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1.
PLoS Genet ; 11(3): e1005083, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25793375

RESUMO

Heterochromatin and associated gene silencing processes play roles in development, genome defense, and chromosome function. In many species, constitutive heterochromatin is decorated with histone H3 tri-methylated at lysine 9 (H3K9me3) and cytosine methylation. In Neurospora crassa, a five-protein complex, DCDC, catalyzes H3K9 methylation, which then directs DNA methylation. Here, we identify and characterize a gene important for DCDC function, dim-3 (defective in methylation-3), which encodes the nuclear import chaperone NUP-6 (Importin α). The critical mutation in dim-3 results in a substitution in an ARM repeat of NUP-6 and causes a substantial loss of H3K9me3 and DNA methylation. Surprisingly, nuclear transport of all known proteins involved in histone and DNA methylation, as well as a canonical transport substrate, appear normal in dim-3 strains. Interactions between DCDC members also appear normal, but the nup-6(dim-3) allele causes the DCDC members DIM-5 and DIM-7 to mislocalize from heterochromatin and NUP-6dim-3 itself is mislocalized from the nuclear envelope, at least in conidia. GCN-5, a member of the SAGA histone acetyltransferase complex, also shows altered localization in dim-3, raising the possibility that NUP-6 is necessary to localize multiple chromatin complexes following nucleocytoplasmic transport.


Assuntos
Metilação de DNA/genética , Proteínas Fúngicas/genética , Heterocromatina/genética , Histona-Lisina N-Metiltransferase/genética , alfa Carioferinas/genética , Citosina/metabolismo , Inativação Gênica , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/genética , Histonas/metabolismo , Lisina/genética , Lisina/metabolismo , Mutação , Neurospora crassa/genética , alfa Carioferinas/metabolismo
2.
Eukaryot Cell ; 14(1): 25-8, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25362134

RESUMO

The cullin-4 (CUL4) complex DCDC (DIM-5/-7/-9/CUL4/DDB1 complex) is essential for DNA methylation and heterochromatin formation in Neurospora crassa. Cullins form the scaffold of cullin-RING E3 ubiquitin ligases (CRLs) and are modified by the covalent attachment of NEDD8, a ubiquitin-like protein that regulates the stability and activity of CRLs. We report that neddylation is not required for CUL4-dependent DNA methylation or heterochromatin formation but is required for the DNA repair functions. Moreover, the RING domain protein RBX1 and a segment of the CUL4 C terminus that normally interacts with RBX1, the E2 ligase, CAND1, and CSN are dispensable for DNA methylation and heterochromatin formation by DCDC. Our study provides evidence for the noncanonical functions of core CRL components.


Assuntos
Proteínas Culina/metabolismo , Proteínas Fúngicas/metabolismo , Heterocromatina/metabolismo , Neurospora crassa/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Culina/química , Proteínas Culina/genética , Metilação de DNA , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Heterocromatina/genética , Neurospora crassa/genética , Ligação Proteica , Estrutura Terciária de Proteína , Fatores de Transcrição/metabolismo , Ubiquitinas/metabolismo
3.
PLoS One ; 7(8): e43210, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22952647

RESUMO

Despite the known relevance of genomic structural variants to pathogen behavior, cancer, development, and evolution, certain repeat based structural variants may evade detection by existing high-throughput techniques. Here, we present ruler arrays, a technique to detect genomic structural variants including insertions and deletions (indels), duplications, and translocations. A ruler array exploits DNA polymerase's processivity to detect physical distances between defined genomic sequences regardless of the intervening sequence. The method combines a sample preparation protocol, tiling genomic microarrays, and a new computational analysis. The analysis of ruler array data from two genomic samples enables the identification of structural variation between the samples. In an empirical test between two closely related haploid strains of yeast ruler arrays detected 78% of the structural variants larger than 100 bp.


