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1.
Genes Genet Syst ; 992024 Apr 18.
Artículo en Inglés | MEDLINE | ID: mdl-38447993

RESUMEN

The budding yeast Saccharomyces cerevisiae is an excellent model organism for studying chromatin regulation with high-resolution genome-wide analyses. Since newly generated genome-wide data are often compared with publicly available datasets, expanding our dataset repertoire will be beneficial for the field. Information on transcription start sites (TSSs) determined at base pair resolution is essential for elucidating mechanisms of transcription and related chromatin regulation, yet no datasets that cover two different cell types are available. Here, we present a CAGE (cap analysis of gene expression) dataset for a-cells and α-cells grown in defined and rich media. Cell type-specific genes were differentially expressed as expected, ensuring the reliability of the data. Some of the differentially expressed TSSs were medium-specific or detected due to unrecognized chromosome rearrangement. By comparing the CAGE data with a high-resolution nucleosome map, major TSSs were primarily found in +1 nucleosomes, with a peak approximately 30 bp from the promoter-proximal end of the nucleosome. The dataset is available at DDBJ/GEA.


Asunto(s)
Estudio de Asociación del Genoma Completo , Nucleosomas , Reproducibilidad de los Resultados , Cromatina/metabolismo , Saccharomyces cerevisiae/genética
2.
BMC Bioinformatics ; 22(1): 322, 2021 Jun 13.
Artículo en Inglés | MEDLINE | ID: mdl-34120589

RESUMEN

BACKGROUND: Assessing the nucleosome-forming potential of specific DNA sequences is important for understanding complex chromatin organization. Methods for predicting nucleosome positioning include bioinformatics and biophysical approaches. An advantage of bioinformatics methods, which are based on in vivo nucleosome maps, is the use of natural sequences that may contain previously unknown elements involved in nucleosome positioning in vivo. The accuracy of such prediction attempts reflects the genomic coordinate resolution of the nucleosome maps applied. Nucleosome maps are constructed using micrococcal nuclease digestion followed by high-throughput sequencing (MNase-seq). However, as MNase has a strong preference for A/T-rich sequences, MNase-seq may not be appropriate for this purpose. In addition to MNase-seq-based maps, base pair-resolution chemical maps of in vivo nucleosomes from three different species (budding and fission yeasts, and mice) are currently available. However, these chemical maps have yet to be integrated into publicly available computational methods. RESULTS: We developed a Bioconductor package (named nuCpos) to demonstrate the superiority of chemical maps in predicting nucleosome positioning. The accuracy of chemical map-based prediction in rotational settings was higher than that of the previously developed MNase-seq-based approach. With our method, predicted nucleosome occupancy reasonably matched in vivo observations and was not affected by A/T nucleotide frequency. Effects of genetic alterations on nucleosome positioning that had been observed in living yeast cells could also be predicted. nuCpos calculates individual histone binding affinity (HBA) scores for given 147-bp sequences to examine their suitability for nucleosome formation. We also established local HBA as a new parameter to predict nucleosome formation, which was calculated for 13 overlapping nucleosomal DNA subsequences. HBA and local HBA scores for various sequences agreed well with previous in vitro and in vivo studies. Furthermore, our results suggest that nucleosomal subsegments that are disfavored in different rotational settings contribute to the defined positioning of nucleosomes. CONCLUSIONS: Our results demonstrate that chemical map-based statistical models are beneficial for studying nucleosomal DNA features. Studies employing nuCpos software can enhance understanding of chromatin regulation and the interpretation of genetic alterations and facilitate the design of artificial sequences.


Asunto(s)
Biología Computacional , Nucleasa Microcócica , Nucleosomas , Programas Informáticos , Animales , Cromatina , ADN , Secuenciación de Nucleótidos de Alto Rendimiento , Ratones , Nucleosomas/genética
3.
Biochem Biophys Res Commun ; 556: 179-184, 2021 06 04.
Artículo en Inglés | MEDLINE | ID: mdl-33839413

