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1.
Am J Hum Genet ; 108(7): 1283-1300, 2021 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-34214447

RESUMO

Most rare clinical missense variants cannot currently be classified as pathogenic or benign. Deficiency in human 5,10-methylenetetrahydrofolate reductase (MTHFR), the most common inherited disorder of folate metabolism, is caused primarily by rare missense variants. Further complicating variant interpretation, variant impacts often depend on environment. An important example of this phenomenon is the MTHFR variant p.Ala222Val (c.665C>T), which is carried by half of all humans and has a phenotypic impact that depends on dietary folate. Here we describe the results of 98,336 variant functional-impact assays, covering nearly all possible MTHFR amino acid substitutions in four folinate environments, each in the presence and absence of p.Ala222Val. The resulting atlas of MTHFR variant effects reveals many complex dependencies on both folinate and p.Ala222Val. MTHFR atlas scores can distinguish pathogenic from benign variants and, among individuals with severe MTHFR deficiency, correlate with age of disease onset. Providing a powerful tool for understanding structure-function relationships, the atlas suggests a role for a disordered loop in retaining cofactor at the active site and identifies variants that enable escape of inhibition by S-adenosylmethionine. Thus, a model based on eight MTHFR variant effect maps illustrates how shifting landscapes of environment- and genetic-background-dependent missense variation can inform our clinical, structural, and functional understanding of MTHFR deficiency.


Assuntos
Metilenotetra-Hidrofolato Redutase (NADPH2)/genética , Mutação de Sentido Incorreto , Substituição de Aminoácidos , Análise Mutacional de DNA , Diploide , Biblioteca Gênica , Genótipo , Humanos , Metilenotetra-Hidrofolato Redutase (NADPH2)/deficiência , Metilenotetra-Hidrofolato Redutase (NADPH2)/fisiologia , Saccharomyces cerevisiae/genética
2.
Mol Syst Biol ; 16(9): e9828, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32939983

RESUMO

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.

3.
Nature ; 580(7803): 402-408, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32296183

RESUMO

Global insights into cellular organization and genome function require comprehensive understanding of the interactome networks that mediate genotype-phenotype relationships1,2. Here we present a human 'all-by-all' reference interactome map of human binary protein interactions, or 'HuRI'. With approximately 53,000 protein-protein interactions, HuRI has approximately four times as many such interactions as there are high-quality curated interactions from small-scale studies. The integration of HuRI with genome3, transcriptome4 and proteome5 data enables cellular function to be studied within most physiological or pathological cellular contexts. We demonstrate the utility of HuRI in identifying the specific subcellular roles of protein-protein interactions. Inferred tissue-specific networks reveal general principles for the formation of cellular context-specific functions and elucidate potential molecular mechanisms that might underlie tissue-specific phenotypes of Mendelian diseases. HuRI is a systematic proteome-wide reference that links genomic variation to phenotypic outcomes.


Assuntos
Proteoma/metabolismo , Espaço Extracelular/metabolismo , Humanos , Especificidade de Órgãos , Mapeamento de Interação de Proteínas
4.
Cell Syst ; 10(1): 25-38.e10, 2020 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-31668799

RESUMO

Many traits are complex, depending non-additively on variant combinations. Even in model systems, such as the yeast S. cerevisiae, carrying out the high-order variant-combination testing needed to dissect complex traits remains a daunting challenge. Here, we describe "X-gene" genetic analysis (XGA), a strategy for engineering and profiling highly combinatorial gene perturbations. We demonstrate XGA on yeast ABC transporters by engineering 5,353 strains, each deleted for a random subset of 16 transporters, and profiling each strain's resistance to 16 compounds. XGA yielded 85,648 genotype-to-resistance observations, revealing high-order genetic interactions for 13 of the 16 transporters studied. Neural networks yielded intuitive functional models and guided exploration of fluconazole resistance, which was influenced non-additively by five genes. Together, our results showed that highly combinatorial genetic perturbation can functionally dissect complex traits, supporting pursuit of analogous strategies in human cells and other model systems.


