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1.
Nucleic Acids Res ; 48(19): 10877-10889, 2020 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-33010153

RESUMO

The Saccharomyces cerevisiae HO gene is a model regulatory system with complex transcriptional regulation. Budding yeast divide asymmetrically and HO is expressed only in mother cells where a nucleosome eviction cascade along the promoter during the cell cycle enables activation. HO expression in daughter cells is inhibited by high concentration of Ash1 in daughters. To understand how Ash1 represses transcription, we used a myo4 mutation which boosts Ash1 accumulation in both mothers and daughters and show that Ash1 inhibits promoter recruitment of SWI/SNF and Gcn5. We show Ash1 is also required for the efficient nucleosome repopulation that occurs after eviction, and the strongest effects of Ash1 are seen when Ash1 has been degraded and at promoter locations distant from where Ash1 bound. Additionally, we defined a specific nucleosome/nucleosome-depleted region structure that restricts HO activation to one of two paralogous DNA-binding factors. We also show that nucleosome eviction occurs bidirectionally over a large distance. Significantly, eviction of the more distant nucleosomes is dependent upon the FACT histone chaperone, and FACT is recruited to these regions when eviction is beginning. These last observations, along with ChIP experiments involving the SBF factor, suggest a long-distance loop transiently forms at the HO promoter.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo II , Regulação Fúngica da Expressão Gênica , Proteínas de Grupo de Alta Mobilidade/metabolismo , Proteínas Repressoras/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Fatores de Elongação da Transcrição/metabolismo , Proteínas de Transporte/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo II/genética , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Histona Acetiltransferases/metabolismo , Nucleossomos/metabolismo , Regiões Promotoras Genéticas , Proteínas de Saccharomyces cerevisiae/genética
2.
Genetics ; 215(2): 407-420, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32327563

RESUMO

Mediator is an essential, multisubunit complex that functions as a transcriptional coactivator in yeast and other eukaryotic organisms. Mediator has four conserved modules, Head, Middle, Tail, and Kinase, and has been implicated in nearly all aspects of gene regulation. The Tail module has been shown to recruit the Mediator complex to the enhancer or upstream activating sequence (UAS) regions of genes via interactions with transcription factors, and the Kinase module facilitates the transition of Mediator from the UAS/enhancer to the preinitiation complex via protein phosphorylation. Here, we analyze expression of the Saccharomyces cerevisiaeHO gene using a sin4 Mediator Tail mutation that separates the Tail module from the rest of the complex; the sin4 mutation permits independent recruitment of the Tail module to promoters without the rest of Mediator. Significant increases in recruitment of the SWI/SNF and SAGA coactivators to the HO promoter UAS were observed in a sin4 mutant, along with increased gene activation. These results are consistent with recent studies that have suggested that the Kinase module functions negatively to inhibit activation by the Tail. However, we found that Kinase module mutations did not mimic the effect of a sin4 mutation on HO expression. This suggests that at HO the core Mediator complex (Middle and Head modules) must play a role in limiting Tail binding to the promoter UAS and gene activation. We propose that the core Mediator complex helps modulate Mediator binding to the UAS regions of genes to limit coactivator recruitment and ensure proper regulation of gene transcription.


Assuntos
Regulação Fúngica da Expressão Gênica , Complexo Mediador/metabolismo , RNA Polimerase II/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Transcrição Gênica , Ativação Transcricional , Complexo Mediador/genética , Regiões Promotoras Genéticas , RNA Polimerase II/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética
3.
Proc Natl Acad Sci U S A ; 115(38): 9351-9358, 2018 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-30201707