Assuntos
Genoma , Variação Estrutural do Genoma , Haploidia , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Hibridização Genômica Comparativa , DNA Polimerase Dirigida por DNA/metabolismo , Proteínas Fúngicas/genética , Genômica/métodos , Modelos Genéticos , Modelos Estatísticos , Polimorfismo de Nucleotídeo Único , Probabilidade , Saccharomyces cerevisiae/genética
4.
Mol Cell ; 45(4): 470-82, 2012 Feb 24.
Artigo em Inglês | MEDLINE | ID: mdl-22264825

RESUMO

Mechanisms through which long intergenic noncoding RNAs (ncRNAs) exert regulatory effects on eukaryotic biological processes remain largely elusive. Most studies of these phenomena rely on methods that measure average behaviors in cell populations, lacking resolution to observe the effects of ncRNA transcription on gene expression in a single cell. Here, we combine quantitative single-molecule RNA FISH experiments with yeast genetics and computational modeling to gain mechanistic insights into the regulation of the Saccharomyces cerevisiae protein-coding gene FLO11 by two intergenic ncRNAs, ICR1 and PWR1. Direct detection of FLO11 mRNA and these ncRNAs in thousands of individual cells revealed alternative expression states and provides evidence that ICR1 and PWR1 contribute to FLO11's variegated transcription, resulting in Flo11-dependent phenotypic heterogeneity in clonal cell populations by modulating recruitment of key transcription factors to the FLO11 promoter.


Assuntos
Regulação Fúngica da Expressão Gênica , Glicoproteínas de Membrana/genética , RNA não Traduzido/fisiologia , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Fatores de Transcrição/metabolismo , DNA Intergênico , Hibridização in Situ Fluorescente , Modelos Genéticos , Regiões Promotoras Genéticas , RNA Mensageiro/metabolismo , Análise de Célula Única , Fatores de Transcrição/fisiologia
5.
J Comput Biol ; 18(3): 295-303, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21385035

RESUMO

STEREO is a novel algorithm that discovers cis-regulatory RNA interactions by assembling complete and potentially overlapping same-strand RNA transcripts from tiling expression data. STEREO first identifies coherent segments of transcription and then discovers individual transcripts that are consistent with the observed segments given intensity and shape constraints. We used STEREO to identify 1446 regions of overlapping transcription in two strains of yeast, including transcripts that comprise a new form of molecular toggle switch that controls gene variegation.


Assuntos
Algoritmos , Regulação Fúngica da Expressão Gênica , Genômica/métodos , RNA Fúngico/genética , Saccharomyces cerevisiae/genética , Transcrição Gênica , Simulação por Computador , Modelos Genéticos , Fosfoglicerato Desidrogenase/genética , Regiões Promotoras Genéticas , Proteínas de Saccharomyces cerevisiae/genética
6.
Proc Natl Acad Sci U S A ; 106(43): 18321-6, 2009 Oct 27.
Artigo em Inglês | MEDLINE | ID: mdl-19805129

RESUMO

The identification of specific functional roles for the numerous long noncoding (nc)RNAs found in eukaryotic transcriptomes is currently a matter of intense study amid speculation that these ncRNAs have key regulatory roles. We have identified a pair of cis-interfering ncRNAs in yeast that contribute to the control of variegated gene expression at the FLO11 locus by implementing a regulatory circuit that toggles between two stable states. These capped, polyadenylated ncRNAs are transcribed across the large intergenic region upstream of the FLO11 ORF. As with mammalian long intervening (li)ncRNAs, these yeast ncRNAs (ICR1 and PWR1) are themselves regulated by transcription factors (Sfl1 and Flo8) and chromatin remodelers (Rpd3L) that are key elements in phenotypic transitions in yeast. The mechanism that we describe explains the unanticipated role of a histone deacetylase complex in activating gene expression, because Rpd3L mutants force the ncRNA circuit into a state that silences the expression of the adjacent variegating gene.