Assuntos
Transporte Biológico/genética , Proteínas de Membrana Transportadoras/genética , Humanos
5.
Mol Syst Biol ; 14(5): e7985, 2018 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-29807908

RESUMO

Condition-dependent genetic interactions can reveal functional relationships between genes that are not evident under standard culture conditions. State-of-the-art yeast genetic interaction mapping, which relies on robotic manipulation of arrays of double-mutant strains, does not scale readily to multi-condition studies. Here, we describe barcode fusion genetics to map genetic interactions (BFG-GI), by which double-mutant strains generated via en masse "party" mating can also be monitored en masse for growth to detect genetic interactions. By using site-specific recombination to fuse two DNA barcodes, each representing a specific gene deletion, BFG-GI enables multiplexed quantitative tracking of double mutants via next-generation sequencing. We applied BFG-GI to a matrix of DNA repair genes under nine different conditions, including methyl methanesulfonate (MMS), 4-nitroquinoline 1-oxide (4NQO), bleomycin, zeocin, and three other DNA-damaging environments. BFG-GI recapitulated known genetic interactions and yielded new condition-dependent genetic interactions. We validated and further explored a subnetwork of condition-dependent genetic interactions involving MAG1, SLX4, and genes encoding the Shu complex, and inferred that loss of the Shu complex leads to an increase in the activation of the checkpoint protein kinase Rad53.


Assuntos
Mapeamento Cromossômico , Código de Barras de DNA Taxonômico , Dano ao DNA , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Reparo do DNA , Epistasia Genética , Deleção de Genes , Loci Gênicos , Sequenciamento de Nucleotídeos em Larga Escala , Metanossulfonato de Metila , Modelos Teóricos , Regiões Promotoras Genéticas , Reprodutibilidade dos Testes
6.
Front Microbiol ; 8: 2194, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29163459

RESUMO

An extensive repertoire of molecular tools is available for genetic analysis in laboratory strains of S. cerevisiae. Although this has widely contributed to the interpretation of gene functionality within haploid laboratory isolates, the genetics of metabolism in commercially-relevant polyploid yeast strains is still poorly understood. Genetic engineering in industrial yeasts is undergoing major changes due to Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated protein (Cas) engineering approaches. Here we apply the CRISPR/Cas9 system to two commercial "starter" strains of S. cerevisiae (EC1118, AWRI796), eliminating the CAN1 arginine permease pathway to generate strains with reduced urea production (18.5 and 35.5% for EC1118 and AWRI796, respectively). In a wine-model environment based on two grape musts obtained from Chardonnay and Cabernet Sauvignon cultivars, both S. cerevisiae starter strains and CAN1 mutants completed the must fermentation in 8-12 days. However, recombinant strains carrying the can1 mutation failed to produce urea, suggesting that the genetic modification successfully impaired the arginine metabolism. In conclusion, the reduction of urea production in a wine-model environment confirms that the CRISPR/Cas9 system has been successfully established in S. cerevisiae wine yeasts.

7.
ACS Cent Sci ; 2(10): 687-701, 2016 Oct 26.
Artigo em Inglês | MEDLINE | ID: mdl-27800551

RESUMO

The development of new antimalarial compounds remains a pivotal part of the strategy for malaria elimination. Recent large-scale phenotypic screens have provided a wealth of potential starting points for hit-to-lead campaigns. One such public set is explored, employing an open source research mechanism in which all data and ideas were shared in real time, anyone was able to participate, and patents were not sought. One chemical subseries was found to exhibit oral activity but contained a labile ester that could not be replaced without loss of activity, and the original hit exhibited remarkable sensitivity to minor structural change. A second subseries displayed high potency, including activity within gametocyte and liver stage assays, but at the cost of low solubility. As an open source research project, unexplored avenues are clearly identified and may be explored further by the community; new findings may be cumulatively added to the present work.