RESUMO

Genome editing with CRISPR-Cas nucleases has been applied successfully to a wide range of cells and organisms. There is, however, considerable variation in the efficiency of cleavage and outcomes at different genomic targets, even within the same cell type. Some of this variability is likely due to the inherent quality of the interaction between the guide RNA and the target sequence, but some may also reflect the relative accessibility of the target. We investigated the influence of chromatin structure, particularly the presence or absence of nucleosomes, on cleavage by the Streptococcus pyogenes Cas9 protein. At multiple target sequences in two promoters in the yeast genome, we find that Cas9 cleavage is strongly inhibited when the DNA target is within a nucleosome. This inhibition is relieved when nucleosomes are depleted. Remarkably, the same is not true of zinc-finger nucleases (ZFNs), which cleave equally well at nucleosome-occupied and nucleosome-depleted sites. These results have implications for the choice of specific targets for genome editing, both in research and in clinical and other practical applications.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes/métodos , Genômica/métodos , Nucleossomos/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Sítios de Ligação/genética , Proteína 9 Associada à CRISPR , Cromatina/genética , Cromatina/metabolismo , DNA Fúngico/genética , DNA Fúngico/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo II/genética , Endonucleases/metabolismo , Nucleossomos/metabolismo , Regiões Promotoras Genéticas/genética , Proteínas de Saccharomyces cerevisiae/genética , Nucleases de Dedos de Zinco/metabolismo
4.
Proc Natl Acad Sci U S A ; 113(34): 9575-80, 2016 08 23.
Artigo em Inglês | MEDLINE | ID: mdl-27506791

RESUMO

The yeast HO endonuclease is expressed in late G1 in haploid mother cells to initiate mating-type interconversion. Cells can be arrested in G1 by nutrient deprivation or by pheromone exposure, but cells that resume cycling after nutrient deprivation or cyclin-dependent kinase (CDK) inactivation express HO in the first cell cycle, whereas HO is not expressed until the second cycle after release from pheromone arrest. Here, we show that transcription of a long noncoding RNA (lncRNA) mediates this differential response. The SBF and Mediator factors remain bound to the inactive promoter during arrest due to CDK inactivation, and these bound factors allow the cell to remember a transcriptional decision made before arrest. If the presence of mating pheromone indicates that this decision is no longer appropriate, a lncRNA originating at -2700 upstream of the HO gene is induced, and the transcription machinery displaces promoter-bound SBF, preventing HO transcription in the subsequent cell cycle. Further, we find that the displaced SBF is blocked from rebinding due to incorporation of its recognition sites within nucleosomes. Expressing the pHO-lncRNA in trans is ineffective, indicating that transcription in cis is required. Factor displacement during lncRNA transcription could be a general mechanism for regulating memory of previous events at promoters.


Assuntos
Endonucleases/genética , Regulação Fúngica da Expressão Gênica , Regiões Promotoras Genéticas , RNA Fúngico/genética , RNA Longo não Codificante/genética , Saccharomyces cerevisiae/genética , Sequência de Bases , Sítios de Ligação , Endonucleases/metabolismo , Pontos de Checagem da Fase G1 do Ciclo Celular , Complexo Mediador/genética , Complexo Mediador/metabolismo , Nucleossomos/genética , Nucleossomos/metabolismo , Ligação Proteica , RNA Fúngico/biossíntese , RNA Longo não Codificante/biossíntese , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição Gênica
5.
Genetics ; 202(2): 551-63, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26627840

RESUMO

Nucleosome-depleted regions (NDRs) are present immediately adjacent to the transcription start site in most eukaryotic promoters. Here we show that NDRs in the upstream promoter region can profoundly affect gene regulation. Chromatin at the yeast HO promoter is highly repressive and numerous coactivators are required for expression. We modified the HO promoter with segments from the well-studied CLN2 NDR, creating chimeric promoters differing in nucleosome occupancy but with binding sites for the same activator, SBF. Nucleosome depletion resulted in substantial increases in both factor binding and gene expression and allowed activation from a much longer distance, probably by allowing recruited coactivators to act further downstream. Nucleosome depletion also affected sequential activation of the HO promoter; HO activation typically requires the ordered recruitment of activators first to URS1, second to the left-half of URS2 (URS2-L), and finally to the right-half of URS2 (URS2-R), with each region representing distinct gates that must be unlocked to achieve activation. The absence of nucleosomes at URS2-L resulted in promoters no longer requiring both the URS1 and URS2-L gates, as either gate alone is now sufficient to promote binding of the SBF factor to URS2-R. Furthermore, nucleosome depletion at URS2 altered the timing of HO expression and bypassed the regulation that restricts expression to mother cells. Our results reveal insight into how nucleosomes can create a requirement for ordered recruitment of factors to facilitate complex transcriptional regulation.


