (Cdk1) and casein kinase 2 (CK2). Outside S phase, integrity of the AAA-ATPase domain is required for Yta7 boundary function, as defined by correct positioning of the histone chaperone Rtt106 and the chromatin remodeling complex RSC. Conversely, in S phase, Yta7 is hyperphosphorylated, causing its release from HTA1 chromatin and productive transcription. Most importantly, abrogation of Yta7 phosphorylation results in constitutive attachment of Yta7 to HTA1 chromatin, preventing efficient transcription post-recruitment of RNA polymerase II (RNAPII). Our study identified the chromatin boundary protein Yta7 as a key regulator that links S-phase kinases with RNAPII function at cell cycle-regulated histone gene promoters.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Cromatina/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Histonas/genética , Fase S/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Transcripción Genética , Proteína Quinasa CDC2/genética , Proteína Quinasa CDC2/metabolismo , Quinasa de la Caseína II/genética , Quinasa de la Caseína II/metabolismo , Proteínas Cromosómicas no Histona/genética , Histonas/metabolismo , Fosforilación , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Existing computational pipelines for quantitative analysis of high-content microscopy data rely on traditional machine learning approaches that fail to accurately classify more than a single dataset without substantial tuning and training, requiring extensive analysis. Here, we demonstrate that the application of deep learning to biological image data can overcome the pitfalls associated with conventional machine learning classifiers. Using a deep convolutional neural network (DeepLoc) to analyze yeast cell images, we show improved performance over traditional approaches in the automated classification of protein subcellular localization. We also demonstrate the ability of DeepLoc to classify highly divergent image sets, including images of pheromone-arrested cells with abnormal cellular morphology, as well as images generated in different genetic backgrounds and in different laboratories. We offer an open-source implementation that enables updating DeepLoc on new microscopy datasets. This study highlights deep learning as an important tool for the expedited analysis of high-content microscopy data.
Asunto(s)
Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/ultraestructura , Biología de Sistemas/métodos , Aprendizaje Automático , Microscopía , Redes Neurales de la Computación , Saccharomyces cerevisiae/metabolismoRESUMEN
The genetic basis of heritable traits has been studied for decades. Although recent mapping efforts have elucidated genetic determinants of transcript levels, mapping of protein abundance has lagged. Here, we analyze levels of 4084 GFP-tagged yeast proteins in the progeny of a cross between a laboratory and a wild strain using flow cytometry and high-content microscopy. The genotype of trans variants contributed little to protein level variation between individual cells but explained >50% of the variance in the population's average protein abundance for half of the GFP fusions tested. To map trans-acting factors responsible, we performed flow sorting and bulk segregant analysis of 25 proteins, finding a median of five protein quantitative trait loci (pQTLs) per GFP fusion. Further, we find that cis-acting variants predominate; the genotype of a gene and its surrounding region had a large effect on protein level six times more frequently than the rest of the genome combined. We present evidence for both shared and independent genetic control of transcript and protein abundance: More than half of the expression QTLs (eQTLs) contribute to changes in protein levels of regulated genes, but several pQTLs do not affect their cognate transcript levels. Allele replacements of genes known to underlie trans eQTL hotspots confirmed the correlation of effects on mRNA and protein levels. This study represents the first genome-scale measurement of genetic contribution to protein levels in single cells and populations, identifies more than a hundred trans pQTLs, and validates the propagation of effects associated with transcript variation to protein abundance.
Asunto(s)
Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Mapeo Cromosómico , Evolución Molecular , Expresión Génica , Frecuencia de los Genes , Genotipo , Sitios de Carácter Cuantitativo , ARN de Hongos/genética , ARN de Hongos/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Systematic functional genomics approaches were used to map a network centered on the small ubiquitin-related modifier (SUMO) system. Over 250 physical interactions were identified using the SUMO protein as bait in affinity purification-mass spectrometry and yeast two-hybrid screens. More than 500 genes and 1400 synthetic genetic interactions were mapped by synthetic genetic array (SGA) analysis using eight different SUMO pathway query genes. The resultant global SUMO network highlights its role in 15 major biological processes and better defines functional relationships between the different components of the SUMO pathway. Using this information-rich resource, we have identified roles for the SUMO system in the function of the AAA ATPase Cdc48p, the regulation of lipid metabolism, localization of the ATP-dependent endonuclease Dna2p, and recovery from the DNA-damage checkpoint.
Asunto(s)
Redes Reguladoras de Genes , Mapeo de Interacción de Proteínas/métodos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/metabolismo , Núcleo Celular , Cromatografía de Afinidad , Daño del ADN , Reparación del ADN , Replicación del ADN , Genes Fúngicos , Metabolismo de los Lípidos , Espectrometría de Masas , Unión Proteica , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Técnicas del Sistema de Dos Híbridos , Ubiquitina/metabolismoRESUMEN
We describe a fluorescent reporter system that exploits the functional genomic tools available in budding yeast to systematically assess consequences of genetic perturbations on gene expression. We used our Reporter-Synthetic Genetic Array (R-SGA) method to screen for regulators of core histone gene expression. We discovered that the histone chaperone Rtt106 functions in a pathway with two other chaperones, Asf1 and the HIR complex, to create a repressive chromatin structure at core histone promoters. We found that activation of histone (HTA1) gene expression involves both relief of Rtt106-mediated repression by the activity of the histone acetyltransferase Rtt109 and restriction of Rtt106 to the promoter region by the bromodomain-containing protein Yta7. We propose that the maintenance of Asf1/HIR/Rtt106-mediated repressive chromatin domains is the primary mechanism of cell-cycle regulation of histone promoters. Our data suggest that this pathway may represent a chromatin regulatory mechanism that is broadly used across the genome.