8.
Science ; 354(6312)2016 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-27811238

RESUMO

Genetic suppression occurs when the phenotypic defects caused by a mutation in a particular gene are rescued by a mutation in a second gene. To explore the principles of genetic suppression, we examined both literature-curated and unbiased experimental data, involving systematic genetic mapping and whole-genome sequencing, to generate a large-scale suppression network among yeast genes. Most suppression pairs identified novel relationships among functionally related genes, providing new insights into the functional wiring diagram of the cell. In addition to suppressor mutations, we identified frequent secondary mutations,in a subset of genes, that likely cause a delay in the onset of stationary phase, which appears to promote their enrichment within a propagating population. These findings allow us to formulate and quantify general mechanisms of genetic suppression.


Assuntos
Redes Reguladoras de Genes , Genes Fúngicos , Genes Supressores , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Supressão Genética , Fenômenos Fisiológicos Celulares/genética , Mapeamento Cromossômico
9.
Mol Syst Biol ; 12(4): 863, 2016 Apr 22.
Artigo em Inglês | MEDLINE | ID: mdl-27107012

RESUMO

High-throughput binary protein interaction mapping is continuing to extend our understanding of cellular function and disease mechanisms. However, we remain one or two orders of magnitude away from a complete interaction map for humans and other major model organisms. Completion will require screening at substantially larger scales with many complementary assays, requiring further efficiency gains in proteome-scale interaction mapping. Here, we report Barcode Fusion Genetics-Yeast Two-Hybrid (BFG-Y2H), by which a full matrix of protein pairs can be screened in a single multiplexed strain pool. BFG-Y2H uses Cre recombination to fuse DNA barcodes from distinct plasmids, generating chimeric protein-pair barcodes that can be quantified via next-generation sequencing. We applied BFG-Y2H to four different matrices ranging in scale from ~25 K to 2.5 M protein pairs. The results show that BFG-Y2H increases the efficiency of protein matrix screening, with quality that is on par with state-of-the-art Y2H methods.


Assuntos
Centrossomo/metabolismo , Mapeamento de Interação de Proteínas/métodos , Proteoma/metabolismo , Saccharomyces cerevisiae/genética , Cromossomos Humanos/metabolismo , Biblioteca Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Ligação Proteica , Técnicas do Sistema de Duplo-Híbrido
10.
Oncotarget ; 6(29): 26909-21, 2015 Sep 29.
Artigo em Inglês | MEDLINE | ID: mdl-26353928

RESUMO

The mevalonate (MVA) pathway is often dysregulated or overexpressed in many cancers suggesting tumor dependency on this classic metabolic pathway. Statins, which target the rate-limiting enzyme of this pathway, 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), are promising agents currently being evaluated in clinical trials for anti-cancer efficacy. To uncover novel targets that potentiate statin-induced apoptosis when knocked down, we carried out a pooled genome-wide short hairpin RNA (shRNA) screen. Genes of the MVA pathway were amongst the top-scoring targets, including sterol regulatory element binding transcription factor 2 (SREBP2), 3-hydroxy-3-methylglutaryl-coenzyme A synthase 1 (HMGCS1) and geranylgeranyl diphosphate synthase 1 (GGPS1). Each gene was independently validated and shown to significantly sensitize A549 cells to statin-induced apoptosis when knocked down. SREBP2 knockdown in lung and breast cancer cells completely abrogated the fluvastatin-induced upregulation of sterol-responsive genes HMGCR and HMGCS1. Knockdown of SREBP2 alone did not affect three-dimensional growth of lung and breast cancer cells, yet in combination with fluvastatin cell growth was disrupted. Taken together, these results show that directly targeting multiple levels of the MVA pathway, including blocking the sterol-feedback loop initiated by statin treatment, is an effective and targetable anti-tumor strategy.


