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1.
PLoS Genet ; 13(2): e1006612, 2017 02.
Article in English | MEDLINE | ID: mdl-28166236

ABSTRACT

Sumoylation regulates a wide range of essential cellular functions through diverse mechanisms that remain to be fully understood. Using S. cerevisiae, a model organism with a single essential SUMO gene (SMT3), we developed a library of >250 mutant strains with single or multiple amino acid substitutions of surface or core residues in the Smt3 protein. By screening this library using plate-based assays, we have generated a comprehensive structure-function based map of Smt3, revealing essential amino acid residues and residues critical for function under a variety of genotoxic and proteotoxic stress conditions. Functionally important residues mapped to surfaces affecting Smt3 precursor processing and deconjugation from protein substrates, covalent conjugation to protein substrates, and non-covalent interactions with E3 ligases and downstream effector proteins containing SUMO-interacting motifs. Lysine residues potentially involved in formation of polymeric chains were also investigated, revealing critical roles for polymeric chains, but redundancy in specific chain linkages. Collectively, our findings provide important insights into the molecular basis of signaling through sumoylation. Moreover, the library of Smt3 mutants represents a valuable resource for further exploring the functions of sumoylation in cellular stress response and other SUMO-dependent pathways.


Subject(s)
Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae/genetics , Small Ubiquitin-Related Modifier Proteins/genetics , Structure-Activity Relationship , Sumoylation/drug effects , Amino Acid Substitution/genetics , DNA Mutational Analysis , Mutagenesis/genetics , Mutagens/toxicity , Protein Binding , Saccharomyces cerevisiae/chemistry , Ubiquitin-Protein Ligases/genetics
2.
G3 (Bethesda) ; 6(4): 881-92, 2016 04 07.
Article in English | MEDLINE | ID: mdl-26837954

ABSTRACT

The yeast pheromone response pathway serves as a valuable model of eukaryotic mitogen-activated protein kinase (MAPK) pathways, and transcription of their downstream targets. Here, we describe application of a screening method combining two technologies: fluorescence-activated cell sorting (FACS), and barcode analysis by sequencing (Bar-Seq). Using this screening method, and pFUS1-GFP as a reporter for MAPK pathway activation, we readily identified mutants in known mating pathway components. In this study, we also include a comprehensive analysis of the FUS1 induction properties of known mating pathway mutants by flow cytometry, featuring single cell analysis of each mutant population. We also characterized a new source of false positives resulting from the design of this screen. Additionally, we identified a deletion mutant, sub1Δ, with increased basal expression of pFUS1-GFP. Here, in the first ChIP-Seq of Sub1, our data shows that Sub1 binds to the promoters of about half the genes in the genome (tripling the 991 loci previously reported), including the promoters of several pheromone-inducible genes, some of which show an increase upon pheromone induction. Here, we also present the first RNA-Seq of a sub1Δ mutant; the majority of genes have no change in RNA, but, of the small subset that do, most show decreased expression, consistent with biochemical studies implicating Sub1 as a positive transcriptional regulator. The RNA-Seq data also show that certain pheromone-inducible genes are induced less in the sub1Δ mutant relative to the wild type, supporting a role for Sub1 in regulation of mating pathway genes. The sub1Δ mutant has increased basal levels of a small subset of other genes besides FUS1, including IMD2 and FIG1, a gene encoding an integral membrane protein necessary for efficient mating.


Subject(s)
DNA Barcoding, Taxonomic , Gene Expression Regulation, Fungal/drug effects , Genes, Fungal , Pheromones/pharmacology , Yeasts/drug effects , Yeasts/genetics , Gene Expression , Gene Knockout Techniques , Genes, Reporter , Genetic Vectors/genetics , Mating Factor/genetics , Mating Factor/metabolism , Models, Biological , Mutation , Yeasts/classification , Yeasts/metabolism
3.
Methods Mol Biol ; 416: 221-47, 2008.
Article in English | MEDLINE | ID: mdl-18392971