Assuntos
Regulação Fúngica da Expressão Gênica , Nucleossomos/genética , Nucleossomos/metabolismo , Regiões Promotoras Genéticas , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Ciclo Celular/genética , Cromatina/genética , Cromatina/metabolismo , Ordem dos Genes , Ligação Proteica , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica
6.
Mol Cell Biol ; 35(4): 688-98, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25512608

RESUMO

Promoters often contain multiple binding sites for a single factor. The yeast HO gene contains nine highly conserved binding sites for the SCB (Swi4/6-dependent cell cycle box) binding factor (SBF) complex (composed of Swi4 and Swi6) in the 700-bp upstream regulatory sequence 2 (URS2) promoter region. Here, we show that the distal and proximal SBF sites in URS2 function differently. Chromatin immunoprecipitation (ChIP) experiments show that SBF binds preferentially to the left side of URS2 (URS2-L), despite equivalent binding to the left-half and right-half SBF sites in vitro. SBF binding at URS2-L sites depends on prior chromatin remodeling events at the upstream URS1 region. These signals from URS1 influence chromatin changes at URS2 but only at sites within a defined distance. SBF bound at URS2-L, however, is unable to activate transcription but instead facilitates SBF binding to sites in the right half (URS2-R), which are required for transcriptional activation. Factor binding at HO, therefore, follows a temporal cascade, with SBF bound at URS2-L serving to relay a signal from URS1 to the SBF sites in URS2-R that ultimately activate gene expression. Taken together, we describe a novel property of a transcription factor that can have two distinct roles in gene activation, depending on its location within a promoter.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Regulação Fúngica da Expressão Gênica , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Sítios de Ligação , Montagem e Desmontagem da Cromatina , Imunoprecipitação da Cromatina , Proteínas de Ligação a DNA/genética , Desoxirribonucleases de Sítio Específico do Tipo II/genética , Regiões Promotoras Genéticas , Ligação Proteica , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Transdução de Sinais , Fatores de Tempo , Fatores de Transcrição/genética , Transcrição Gênica
7.
Genetics ; 190(2): 523-35, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22135353

RESUMO

Extensive mutagenesis via massive recoding of retrotransposon Ty1 produced a synthetic codon-optimized retrotransposon (CO-Ty1). CO-Ty1 is defective for retrotransposition, suggesting a sequence capable of down-regulating retrotransposition. We mapped this sequence to a critical ~20-bp region within CO-Ty1 reverse transcriptase (RT) and confirmed that it reduced Ty1 transposition, protein, and RNA levels. Repression was not Ty1 specific; when introduced immediately downstream of the green fluorescent protein (GFP) stop codon, GFP expression was similarly reduced. Rap1p mediated this down-regulation, as shown by mutagenesis and chromatin immunoprecipitation. A regular threefold drop is observed in different contexts, suggesting utility for synthetic circuits. A large reduction of RNAP II occupancy on the CO-Ty1 construct was observed 3' to the identified Rap1p site and a novel 3' truncated RNA species was observed. We propose a novel mechanism of transcriptional regulation by Rap1p whereby it serves as a transcriptional roadblock when bound to transcription unit sequences.