Assuntos
Apoptose , Regulação Neoplásica da Expressão Gênica , Ácido Mevalônico/metabolismo , Neoplasias/patologia , Interferência de RNA , Antineoplásicos/química , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Dimetilaliltranstransferase/genética , Farnesiltranstransferase/genética , Ácidos Graxos Monoinsaturados/química , Feminino , Fluvastatina , Geraniltranstransferase/genética , Humanos , Hidroximetilglutaril-CoA Redutases/metabolismo , Hidroximetilglutaril-CoA Sintase/genética , Indóis/química , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/metabolismo , RNA Interferente Pequeno/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Proteína de Ligação a Elemento Regulador de Esterol 2/genética
11.
Apoptosis ; 20(7): 948-59, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25832785

RESUMO

To identify new biological vulnerabilities in acute myeloid leukemia, we screened a library of natural products for compounds cytotoxic to TEX leukemia cells. This screen identified the novel small molecule Deoxysappanone B 7,4' dimethyl ether (Deox B 7,4), which possessed nanomolar anti-leukemic activity. To determine the anti-leukemic mechanism of action of Deox B 7,4, we conducted a genome-wide screen in Saccharomyces cerevisiae and identified enrichment of genes related to mitotic cell cycle as well as vacuolar acidification, therefore pointing to microtubules and vacuolar (V)-ATPase as potential drug targets. Further investigations into the mechanisms of action of Deox B 7,4 and a related analogue revealed that these compounds were reversible microtubule inhibitors that bound near the colchicine site. In addition, Deox B 7,4 and its analogue increased lysosomal V-ATPase activity and lysosome acidity. The effects on microtubules and lysosomes were functionally important for the anti-leukemic effects of these drugs. The lysosomal effects were characteristic of select microtubule inhibitors as only the Deox compounds and nocodazole, but not colchicine, vinca alkaloids or paclitaxel, altered lysosome acidity and induced lysosomal disruption. Thus, our data highlight a new mechanism of action of select microtubule inhibitors on lysosomal function.


Assuntos
Cromonas/farmacologia , Guaiacol/análogos & derivados , Leucemia Mieloide Aguda/metabolismo , Lisossomos/efeitos dos fármacos , Moduladores de Tubulina/farmacologia , Animais , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Guaiacol/farmacologia , Humanos , Leucemia Mieloide Aguda/patologia , Lisossomos/química , Lisossomos/metabolismo , Camundongos , Saccharomyces cerevisiae , ATPases Vacuolares Próton-Translocadoras/metabolismo
12.
Biomed Res Int ; 2015: 976458, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25667933

RESUMO

Spaceflight is a unique environment with profound effects on biological systems including tissue redistribution and musculoskeletal stresses. However, the more subtle biological effects of spaceflight on cells and organisms are difficult to measure in a systematic, unbiased manner. Here we test the utility of the molecularly barcoded yeast deletion collection to provide a quantitative assessment of the effects of microgravity on a model organism. We developed robust hardware to screen, in parallel, the complete collection of ~4800 homozygous and ~5900 heterozygous (including ~1100 single-copy deletions of essential genes) yeast deletion strains, each carrying unique DNA that acts as strain identifiers. We compared strain fitness for the homozygous and heterozygous yeast deletion collections grown in spaceflight and ground, as well as plus and minus hyperosmolar sodium chloride, providing a second additive stressor. The genome-wide sensitivity profiles obtained from these treatments were then queried for their similarity to a compendium of drugs whose effects on the yeast collection have been previously reported. We found that the effects of spaceflight have high concordance with the effects of DNA-damaging agents and changes in redox state, suggesting mechanisms by which spaceflight may negatively affect cell fitness.


Assuntos
Deleção de Sequência/genética , Leveduras/genética , Leveduras/fisiologia , DNA Fúngico/genética , Estudos de Avaliação como Assunto , Voo Espacial/métodos , Ausência de Peso
13.
Science ; 344(6180): 208-11, 2014 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-24723613

RESUMO

Genome-wide characterization of the in vivo cellular response to perturbation is fundamental to understanding how cells survive stress. Identifying the proteins and pathways perturbed by small molecules affects biology and medicine by revealing the mechanisms of drug action. We used a yeast chemogenomics platform that quantifies the requirement for each gene for resistance to a compound in vivo to profile 3250 small molecules in a systematic and unbiased manner. We identified 317 compounds that specifically perturb the function of 121 genes and characterized the mechanism of specific compounds. Global analysis revealed that the cellular response to small molecules is limited and described by a network of 45 major chemogenomic signatures. Our results provide a resource for the discovery of functional interactions among genes, chemicals, and biological processes.