RESUMEN

Trinucleotide repeat sequences (TRSs), consisting of 10 unique classes of repeats in DNA, are members of microsatellites and abundantly and non-randomly distributed in many eukaryotic genomes. The lengths of TRSs are mutable, and the expansions of several TRSs are implicated in hereditary neurological diseases. However, the underlying causes of the biased distribution and the dynamic properties of TRSs in the genome remain elusive. Here, we examined the effects of TRSs on nucleosome formation in vivo by histone H4-S47C site-directed chemical cleavages, using well-defined yeast minichromosomes in which each of the ten TRS classes resided in the central region of a positioned nucleosome. We showed that (AAT)12 and (ACT)12 act as strong nucleosome-promoting sequences, while (AGG)12 and (CCG)12 act as nucleosome-excluding sequences in vivo. The local histone binding affinity scores support the idea that nucleosome formation in TRSs, except for (AGG)12, is mainly determined by the affinity for the histone octamers. Overall, our study presents a framework for understanding the nucleosome-forming abilities of TRSs.


Asunto(s)
Nucleosomas/química , Nucleosomas/genética , Saccharomyces cerevisiae/genética , Repeticiones de Trinucleótidos/genética , Secuencia de Bases/genética , Cromosomas Fúngicos/química , Cromosomas Fúngicos/genética , Cromosomas Fúngicos/metabolismo , ADN/química , ADN/genética , ADN/metabolismo , Histonas/metabolismo , Nucleosomas/metabolismo , Saccharomyces cerevisiae/metabolismo
4.
Biol Pharm Bull ; 42(2): 289-294, 2019 Feb 01.
Artículo en Inglés | MEDLINE | ID: mdl-30531092

RESUMEN

In eukaryotic genomes, the nucleosome is the structural and functional unit, and its position and dynamics are important for gene expression control and epigenetic regulation. Epigenetics is an important mechanism in development and homeostasis, and aberrant epigenetics is a common feature in cancer. Although understanding the mechanistic basis that determines nucleosome positioning in vivo is important for elucidating chromatin function and epigenetic regulation, a suitable experimental system to examine such mechanisms is still being developed. Herein, we examined nucleosome organization in yeast minichromosomes, using a parallel mapping method we previously developed that involve site-directed chemical cleavage and micrococcal nuclease digestion. This parallel mapping is capable of revealing the differences in the occupancy and the stability of individual nucleosomes in the minichromosome. Based on the previously characterized minichromosome, we engineered a set of new minichromosomes, aimed at strengthening the positioning of the nucleosomes. The site-directed chemical mapping method demonstrated that the nucleosome positioning in the newly designed yeast minichromosome system was significantly more stable. This system will be useful for elucidating the determinants of nucleosome organization, such as DNA sequences and/or nucleosome binding proteins, and for determining the relationships between nucleosome dynamics and epigenetic regulation, which are targets for therapeutic agents.


Asunto(s)
Nucleosomas/genética , Saccharomyces cerevisiae/genética , Levaduras/genética , Cromatina/genética , Cromatina/metabolismo , Mapeo Cromosómico/métodos , Cromosomas Fúngicos , Nucleasa Microcócica/metabolismo , Nucleosomas/metabolismo , Saccharomyces cerevisiae/metabolismo , Levaduras/metabolismo
5.
PLoS One ; 12(10): e0186974, 2017.
Artículo en Inglés | MEDLINE | ID: mdl-29073207

RESUMEN

Micrococcal nuclease (MNase) has been widely used for analyses of nucleosome locations in many organisms. However, due to its sequence preference, the interpretations of the positions and occupancies of nucleosomes using MNase have remained controversial. Next-generation sequencing (NGS) has also been utilized for analyses of MNase-digests, but some technical biases are commonly present in the NGS experiments. Here, we established a gel-based method to map nucleosome positions in Saccharomyces cerevisiae, using isolated nuclei as the substrate for the histone H4 S47C-site-directed chemical cleavage in parallel with MNase digestion. The parallel mapping allowed us to compare the chemically and enzymatically cleaved sites by indirect end-labeling and primer extension mapping, and thus we could determine the nucleosome positions and the sizes of the nucleosome-free regions (or nucleosome-depleted regions) more accurately, as compared to nucleosome mapping by MNase alone. The analysis also revealed that the structural features of the nucleosomes flanked by the nucleosome-free region were different from those within regularly arrayed nucleosomes, showing that the structures and dynamics of individual nucleosomes strongly depend on their locations. Moreover, we demonstrated that the parallel mapping results were generally consistent with the previous genome-wide chemical mapping and MNase-Seq results. Thus, the gel-based parallel mapping will be useful for the analysis of a specific locus under various conditions.