Assuntos
Regulação Fúngica da Expressão Gênica , Histona Desacetilases/metabolismo , Glicoproteínas de Membrana/genética , RNA Fúngico/genética , RNA não Traduzido/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Montagem e Desmontagem da Cromatina , Histona Desacetilases/genética , Fases de Leitura Aberta , Regiões Promotoras Genéticas , Ligação Proteica , Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
7.
Cell Host Microbe ; 2(1): 55-67, 2007 Jul 12.
Artigo em Inglês | MEDLINE | ID: mdl-18005717

RESUMO

Innate immunity depends upon recognition of surface features common to broad groups of pathogens. The glucose polymer beta-glucan has been implicated in fungal immune recognition. Fungal walls have two kinds of beta-glucan: beta-1,3-glucan and beta-1,6-glucan. Predominance of beta-1,3-glucan has led to the presumption that it is the key immunological determinant for neutrophils. Examining various beta-glucans for their ability to stimulate human neutrophils, we find that the minor cell wall component beta-1,6-glucan mediates neutrophil activity more efficiently than beta-1,3-glucan, as measured by engulfment, production of reactive oxygen species, and expression of heat shock proteins. Neutrophils rapidly ingest beads coated with beta-1,6-glucan while ignoring those coated with beta-1,3-glucan. Complement factors C3b/C3d are deposited on beta-1,6-glucan more readily than on beta-1,3-glucan. Beta-1,6-glucan is also important for efficient engulfment of the human pathogen Candida albicans. These unique stimulatory effects offer potential for directed stimulation of neutrophils in a therapeutic context.


Assuntos
Candida/fisiologia , Parede Celular/fisiologia , Neutrófilos/fisiologia , Fagocitose/fisiologia , Regulação da Expressão Gênica , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/fisiologia , Humanos , Neutrófilos/microbiologia , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase , beta-Glucanas/farmacologia
8.
Cell ; 127(4): 735-45, 2006 Nov 17.
Artigo em Inglês | MEDLINE | ID: mdl-17110333

RESUMO

Entry into meiosis is a key developmental decision. We show here that meiotic entry in Saccharomyces cerevisiae is controlled by antisense-mediated regulation of IME4, a gene required for initiating meiosis. In MAT a/alpha diploids the antisense IME4 transcript is repressed by binding of the a1/alpha2 heterodimer at a conserved site located downstream of the IME4 coding sequence. MAT a/alpha diploids that produce IME4 antisense transcript have diminished sense transcription and fail to initiate meiosis. Haploids that produce the sense transcript have diminished antisense transcription and manifest several diploid phenotypes. Our data are consistent with transcription interference as a regulatory mechanism at the IME4 locus that determines cell fate.


Assuntos
Linhagem da Célula , RNA Antissenso/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Transcrição Gênica , Sequência de Bases , Sítios de Ligação , Sequência Conservada , Dimerização , Diploide , Regulação Fúngica da Expressão Gênica , Genes Fúngicos Tipo Acasalamento/genética , Haploidia , Modelos Genéticos , Dados de Sequência Molecular , Mutação/genética , Fases de Leitura Aberta/genética , Fenótipo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
9.
Plant J ; 45(2): 166-79, 2006 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-16367962

RESUMO

Copines are calcium-dependent membrane-binding proteins that are highly conserved among protozoa, plants, nematodes and mammals. Although they are implicated in membrane trafficking and signal transduction, the functions of these proteins are not well understood. The Arabidopsis copine gene BON1/CPN1 was previously shown to negatively regulate a disease resistance (R) gene SNC1. Here we report that in Arabidopsis, as in other organisms, there is a family of copine genes, BON1, 2 and 3. Using double and triple mutant combinations we show that these three copine genes have overlapping functions essential for the viability of plants. The loss of function of BON1 combined with that of BON2 or BON3 leads to extensive cell death phenotypes resembling the hypersensitive response (HR) in defense responses. The resulting lethality can be suppressed by mutations in PAD4 or EDS1 which are required for R gene signaling and cell death control. Accession-dependent phenotypes of the mutant combinations suggest that the BON/CPN genes may together repress several R genes other than SNC1. Moreover, the mutant combinations exhibit developmental defects when R-gene-mediated defense responses are largely suppressed in pad4 and eds1 mutants. Thus, the copine family in Arabidopsis may have effects in promoting growth and development in addition to repressing cell death, and these two processes might be intricately intertwined.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/citologia , Proteínas de Transporte/genética , Morte Celular/genética , Divisão Celular/genética , Genes de Plantas , Proteínas de Membrana/genética , Família Multigênica , Sequência de Aminoácidos , Proteínas de Arabidopsis/química , Proteínas de Ligação ao Cálcio , Proteínas de Transporte/química , Genes Letais , Proteínas de Membrana/química , Dados de Sequência Molecular , Mutação , Homologia de Sequência de Aminoácidos
10.
Proc Natl Acad Sci U S A ; 101(12): 4153-7, 2004 Mar 23.
Artigo em Inglês | MEDLINE | ID: mdl-15010530