Assuntos
Células/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos/métodos , Resistência a Medicamentos/genética , Redes Reguladoras de Genes , Estudo de Associação Genômica Ampla/métodos , Bibliotecas de Moléculas Pequenas/farmacologia , Linhagem Celular Tumoral , Haploinsuficiência , Humanos , Farmacogenética , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética
14.
PLoS Genet ; 9(5): e1003479, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23658529

RESUMO

Nucleosomes in all eukaryotes examined to date adopt a characteristic architecture within genes and play fundamental roles in regulating transcription, yet the identity and precise roles of many of the trans-acting factors responsible for the establishment and maintenance of this organization remain to be identified. We profiled a compendium of 50 yeast strains carrying conditional alleles or complete deletions of genes involved in transcriptional regulation, histone biology, and chromatin remodeling, as well as compounds that target transcription and histone deacetylases, to assess their respective roles in nucleosome positioning and transcription. We find that nucleosome patterning in genes is affected by many factors, including the CAF-1 complex, Spt10, and Spt21, in addition to previously reported remodeler ATPases and histone chaperones. Disruption of these factors or reductions in histone levels led genic nucleosomes to assume positions more consistent with their intrinsic sequence preferences, with pronounced and specific shifts of the +1 nucleosome relative to the transcription start site. These shifts of +1 nucleosomes appear to have functional consequences, as several affected genes in Ino80 mutants exhibited altered expression responses. Our parallel expression profiling compendium revealed extensive transcription changes in intergenic and antisense regions, most of which occur in regions with altered nucleosome occupancy and positioning. We show that the nucleosome-excluding transcription factors Reb1, Abf1, Tbf1, and Rsc3 suppress cryptic transcripts at their target promoters, while a combined analysis of nucleosome and expression profiles identified 36 novel transcripts that are normally repressed by Tup1/Cyc8. Our data confirm and extend the roles of chromatin remodelers and chaperones as major determinants of genic nucleosome positioning, and these data provide a valuable resource for future studies.


Assuntos
Montagem e Desmontagem da Cromatina/genética , Chaperonas de Histonas/genética , Saccharomyces cerevisiae/genética , Transcrição Genética , Adenosina Trifosfatases/genética , Cromatina , Fator 1 de Modelagem da Cromatina/genética , Proteínas de Ligação a DNA/genética , Regulação Fúngica da Expressão Gênica , Histona Acetiltransferases/genética , Nucleossomos , Regiões Promotoras Genéticas , Proteínas de Saccharomyces cerevisiae/genética , Transativadores/genética , Transativadores/metabolismo , Fatores de Transcrição/genética
15.
J Clin Invest ; 123(1): 315-28, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23202731

RESUMO

Despite efforts to understand and treat acute myeloid leukemia (AML), there remains a need for more comprehensive therapies to prevent AML-associated relapses. To identify new therapeutic strategies for AML, we screened a library of on- and off-patent drugs and identified the antimalarial agent mefloquine as a compound that selectively kills AML cells and AML stem cells in a panel of leukemia cell lines and in mice. Using a yeast genome-wide functional screen for mefloquine sensitizers, we identified genes associated with the yeast vacuole, the homolog of the mammalian lysosome. Consistent with this, we determined that mefloquine disrupts lysosomes, directly permeabilizes the lysosome membrane, and releases cathepsins into the cytosol. Knockdown of the lysosomal membrane proteins LAMP1 and LAMP2 resulted in decreased cell viability, as did treatment of AML cells with known lysosome disrupters. Highlighting a potential therapeutic rationale for this strategy, leukemic cells had significantly larger lysosomes compared with normal cells, and leukemia-initiating cells overexpressed lysosomal biogenesis genes. These results demonstrate that lysosomal disruption preferentially targets AML cells and AML progenitor cells, providing a rationale for testing lysosomal disruption as a novel therapeutic strategy for AML.