ABSTRACT

Comprehensive collections of open reading frame (ORF) deletion mutant strains exist for the budding yeast Saccharomyces cerevisiae. With great prescience, these strains were designed with short molecular bar codes or TAGs that uniquely mark each deletion allele, flanked by shared priming sequences. These features have enabled researchers to handle yeast mutant collections as complex pools of approximately 6000 strains. The presence of any individual mutant within a pool can be assessed indirectly by measuring the relative abundance of its corresponding TAG(s) in genomic DNA prepared from the pool. This is readily accomplished by wholesale polymerase chain reaction (PCR) amplification of the TAGs using fluorescent oligonucleotide primers that recognize the common flanking sequences, followed by hybridization of the labeled PCR products to a TAG oligonucleotide microarray. Here we describe a method-diploid-based synthetic lethality analysis by microarray (dSLAM)-whereby such pools can be manipulated to rapidly construct and assess the fitness of 6000 double-mutant strains in a single experiment. Analysis of double-mutant strains is of growing importance in defining the spectrum of essential cellular functionalities and in understanding how these functionalities interrelate.


Subject(s)
Gene Expression Regulation, Fungal/physiology , Genes, Lethal , Mutation/physiology , Oligonucleotide Array Sequence Analysis/methods , Saccharomyces cerevisiae/genetics , Gene Deletion , Gene Expression Profiling/methods , Gene Expression Regulation, Fungal/genetics , Genome, Fungal
4.
Genes Dev ; 20(8): 966-76, 2006 Apr 15.
Article in English | MEDLINE | ID: mdl-16598039

ABSTRACT

Covalent histone post-translational modifications such as acetylation, methylation, phosphorylation, and ubiquitylation play pivotal roles in regulating many cellular processes, including transcription, response to DNA damage, and epigenetic control. Although positive-acting post-translational modifications have been studied in Saccharomyces cerevisiae, histone modifications that are associated with transcriptional repression have not been shown to occur in this yeast. Here, we provide evidence that histone sumoylation negatively regulates transcription in S. cerevisiae. We show that all four core histones are sumoylated and identify specific sites of sumoylation in histones H2A, H2B, and H4. We demonstrate that histone sumoylation sites are involved directly in transcriptional repression. Further, while histone sumoylation occurs at all loci tested throughout the genome, slightly higher levels occur proximal to telomeres. We observe a dynamic interplay between histone sumoylation and either acetylation or ubiquitylation, where sumoylation serves as a potential block to these activating modifications. These results indicate that sumoylation is the first negative histone modification to be identified in S. cerevisiae and further suggest that sumoylation may serve as a general dynamic mark to oppose transcription.


Subject(s)
Histones/metabolism , SUMO-1 Protein/metabolism , Saccharomyces cerevisiae/metabolism , Acetylation , Amino Acid Sequence , Blotting, Western , Immunoprecipitation , Protein Processing, Post-Translational , Saccharomyces cerevisiae/genetics , Telomere , Ubiquitin/metabolism
5.
Trends Genet ; 22(1): 56-63, 2006 Jan.
Article in English | MEDLINE | ID: mdl-16309778

ABSTRACT

The Saccharomyces genome-deletion project created >5900 'molecularly barcoded' yeast knockout mutants (YKO mutants). The YKO mutant collections have facilitated large-scale analyses of a multitude of mutant phenotypes. For example, both synthetic genetic array (SGA) and synthetic-lethality analysis by microarray (SLAM) methods have been used for synthetic-lethality screens. Global analysis of synthetic lethality promises to identify cellular pathways that 'buffer' each other biologically. The combination of global synthetic-lethality analysis, together with global protein-protein interaction analyses, mRNA expression profiling and functional profiling will, in principle, enable construction of a cellular 'wiring diagram' that will help frame a deeper understanding of human biology and disease.


Subject(s)
Saccharomyces cerevisiae/genetics , Chromosome Mapping , Gene Deletion , Gene Expression Profiling , Genes, Fungal , Genes, Lethal , Genetic Techniques , Genome, Fungal , Oligonucleotide Array Sequence Analysis , RNA, Fungal/genetics , RNA, Messenger/genetics
6.
Mol Cell Biol ; 23(9): 3202-15, 2003 May.
Article in English | MEDLINE | ID: mdl-12697820

ABSTRACT

The accurate segregation of chromosomes requires the kinetochore, a complex protein machine that assembles onto centromeric DNA to mediate attachment of replicated sister chromatids to the mitotic spindle apparatus. This study reveals an important role for the yeast RSC ATP-dependent chromatin-remodeling complex at the kinetochore in chromosome transmission. Mutations in genes encoding two core subunits of RSC, the ATPase Sth1p and the Snf5p homolog Sfh1p, interact genetically with mutations in genes encoding kinetochore proteins and with a mutation in centromeric DNA. RSC also interacts genetically and physically with the histone and histone variant components of centromeric chromatin. Importantly, RSC is localized to centromeric and centromere-proximal chromosomal regions, and its association with these loci is dependent on Sth1p. Both sth1 and sfh1 mutants exhibit altered centromeric and centromere-proximal chromatin structure and increased missegregation of authentic chromosomes. Finally, RSC is not required for centromeric deposition of the histone H3 variant Cse4p, suggesting that RSC plays a role in reconfiguring centromeric and flanking nucleosomes following Cse4p recruitment for proper chromosome transmission.