Asunto(s)
Mapeo Cromosómico/métodos , Radical Hidroxilo/metabolismo , Nucleasa Microcócica/metabolismo , Nucleosomas/genética , Nucleosomas/metabolismo , Isomerasas Aldosa-Cetosa/genética , ADN de Hongos/genética , Sitios Genéticos/genética , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
6.
Biochem Biophys Res Commun ; 476(2): 57-62, 2016 07 22.
Artículo en Inglés | MEDLINE | ID: mdl-27208777

RESUMEN

Nucleosome-depleted regions (NDRs) (also called nucleosome-free regions or NFRs) are often found in the promoter regions of many yeast genes, and are formed by multiple mechanisms, including the binding of activators and enhancers, the actions of chromatin remodeling complexes, and the specific DNA sequences themselves. However, it remains unclear whether NDR formation per se is essential for transcriptional activation. Here, we examined the relationship between nucleosome organization and gene expression using a defined yeast reporter system, consisting of the CYC1 minimal core promoter and the lacZ gene. We introduced simple repeated sequences that should be either incorporated in nucleosomes or excluded from nucleosomes in the site upstream of the TATA boxes. The (CTG)12, (GAA)12 and (TGTAGG)6 inserts were incorporated into a positioned nucleosome in the core promoter region, and did not affect the reporter gene expression. In contrast, the insertion of (CGG)12, (TTAGGG)6, (A)34 or (CG)8 induced lacZ expression by 10-20 fold. Nucleosome mapping analyses revealed that the inserts that induced the reporter gene expression prevented nucleosome formation, and created an NDR upstream of the TATA boxes. Thus, our results demonstrated that NDR formation dictated by DNA sequences is sufficient for transcriptional activation from the core promoter in vivo.


Asunto(s)
ADN de Hongos/genética , Regulación Fúngica de la Expresión Génica , Nucleosomas/genética , Regiones Promotoras Genéticas , Saccharomyces cerevisiae/genética , Activación Transcripcional , Secuencia de Bases , Cromosomas Fúngicos/química , Cromosomas Fúngicos/genética , ADN de Hongos/química , Operón Lac , Nucleosomas/química , Saccharomyces cerevisiae/citología , TATA Box
7.
Biomol Concepts ; 6(1): 67-75, 2015 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-25720088

RESUMEN

Telomeres are DNA-protein complexes located at the ends of linear eukaryotic chromosomes, and are essential for chromosome stability and maintenance. In most organisms, telomeres consist of tandemly repeated sequences of guanine-clusters. In higher eukaryotes, most of the telomeric repeat regions are tightly packaged into nucleosomes, even though telomeric repeats act as nucleosome-disfavoring sequences. Although telomeres were considered to be condensed heterochromatin structures, recent studies revealed that the chromatin structures in telomeres are actually dynamic. The dynamic properties of telomeric chromatin are considered to be important for the structural changes between the euchromatic and heterochromatic states during the cell cycle and in cellular differentiation. We propose that the nucleosome-disfavoring property of telomeric repeats is a crucial determinant for the lability of telomeric nucleosomes, and provides a platform for chromatin dynamics in telomeres. Furthermore, we discuss the influences of telomeric components on the nucleosome organization and chromatin dynamics in telomeres.


Asunto(s)
Cromatina/metabolismo , Histonas/metabolismo , Nucleosomas/química , Nucleosomas/metabolismo , Telómero/metabolismo , ADN/química , Humanos , Isoformas de Proteínas/metabolismo , Procesamiento Proteico-Postraduccional , Proteína 1 de Unión a Repeticiones Teloméricas/metabolismo , Proteína 2 de Unión a Repeticiones Teloméricas/metabolismo , Levaduras
8.
Nucleic Acids Res ; 42(3): 1541-52, 2014 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-24174540