RESUMO

Fungi must recognize plant-specific signals to initiate subsequent morphogenetic events such as filamentation that lead to infection. Here we show that the plant hormone indoleacetic acid (IAA) induces adhesion and filamentation of Saccharomyces cerevisiae. Genome expression profiling of cells treated with IAA identified Yap1, a fungal specific transcription factor, as a key mediator of this response. Strains lacking YAP1 (yap1-1) are hypersensitive to growth on IAA because they accumulate more IAA than can wild type. Members of a family of transporters the amino acid/auxin:proton symport permeases with homology to AUX1, a putative IAA transporter from plants, are up-regulated in the yap1-1 mutant. Deletion of any one of these transporters makes yap1-1 mutants more resistant to IAA by decreasing its uptake. The permease mutants are defective in IAA perception and filamentation. The ability of a fungus to perceive a plant hormone that causes it to differentiate into an invasive form has important implications for plant-pathogen interactions.


Assuntos
Ácidos Indolacéticos/farmacologia , Saccharomyces cerevisiae/crescimento & desenvolvimento , Relação Dose-Resposta a Droga , Perfilação da Expressão Gênica , Hifas/efeitos dos fármacos , Hifas/crescimento & desenvolvimento , Glicoproteínas de Membrana , Proteínas de Membrana/metabolismo , Reguladores de Crescimento de Plantas/farmacologia , Saccharomyces cerevisiae/efeitos dos fármacos , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo
11.
Plant J ; 37(3): 340-53, 2004 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-14731255

RESUMO

Lateral root formation, the primary way plants increase their root mass, displays developmental plasticity in response to environmental changes. The aberrant lateral root formation (alf)4-1 mutation blocks the initiation of lateral roots, thus greatly altering root system architecture. We have positionally cloned the ALF4 gene and have further characterized its phenotype. The encoded ALF4 protein is conserved among plants and has no similarities to proteins from other kingdoms. The gene is present in a single copy in Arabidopsis. Using translational reporters for ALF4 gene expression, we have determined that the ALF4 protein is nuclear localized and that the gene is expressed in most plant tissues; however, ALF4 expression and ALF4's subcellular location are not regulated by auxin. These findings taken together with further genetic and phenotypic characterization of the alf4-1 mutant suggest that ALF4 functions independent from auxin signaling and instead functions in maintaining the pericycle in the mitotically competent state needed for lateral root formation. Our results provide genetic evidence that the pericycle shares properties with meristems and that this tissue plays a central role in creating the developmental plasticity needed for root system development.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Núcleo Celular/genética , Raízes de Plantas/crescimento & desenvolvimento , Processamento Alternativo , Sequência de Aminoácidos , Arabidopsis/citologia , Proteínas de Arabidopsis/química , Sequência de Bases , Clonagem Molecular , Primers do DNA , Mitose , Dados de Sequência Molecular , Reação em Cadeia da Polimerase Via Transcriptase Reversa
12.
Proc Natl Acad Sci U S A ; 100(19): 11007-12, 2003 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-12958213

RESUMO

The transcriptional profiles of yeast cells that have been phagocytosed by either human neutrophils or monocytes were compared by using whole genome arrays. After phagocytosis by neutrophils, both Saccharomyces cerevisiae and Candida albicans respond by inducing genes of the methionine and arginine pathways. Neither of these pathways is induced upon phagocytosis by monocytes. Both fungi show a similar induction of these pathways when transferred from amino acid-rich medium to amino acid-deficient medium. These data suggest that the internal phagosome of the neutrophil is an amino acid-deficient environment.


Assuntos
Aminoácidos/metabolismo , Candida albicans/metabolismo , Neutrófilos/imunologia , Fagocitose , Saccharomyces cerevisiae/metabolismo , Ciclo Celular , Peróxido de Hidrogênio/farmacologia , Neutrófilos/fisiologia , Estresse Oxidativo , Transcrição Gênica/fisiologia
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