Subject(s)
Chromatin/metabolism , Chromosome Segregation , DNA-Binding Proteins/metabolism , Kinetochores/physiology , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/genetics , Transcription Factors/metabolism , Carrier Proteins/genetics , Carrier Proteins/metabolism , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Centromere/chemistry , Centromere/physiology , Chromatin/genetics , Chromatin/ultrastructure , Chromosomal Proteins, Non-Histone , DNA-Binding Proteins/genetics , Histones/genetics , Macromolecular Substances , Membrane Proteins/genetics , Membrane Proteins/metabolism , Mutation , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Phospholipid Transfer Proteins , Saccharomyces cerevisiae Proteins/genetics , Spindle Apparatus , Transcription Factors/genetics
7.
Mol Cell ; 10(4): 843-55, 2002 Oct.
Article in English | MEDLINE | ID: mdl-12419228

ABSTRACT

During a screen to identify c-Jun activators, we isolated a cysteine protease, SuPr-1, that induced c-Jun-dependent transcription independently of c-Jun phosphorylation. SuPr-1 is a member of a new family of proteases that hydrolyze the ubiquitin-like modifier, SUMO-1. SuPr-1 hydrolyzed SUMO-1-modified forms of the promyelocytic leukemia gene product, PML, and altered the subcellular distribution of PML in nuclear PODs (PML oncogenic domains). SuPr-1 also altered the distribution of other nuclear POD-associated proteins, such as CBP and Daxx, that act as transcriptional regulators. SuPr-1 action on transcription was enhanced by PML, and SuPr-1 failed to activate transcription in PML-deficient fibroblasts. Our studies establish an important role for SUMO proteases in transcription.


Subject(s)
Cysteine Endopeptidases/metabolism , Neoplasm Proteins/metabolism , Nuclear Proteins , Transcription Factors/metabolism , Transcriptional Activation , Amino Acid Sequence , Animals , Base Sequence , Cell Line , Cell Nucleus Structures/metabolism , Cysteine Endopeptidases/genetics , HeLa Cells , Humans , Mice , Molecular Sequence Data , Mutation , Neoplasm Proteins/genetics , Phosphorylation , Promoter Regions, Genetic , Promyelocytic Leukemia Protein , Protein Binding , Proto-Oncogene Proteins c-jun/metabolism , Sequence Homology, Nucleic Acid , Substrate Specificity , Transcription Factors/genetics , Tumor Suppressor Proteins
8.
Eur J Biochem ; 269(9): 2300-14, 2002 May.
Article in English | MEDLINE | ID: mdl-11985612

ABSTRACT

The centromere-kinetochore complex is a highly specialized chromatin domain that both mediates and monitors chromosome-spindle interactions responsible for accurate partitioning of sister chromatids to daughter cells. Centromeres are distinguished from adjacent chromatin by specific patterns of histone modification and the presence of a centromere-specific histone H3 variant (e.g. CENP-A). Centromere-proximal regions usually correspond to sites of avid and persistent sister chromatid cohesion mediated by the conserved cohesin complex. In budding yeast, there is a substantial body of evidence indicating centromeres direct formation and/or stabilization of centromere-proximal cohesion. In other organisms, the dependency of cohesion on centromere function is not as clear. Indeed, it appears that pericentromeric heterochromatin recruits cohesion proteins independent of centromere function. Nonetheless, aspects of centromere function are impaired in the absence of sister chromatid cohesion, suggesting the two are interdependent. Here we review the nature of centromeric chromatin, the dynamics and regulation of sister chromatid cohesion, and the relationship between the two.


Subject(s)
Centromere/physiology , Chromatids/physiology , Kinetochores/physiology , Animals , Chromosomal Proteins, Non-Histone/physiology , Heterochromatin/physiology , Humans
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