RESUMEN

Telomeric DNAs consist of tandem repeats of G-clusters such as TTAGGG and TG1-3, which are the human and yeast repeat sequences, respectively. In the yeast Saccharomyces cerevisiae, the telomeric repeats are non-nucleosomal, whereas in humans, they are organized in tightly packaged nucleosomes. However, previous in vitro studies revealed that the binding affinities of human and yeast telomeric repeat sequences to histone octamers in vitro were similar, which is apparently inconsistent with the differences in the human and yeast telomeric chromatin structures. To further investigate the relationship between telomeric sequences and chromatin structure, we examined the effect of telomeric repeats on the formation of positioned nucleosomes in vivo by indirect end-label mapping, primer extension mapping and nucleosome repeat analyses, using a defined minichromosome in yeast cells. We found that the human and yeast telomeric repeat sequences both disfavour nucleosome assembly and alter nucleosome positioning in the yeast minichromosome. We further demonstrated that the G-clusters in the telomeric repeats are required for the nucleosome-disfavouring properties. Thus, our results suggest that this inherent structural feature of the telomeric repeat sequences is involved in the functional dynamics of the telomeric chromatin structure.


Asunto(s)
Nucleosomas/metabolismo , Secuencias Repetitivas de Ácidos Nucleicos , Telómero/química , Humanos , Nucleasa Microcócica , Saccharomyces cerevisiae/genética , Telómero/metabolismo
9.
FEBS J ; 277(21): 4539-48, 2010 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-21040474

RESUMEN

5-Bromodeoxyuridine (BrdU) modulates the expression of particular genes associated with cellular differentiation and senescence when incorporated into DNA instead of thymidine (dThd). To date, a molecular mechanism for this phenomenon remains a mystery in spite of a large number of studies. Recently, we have demonstrated that BrdU disrupts nucleosome positioning on model plasmids mediated by specific AT-tracts in yeast cells. Here we constructed a cognate plasmid that can form an ordered array of nucleosomes determined by an α2 operator and contains the BAR1 gene as an expression marker gene to examine BAR1 expression in dThd-auxotrophic MATα cells under various conditions. In medium containing dThd, BAR1 expression was completely repressed, associated with the formation of the stable array of nucleosomes. Insertion of AT-tracts into a site of the promoter region slightly increased BAR1 expression and slightly destabilized nucleosome positioning dependent on their sequence specificity. In medium containing BrdU, BAR1 expression was further enhanced, associated with more marked disruption of nucleosome positioning on the promoter region. Disruption of nucleosome positioning seems to be sufficient for full expression of the marker gene if necessary transcription factors are supplied. Incorporation of 5-bromouracil into the plasmid did not weaken the binding of the α2/Mcm1 repressor complex to its legitimate binding site, as revealed by an in vivo UV photofootprinting assay. These results suggest that BrdU increases transcription of repressed genes by disruption of nucleosome positioning around their promoters.


Asunto(s)
Bromouracilo/farmacología , Nucleosomas/efectos de los fármacos , Proteínas de Saccharomyces cerevisiae/genética , Transcripción Genética/efectos de los fármacos , Antimetabolitos/farmacología , Ácido Aspártico Endopeptidasas/genética , Northern Blotting , Perfilación de la Expresión Génica , Regulación Fúngica de la Expresión Génica/efectos de los fármacos , Proteína 1 de Mantenimiento de Minicromosoma , Nucleosomas/metabolismo , Regiones Promotoras Genéticas/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/genética
10.
Biochemistry ; 49(11): 2351-8, 2010 Mar 23.
Artículo en Inglés | MEDLINE | ID: mdl-20166733

RESUMEN

Superhelically curved DNA structures can strongly activate transcription in mammalian cells. However, the mechanism underlying the activation has not been clarified. We investigated this mechanism in yeast cells, using 108, 180, and 252 bp synthetic curved DNA segments. Even in the presence of nucleosomes, these DNAs activated transcription from a UAS-deleted CYC1 promoter that is silenced in the presence of nucleosomes. The fold-activations of transcription by these segments, relative to the transcription on the control that lacked such segments, were 51.4, 63.4, and 56.4, respectively. The superhelically curved DNA structures favored nucleosome formation. However, the translational positions of the nucleosomes were dynamic. The high mobility of the nucleosomes on the superhelically curved DNA structures seemed to influence the mobility of the nucleosomes formed on the promoter and eventually enhanced the access to the center region of one TATA sequence. Functioning as a dock for the histone core and allowing nucleosome sliding seem to be the mechanisms underlying the transcriptional activation by superhelically curved DNA structures in chromatin. The present study provides important clues for designing and constructing artificial chromatin modulators, as a tool for chromatin engineering.