Assuntos
Códon , Retroelementos/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Ligação a Telômeros/metabolismo , Fatores de Transcrição/metabolismo , Sequência de Bases , Sítios de Ligação , Regulação Fúngica da Expressão Gênica , Ordem dos Genes , Vetores Genéticos/genética , Modelos Biológicos , Dados de Sequência Molecular , Mutação , Motivos de Nucleotídeos , Domínios e Motivos de Interação entre Proteínas , RNA Mensageiro/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Alinhamento de Sequência , Complexo Shelterina , Proteínas de Ligação a Telômeros/química , Fatores de Transcrição/química , Transcrição Gênica
8.
Nucleic Acids Res ; 38(8): 2603-6, 2010 May.
Artigo em Inglês | MEDLINE | ID: mdl-20211837

RESUMO

GeneDesign is a set of web applications that provides public access to a nucleotide manipulation pipeline for synthetic biology. The server is public and freely accessible, and the source is available for download under the New BSD License. Since GeneDesign was published and made publicly available 3 years ago, we have made its code base more efficient, added several algorithms and modules, updated the restriction enzyme library, added batch processing capabilities, and added several command line modules, all of which we briefly describe here.


Assuntos
Genes Sintéticos , Software , Códon , Gráficos por Computador , Enzimas de Restrição do DNA , Internet
9.
J Virol ; 81(17): 9004-12, 2007 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-17537863

RESUMO

Ty1 reverse transcriptase/RNase H (RT/RH) is exquisitely sensitive to manganese concentrations. Elevated intracellular free Mn(2+) inhibits Ty1 retrotransposition and in vitro Ty1 RT-polymerizing activity. Furthermore, Mn(2+) inhibition is not limited to the Ty1 RT, as this ion similarly inhibits the activities of both avian myeloblastosis virus and human immunodeficiency virus type 1 RTs. To further characterize Mn(2+) inhibition, we generated RT/RH suppressor mutants capable of increased Ty1 transposition in pmr1 Delta cells. PMR1 codes for a P-type ATPase that regulates intracellular calcium and manganese ion homeostasis, and pmr1 mutants accumulate elevated intracellular manganese levels and display 100-fold less transposition than PMR1(+) cells. Mapping of these suppressor mutations revealed, surprisingly, that suppressor point mutations localize not to the RT itself but to the RH domain of the protein. Furthermore, Mn(2+) inhibition of in vitro RT activity is greatly reduced in all the suppressor mutants, whereas RH activity and cleavage specificity remain largely unchanged. These intriguing results reveal that the effect of these suppressor mutations is transmitted to the polymerase domain and suggest biochemical communication between these two domains during reverse transcription.


Assuntos
Manganês/farmacologia , DNA Polimerase Dirigida por RNA/genética , DNA Polimerase Dirigida por RNA/metabolismo , Retroelementos , Ribonuclease H/genética , Ribonuclease H/metabolismo , Sequência de Aminoácidos , Vírus da Mieloblastose Aviária/enzimologia , Análise Mutacional de DNA , Inibidores Enzimáticos/farmacologia , HIV-1/enzimologia , Modelos Moleculares , Dados de Sequência Molecular , Mutação Puntual , Estrutura Terciária de Proteína/genética , DNA Polimerase Dirigida por RNA/química , Ribonuclease H/antagonistas & inibidores , Ribonuclease H/química , Saccharomyces cerevisiae/genética , Supressão Genética
10.
Genome Res ; 16(4): 550-6, 2006 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-16481661

RESUMO

Modern molecular biology has brought many new tools to the geneticist as well as an exponentially expanding database of genomes and new genes for study. Of particular use in the analysis of these genes is the synthetic gene, a nucleotide sequence designed to the specifications of the investigator. Typically, synthetic genes encode the same product as the gene of interest, but the synthetic nucleotide sequence for that protein may contain modifications affecting expression or base composition. Other desirable changes typically involve the revision of restriction sites. Designing synthetic genes by hand is a time-consuming and error-prone process that may involve several computer programs. We have developed a tools environment that combines many modules to provide a platform for rapid synthetic gene design for multikilobase sequences. We have used GeneDesign to successfully design a synthetic Ty1 element and a large variety of other synthetic sequences. GeneDesign has been implemented as a publicly accessible Web-based resource and can be found at http://slam.bs.jhmi.edu/gd.


Assuntos
Engenharia Genética , Retroelementos/genética , Análise de Sequência de DNA , Análise de Sequência de Proteína , Software , Engenharia Genética/métodos , Análise de Sequência de DNA/métodos , Análise de Sequência de Proteína/métodos
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