Assuntos
Membranas Intracelulares/metabolismo , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/metabolismo , Lisossomos/metabolismo , Células-Tronco Neoplásicas/metabolismo , Animais , Antimaláricos/farmacocinética , Antimaláricos/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Feminino , Técnicas de Silenciamento de Genes , Estudo de Associação Genômica Ampla , Humanos , Membranas Intracelulares/patologia , Células K562 , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Proteína 2 de Membrana Associada ao Lisossomo , Glicoproteínas de Membrana Associadas ao Lisossomo/genética , Glicoproteínas de Membrana Associadas ao Lisossomo/metabolismo , Lisossomos/genética , Lisossomos/fisiologia , Masculino , Mefloquina/farmacocinética , Mefloquina/farmacologia , Camundongos , Células-Tronco Neoplásicas/patologia , Permeabilidade/efeitos dos fármacos , Saccharomyces cerevisiae/genética
16.
Mol Cell Biol ; 33(1): 170-82, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23109428

RESUMO

Across diverse eukaryotes, the Paf1 complex (Paf1C) plays critical roles in RNA polymerase II transcription elongation and regulation of histone modifications. Beyond these roles, the human and Saccharomyces cerevisiae Paf1 complexes also interact with RNA 3'-end processing components to affect transcript 3'-end formation. Specifically, the Saccharomyces cerevisiae Paf1C functions with the RNA binding proteins Nrd1 and Nab3 to regulate the termination of at least two small nucleolar RNAs (snoRNAs). To determine how Paf1C-dependent functions regulate snoRNA formation, we used high-density tiling arrays to analyze transcripts in paf1Δ cells and uncover new snoRNA targets of Paf1. Detailed examination of Paf1-regulated snoRNA genes revealed locus-specific requirements for Paf1-dependent posttranslational histone modifications. We also discovered roles for the transcriptional regulators Bur1-Bur2, Rad6, and Set2 in snoRNA 3'-end formation. Surprisingly, at some snoRNAs, this function of Rad6 appears to be primarily independent of its role in histone H2B monoubiquitylation. Cumulatively, our work reveals a broad requirement for the Paf1C in snoRNA 3'-end formation in S. cerevisiae, implicates the participation of transcriptional proteins and histone modifications in this process, and suggests that the Paf1C contributes to the fine tuning of nuanced levels of regulation that exist at individual loci.


Assuntos
Regulação Fúngica da Expressão Gênica , Histonas/metabolismo , Proteínas Nucleares/metabolismo , RNA Nucleolar Pequeno/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Quinases Ciclina-Dependentes/genética , Quinases Ciclina-Dependentes/metabolismo , Ciclinas/genética , Ciclinas/metabolismo , Histonas/genética , Metiltransferases/genética , Metiltransferases/metabolismo , Mutação , Proteínas Nucleares/genética , Processamento de Proteína Pós-Traducional , RNA Nucleolar Pequeno/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteína de Ligação a TATA-Box/genética , Proteína de Ligação a TATA-Box/metabolismo , Fatores de Elongação da Transcrição/genética , Fatores de Elongação da Transcrição/metabolismo , Enzimas de Conjugação de Ubiquitina/genética , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitinação
17.
Elife ; 1: e00078, 2012 Dec 13.
Artigo em Inglês | MEDLINE | ID: mdl-23240084