Asunto(s)
Cromatina/genética , ADN de Hongos/química , ADN de Hongos/metabolismo , ADN Superhelicoidal/química , ADN Superhelicoidal/metabolismo , Saccharomyces cerevisiae/genética , Transcripción Genética , Secuencia de Bases , Cromosomas Fúngicos/genética , ADN de Hongos/genética , ADN Superhelicoidal/genética , Modelos Moleculares , Conformación de Ácido Nucleico , Nucleosomas/metabolismo , Regiones Promotoras Genéticas/genética , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/metabolismo , Homología de Secuencia de Ácido Nucleico , Timidina Quinasa/genética
11.
Proteins ; 75(2): 336-47, 2009 May 01.
Artículo en Inglés | MEDLINE | ID: mdl-18831036

RESUMEN

SUMO E3 ligase of the Siz/PIAS family that promotes sumoylation of target proteins contains SAP motif in its N-terminal region. The SAP motif with a consensus sequence of 35 residues was first proposed to be as a new DNA binding motif found in diverse nuclear proteins involved in chromosomal organization. We have determined solution structures of the SAP domains of SUMO ligases Siz1 from yeast and rice by NMR spectroscopy, showing that the structure of the SAP domain (residues 2-105) of rice Siz1 is a four-helix bundle with an up-down-extended loop-down-up topology, whereas the SAP domain (residues 1-111) of yeast Siz1 is comprised of five helices where the fifth helix alpha5 causes a significant change in the alignment of the four-helix bundle characteristic to the SAP domains of the Siz/PIAS family. We have also demonstrated that both SAP domains have binding ability to an A/T-rich DNA, but that binding affinity of yeast Siz1 SAP is at least by an order of magnitude higher than that of rice Siz1 SAP. Our NMR titration experiments clearly showed that yeast Siz1 SAP uses alpha2-helix for DNA binding more effectively than rice Siz1 SAP, which would result from the dislocation of this helix due to the existence of the extra helix alpha5. In addition, based on the structures of the SAP domains determined here and registered in Protein Data Bank, general features of structures of the SAP domains are discussed in conjunction with equivocal nature of their DNA binding.


Asunto(s)
ADN/metabolismo , Oryza/enzimología , Proteínas de Plantas/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/enzimología , Ubiquitina-Proteína Ligasas/química , ADN/química , Resonancia Magnética Nuclear Biomolecular , Proteínas de Plantas/metabolismo , Unión Proteica , Conformación Proteica , Estructura Terciaria de Proteína , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo
12.
Eukaryot Cell ; 7(6): 949-57, 2008 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-18408055

RESUMEN

Nutrient-sensing kinases play important roles for the yeast Saccharomyces cerevisiae to adapt to new nutrient conditions when the nutrient status changes. Our previous global gene expression analysis revealed that the Pho85 kinase, one of the yeast nutrient-sensing kinases, is involved in the changes in gene expression profiles when yeast cells undergo a diauxic shift. We also found that the stationary phase-specific genes SNZ1 and SNO1, which share a common promoter, are not properly induced when Pho85 is absent. To examine the role of the kinase in SNZ1/SNO1 regulation, we analyzed their expression during the growth of various yeast mutants, including those affecting Pho85 function or lacking the Pho4 transcription factor, an in vivo substrate of Pho85, and tested Pho4 binding by chromatin immunoprecipitation. Pho4 exhibits temporal binding to the SNZ1/SNO1 promoter to down-regulate the promoter activity, and a Deltapho4 mutation advances the timing of SNZ1/SNO1 expression. SNZ2, another member of the SNZ/SNO family, is expressed at an earlier growth stage than SNZ1, and Pho4 does not affect this timing, although Pho85 is required for SNZ2 expression. Thus, Pho4 appears to regulate the different timing of the expression of the SNZ/SNO family members. Pho4 binding to the SNZ1/SNO1 promoter is accompanied by alterations in chromatin structure, and Rpd3 histone deacetylase is required for the proper timing of SNZ1/SNO1 expression, while Asf1 histone chaperone is indispensable for their expression. These results imply that Pho4 plays positive and negative roles in transcriptional regulation, with both cases involving structural changes in its target chromatin.


Asunto(s)
Proteínas de Unión al ADN/metabolismo , Regulación Fúngica de la Expresión Génica , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/metabolismo , Cromatina/metabolismo , Regiones Promotoras Genéticas , Saccharomyces cerevisiae/genética , Transcripción Genética
13.
Biol Pharm Bull ; 31(2): 187-92, 2008 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-18239271

RESUMEN

The analysis of nucleosome positions and transcription factor binding in chromatin is a central issue for understanding the mechanisms of gene expression in eukaryotes. Here, we have developed a footprinting technique, using multi-cycle primer extension with an infrared-fluorescence DNA sequencer, to analyze chromatin structure in isolated yeast nuclei and transcriptional activator binding in living yeast cells. Using this technique, the binding of the yeast activators Hap1 and Hap2/3/4/5 to their cognate sites was detectable as hypersensitive sites by in vivo UV-photofootprinting, and the locations of nucleosomes in yeast minichromosomes were determined by micrococcal nuclease mapping. We also applied this method to determine the position of the nucleosome in the 5S DNA fragment reconstituted in vitro. This technique allowed us to eliminate the use of radioactive materials and to perform experiments on common benches. Thus, the footprinting procedure established in this study will be useful to researchers studying DNA-protein interactions and chromatin structure in vivo and in vitro.


Asunto(s)
ADN de Hongos/química , Nucleosomas/química , Huella de Proteína , Transactivadores/química , Cromatina/química , Cromatina/genética , Cromosomas Fúngicos/genética , Reparación del ADN/fisiología , Replicación del ADN/fisiología , ADN de Hongos/genética , Operón Lac/genética , Nucleosomas/genética , Plásmidos/genética , Recombinación Genética , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Análisis de Secuencia de ADN , Espectrometría de Fluorescencia , Espectrofotometría Infrarroja , Transactivadores/genética
14.
Biochem Biophys Res Commun ; 368(3): 662-9, 2008 Apr 11.
Artículo en Inglés | MEDLINE | ID: mdl-18258180

RESUMEN

5-Bromodeoxyuridine (BrdU) modulates expression of particular genes associated with cellular differentiation and senescence. Our previous studies have suggested an involvement of chromatin structure in this phenomenon. Here, we examined the effect of 5-bromouracil on nucleosome positioning in vivo using TALS plasmid in yeast cells. This plasmid can stably and precisely be assembled nucleosomes aided by the alpha2 repressor complex bound to its alpha2 operator. Insertion of AT-rich sequences into a site near the operator destabilized nucleosome positioning dependent on their length and sequences. Addition of BrdU almost completely disrupted nucleosome positioning through specific AT-tracts. The effective AT-rich sequences migrated faster on polyacrylamide gel electrophoresis, and their mobility was further accelerated by substitution of thymine with 5-bromouracil. Since this property is indicative of a rigid conformation of DNA, our results suggest that 5-bromouracil disrupts nucleosome positioning by inducing A-form-like DNA.


Asunto(s)
Bromouracilo/administración & dosificación , Posicionamiento de Cromosoma/genética , ADN de Forma A/química , ADN de Forma A/genética , Nucleosomas/genética , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/genética , Posicionamiento de Cromosoma/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Conformación de Ácido Nucleico/efectos de los fármacos , Nucleosomas/efectos de los fármacos
15.
Biochem Biophys Res Commun ; 364(3): 583-8, 2007 Dec 21.
Artículo en Inglés | MEDLINE | ID: mdl-17959145

RESUMEN

Nucleosome positioning has been proposed as a mechanism of transcriptional repression. Here, we examined whether nucleosome positioning affects activator binding in living yeast cells. We introduced the cognate Hap1 binding site (UAS1) at a location 24-43 bp, 29-48 bp, or 61-80 bp interior to the edge of a nucleosome positioned by alpha2/Mcm1 in yeast minichromosomes. Hap1 binding to the UAS1 was severely inhibited, not only at the pseudo-dyad but also in the peripheral region of the positioned nucleosome in alpha cells, while it was detectable in a cells, in which the nucleosomes were not positioned. Hap1 binding was restored in alpha cells with tup1 or isw2 mutations, which caused the loss of nucleosome positioning. These results support the mechanism in which alpha2/Mcm1-dependent nucleosome positioning has a regulatory function to limit the access of transcription factors.