RESUMO

The eukaryotic nucleosome is the fundamental unit of chromatin, comprising a protein octamer that wraps ∼147 bp of DNA and has essential roles in DNA compaction, replication and gene expression. Nucleosomes and chromatin have historically been considered to be unique to eukaryotes, yet studies of select archaea have identified homologs of histone proteins that assemble into tetrameric nucleosomes. Here we report the first archaeal genome-wide nucleosome occupancy map, as observed in the halophile Haloferax volcanii. Nucleosome occupancy was compared with gene expression by compiling a comprehensive transcriptome of Hfx. volcanii. We found that archaeal transcripts possess hallmarks of eukaryotic chromatin structure: nucleosome-depleted regions at transcriptional start sites and conserved -1 and +1 promoter nucleosomes. Our observations demonstrate that histones and chromatin architecture evolved before the divergence of Archaea and Eukarya, suggesting that the fundamental role of chromatin in the regulation of gene expression is ancient.DOI:http://dx.doi.org/10.7554/eLife.00078.001.


Assuntos
Regulação da Expressão Gênica em Archaea , Genoma Arqueal , Haloferax volcanii/genética , Histonas/genética , Nucleossomos/química , Fatores de Transcrição/genética , Evolução Biológica , Montagem e Desmontagem da Cromatina , Replicação do DNA , Células Eucarióticas/citologia , Células Eucarióticas/metabolismo , Perfilação da Expressão Gênica , Haloferax volcanii/química , Haloferax volcanii/metabolismo , Histonas/metabolismo , Anotação de Sequência Molecular , Nucleossomos/metabolismo , Regiões Promotoras Genéticas , Fatores de Transcrição/metabolismo , Sítio de Iniciação de Transcrição , Transcrição Genética
18.
Dev Cell ; 23(5): 1059-71, 2012 Nov 13.
Artigo em Inglês | MEDLINE | ID: mdl-23123093

RESUMO

Gametes are among the most highly specialized cells produced during development. Although gametogenesis culminates in transcriptional quiescence in plants and animals, regulatory mechanisms controlling this are unknown. Here, we confirm that gamete differentiation in the single-celled yeast Saccharomyces cerevisiae is accompanied by global transcriptional shutoff following the completion of meiosis. We show that Jhd2, a highly conserved JARID1-family histone H3K4 demethylase, activates protein-coding gene transcription in opposition to this programmed transcriptional shutoff, sustaining the period of productive transcription during spore differentiation. Moreover, using genome-wide nucleosome, H3K4me, and transcript mapping experiments, we demonstrate that JHD2 globally represses intergenic noncoding transcription during this period. The widespread transcriptional defects of JHD2 mutants are associated with precocious differentiation and the production of stress-sensitive spores, demonstrating that Jhd2 regulation of the global postmeiotic transcriptional program is critical for the production of healthy meiotic progeny.


Assuntos
Gametogênese/genética , Gametogênese/fisiologia , Histonas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Epigênese Genética , Genes Fúngicos , Histona Desmetilases com o Domínio Jumonji/genética , Histona Desmetilases com o Domínio Jumonji/metabolismo , Meiose , Metilação , Mutação , Nucleossomos/metabolismo , RNA Fúngico/genética , RNA Fúngico/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Esporos Fúngicos/genética , Esporos Fúngicos/crescimento & desenvolvimento , Esporos Fúngicos/metabolismo , Proteínas de Ligação a Telômeros/genética , Proteínas de Ligação a Telômeros/metabolismo , Fatores de Tempo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição Genética
19.
BMC Genomics ; 13: 267, 2012 Jun 22.
Artigo em Inglês | MEDLINE | ID: mdl-22727066