Asunto(s)
Proteínas de Unión al ADN/genética , Nucleosomas/fisiología , Nucleosomas/ultraestructura , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Transactivadores/genética , Factores de Transcripción/genética , Activación Transcripcional/genética , Proteína 1 de Mantenimiento de Minicromosoma , Unión Proteica
16.
Eukaryot Cell ; 5(11): 1925-33, 2006 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-16980406

RESUMEN

In Saccharomyces cerevisiae, a-cell-specific genes are repressed in MATalpha cells by alpha2/Mcm1, acting in concert with the Ssn6-Tup1 corepressors and the Isw2 chromatin remodeling complex, and nucleosome positioning has been proposed as one mechanism of repression. However, prior studies showed that nucleosome positioning is not essential for repression by alpha2/Mcm1 in artificial reporter plasmids, and the importance of the nucleosome positioning remains questionable. We have tested the function of positioned nucleosomes through alteration of genomic chromatin at the a-cell-specific gene BAR1. We report here that a positioned nucleosome in the BAR1 promoter is disrupted in cis by the insertion of diverse DNA sequences such as poly(dA) . poly(dT) and poly(dC-dG) . poly(dC-dG), leading to inappropriate partial derepression of BAR1. Also, we show that isw2 mutation causes loss of nucleosome positioning in BAR1 in MATalpha cells as well as partial disruption of repression. Thus, nucleosome positioning is required for full repression, but loss of nucleosome positioning is not sufficient to relieve repression completely. Even though disruption of nucleosome positioning by the cis- and trans-acting modulators of chromatin has a modest effect on the level of transcription, it causes significant degradation of the alpha-mating pheromone in MATalpha cells, thereby affecting its cell type identity. Our results illustrate a useful paradigm for analysis of chromatin structural effects at genomic loci.


Asunto(s)
Secuencia de Bases , Regulación Fúngica de la Expresión Génica , Genoma Fúngico , Nucleosomas/metabolismo , Proteínas Represoras/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Factores de Transcripción/metabolismo , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Ácido Aspártico Endopeptidasas/genética , Ácido Aspártico Endopeptidasas/metabolismo , Cromatina/metabolismo , ADN/química , ADN/metabolismo , Proteína 1 de Mantenimiento de Minicromosoma , Conformación de Ácido Nucleico , Proteínas Represoras/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Factores de Transcripción/genética
17.
Nucleic Acids Symp Ser (Oxf) ; (49): 325-6, 2005.
Artículo en Inglés | MEDLINE | ID: mdl-17150765

RESUMEN

Early meiotic gene expression in Saccharomyces cerevisiae is regulated through chromatin alterations. To elucidate chromatin function in meiotic gene expression, we have studied the roles of histone methyltransferases in sporulation. Three histone lysine methyltransferases, Set1p, Set2p and Dot1p, have been identified in S. cerevisiae. We constructed a series of strains carrying set1delta set2delta and dot1delta mutations, and characterized sporulation process of these mutant strains. It was found that set1delta set2delta double and set1delta set2delta dot1delta triple mutations severely impaired spore formation. Because set1delta and set2delta affect this process additively, we suggest that Set1p and Set2p have overlapping functions in this developmental process.


Asunto(s)
N-Metiltransferasa de Histona-Lisina/fisiología , Proteínas de Saccharomyces cerevisiae/fisiología , Saccharomyces cerevisiae/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/fisiología , Eliminación de Gen , N-Metiltransferasa de Histona-Lisina/genética , Meiosis/genética , Metiltransferasas/genética , Metiltransferasas/fisiología , Proteínas Nucleares/genética , Proteínas Nucleares/fisiología , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/fisiología , Proteínas de Saccharomyces cerevisiae/genética , Esporas Fúngicas/crecimiento & desarrollo , Factores de Transcripción/genética , Factores de Transcripción/fisiología
18.
Anal Biochem ; 323(2): 218-23, 2003 Dec 15.
Artículo en Inglés | MEDLINE | ID: mdl-14656528