RESUMO

BACKGROUND: Chitosan oligosaccharide (COS), a deacetylated derivative of chitin, is an abundant, and renewable natural polymer. COS has higher antimicrobial properties than chitosan and is presumed to act by disrupting/permeabilizing the cell membranes of bacteria, yeast and fungi. COS is relatively non-toxic to mammals. By identifying the molecular and genetic targets of COS, we hope to gain a better understanding of the antifungal mode of action of COS. RESULTS: Three different chemogenomic fitness assays, haploinsufficiency (HIP), homozygous deletion (HOP), and multicopy suppression (MSP) profiling were combined with a transcriptomic analysis to gain insight in to the mode of action and mechanisms of resistance to chitosan oligosaccharides. The fitness assays identified 39 yeast deletion strains sensitive to COS and 21 suppressors of COS sensitivity. The genes identified are involved in processes such as RNA biology (transcription, translation and regulatory mechanisms), membrane functions (e.g. signalling, transport and targeting), membrane structural components, cell division, and proteasome processes. The transcriptomes of control wild type and 5 suppressor strains overexpressing ARL1, BCK2, ERG24, MSG5, or RBA50, were analyzed in the presence and absence of COS. Some of the up-regulated transcripts in the suppressor overexpressing strains exposed to COS included genes involved in transcription, cell cycle, stress response and the Ras signal transduction pathway. Down-regulated transcripts included those encoding protein folding components and respiratory chain proteins. The COS-induced transcriptional response is distinct from previously described environmental stress responses (i.e. thermal, salt, osmotic and oxidative stress) and pre-treatment with these well characterized environmental stressors provided little or any resistance to COS. CONCLUSIONS: Overexpression of the ARL1 gene, a member of the Ras superfamily that regulates membrane trafficking, provides protection against COS-induced cell membrane permeability and damage. We found that the ARL1 COS-resistant over-expression strain was as sensitive to Amphotericin B, Fluconazole and Terbinafine as the wild type cells and that when COS and Fluconazole are used in combination they act in a synergistic fashion. The gene targets of COS identified in this study indicate that COS's mechanism of action is different from other commonly studied fungicides that target membranes, suggesting that COS may be an effective fungicide for drug-resistant fungal pathogens.


Assuntos
Quitosana/farmacologia , Saccharomyces cerevisiae/efeitos dos fármacos , Anfotericina B/farmacologia , Antifúngicos/farmacologia , Permeabilidade da Membrana Celular/efeitos dos fármacos , Farmacorresistência Fúngica/efeitos dos fármacos , Fluconazol/farmacologia , Perfilação da Expressão Gênica , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Haploinsuficiência/efeitos dos fármacos , Proteínas Monoméricas de Ligação ao GTP/genética , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Naftalenos/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Terbinafina , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo
20.
PLoS One ; 7(1): e29798, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22253786

RESUMO

Elesclomol is a first-in-class investigational drug currently undergoing clinical evaluation as a novel cancer therapeutic. The potent antitumor activity of the compound results from the elevation of reactive oxygen species (ROS) and oxidative stress to levels incompatible with cellular survival. However, the molecular target(s) and mechanism by which elesclomol generates ROS and subsequent cell death were previously undefined. The cellular cytotoxicity of elesclomol in the yeast S. cerevisiae appears to occur by a mechanism similar, if not identical, to that in cancer cells. Accordingly, here we used a powerful and validated technology only available in yeast that provides critical insights into the mechanism of action, targets and processes that are disrupted by drug treatment. Using this approach we show that elesclomol does not work through a specific cellular protein target. Instead, it targets a biologically coherent set of processes occurring in the mitochondrion. Specifically, the results indicate that elesclomol, driven by its redox chemistry, interacts with the electron transport chain (ETC) to generate high levels of ROS within the organelle and consequently cell death. Additional experiments in melanoma cells involving drug treatments or cells lacking ETC function confirm that the drug works similarly in human cancer cells. This deeper understanding of elesclomol's mode of action has important implications for the therapeutic application of the drug, including providing a rationale for biomarker-based stratification of patients likely to respond in the clinical setting.


Assuntos
Antineoplásicos/farmacologia , Hidrazinas/farmacologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Antineoplásicos/química , Antineoplásicos/uso terapêutico , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Cobre/farmacologia , DNA Mitocondrial/genética , Ensaios de Seleção de Medicamentos Antitumorais , Transporte de Elétrons/efeitos dos fármacos , Humanos , Hidrazinas/química , Hidrazinas/uso terapêutico , Melanoma/tratamento farmacológico , Mutação/genética , Espécies Reativas de Oxigênio/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos
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