RESUMEN

We have synthesized and characterized a thermoreversible hydrogel of multiplied block copolymers, composed of poly(ethylene oxide) and poly(propylene oxide), for DNA electrophoresis. The aqueous solution of block copolymers turned into a hydrogel upon heating at temperatures above 10-11 degrees C, whereas it reverted into a solution upon cooling. Linear double-stranded DNA molecules migrated through the gel matrices at a rate that was inversely proportional to the logarithm of the DNA length. The hydrogel is most effective for separating DNA fragments in the 10- to 2000-bp range. The resolving range lay in-between the effective ranges of polyacrylamide and agarose gel electrophoreses of DNA. The gel slices containing DNA fragments were liquefied by cooling on ice, and the DNA was precipitated with ethanol. No contaminants that inhibit enzymatic reactions were found in the DNA recovered from the hydrogel. Plasmid DNA recovered from the hydrogel was recircularized with T4 DNA ligase and yielded highly efficient Escherichia coli transformation. Therefore, thermoreversible gel electrophoresis will be a useful method for DNA separation and isolation in recombinant DNA technology.


Asunto(s)
ADN/aislamiento & purificación , Electroforesis/métodos , Hidrogel de Polietilenoglicol-Dimetacrilato/química , Polietilenglicoles/química , Polímeros/química , Glicoles de Propileno/química , Electroquímica , Electroforesis/instrumentación , Electroforesis en Gel de Poliacrilamida , Escherichia coli/genética , Polietilenglicoles/síntesis química , Polímeros/síntesis química , Glicoles de Propileno/síntesis química , Temperatura , Transformación Genética , Viscosidad
19.
Nucleic Acids Res ; 31(24): 7199-207, 2003 Dec 15.
Artículo en Inglés | MEDLINE | ID: mdl-14654695

RESUMEN

Hho1p is assumed to serve as a linker histone in Saccharomyces cerevisiae and, notably, it possesses two putative globular domains, designated HD1 (residues 41-118) and HD2 (residues 171-252), that are homologous to histone H5 from chicken erythrocytes. We have determined the three-dimensional structure of globular domain HD1 with high precision by heteronuclear magnetic resonance spectroscopy. The structure had a winged helix-turn-helix motif composed of an alphabetaalphaalphabetabeta fold and closely resembled the structure of the globular domain of histone H5. Interestingly, the second globular domain, HD2, in Hho1p was unstructured under physiological conditions. Gel mobility assay demonstrated that Hho1p preferentially binds to supercoiled DNA over linearized DNA. Furthermore, NMR analysis of the complex of a deletion mutant protein (residues 1-118) of Hho1p with a linear DNA duplex revealed that four regions within the globular domain HD1 are involved in the DNA binding. The above results suggested that Hho1p possesses properties similar to those of linker histones in higher eukaryotes in terms of the structure and binding preference towards supercoiled DNA.


Asunto(s)
Secuencias Hélice-Giro-Hélice , Histonas/química , Resonancia Magnética Nuclear Biomolecular , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/química , Secuencia de Aminoácidos , Dicroismo Circular , ADN Superhelicoidal/química , ADN Superhelicoidal/metabolismo , Histonas/genética , Histonas/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Docilidad , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Eliminación de Secuencia/genética
20.
Artículo en Inglés | MEDLINE | ID: mdl-12906901

RESUMEN

Single-strand DNA binding protein (SSB) from Escherichia coli lysate was purified by counter-current chromatography (CCC) using the ammonium sulfate precipitation method in a coiled column. About 5 ml of E. coli lysate was separated by CCC using a polymer phase system composed of 16% (w/w) polyethylene glycol (PEG) 1000 and 17% (w/w) ammonium sulfate aqueous polymer two-phase solvent system. The precipitation of proteins in the lysate took place in the CCC column, and the SSB protein was eluted in the fraction 51-56. Many other impurities were either eluted immediately after the solvent front or precipitated in the column. The identities of the proteins in the fractions and in the precipitate were confirmed by SDS-polyacrylamide gel electrophoresis with Coomassie Brilliant Blue staining.


Asunto(s)
Distribución en Contracorriente/métodos , Proteínas de Unión al ADN/aislamiento & purificación , Proteínas de Escherichia coli/aislamiento & purificación , Escherichia coli/química , Precipitación Química , Electroforesis en Gel de Poliacrilamida
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