Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 11 de 11
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Epigenetics ; 15(6-7): 702-714, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31900031

RESUMO

The transcriptional program that dictates haematopoietic cell fate and differentiation requires an epigenetic regulatory and memory function, provided by a network of epigenetic factors that regulate DNA methylation, post-translational histone modifications and chromatin structure. Disturbed epigenetic regulation causes perturbations in the blood cell differentiation program that results in various types of haematopoietic disorders. Thus, accurate epigenetic regulation is essential for functional haematopoiesis. In this study, we used a CRISPR-Cas9 screening approach to identify new epigenetic regulators in myeloid differentiation. We designed a Chromatin-UMI CRISPR guide library targeting 1092 epigenetic regulators. Phorbol 12-myristate 13-acetate (PMA) treatment of the chronic myeloid leukaemia cell line K-562 was used as a megakaryocytic myeloid differentiation model. Both previously described developmental epigenetic regulators and novel factors were identified in our screen. In this study, we validated and characterized a role for the chromatin remodeller CHD2 in myeloid proliferation and megakaryocytic differentiation.


Assuntos
Proteínas de Ligação a DNA/genética , Mielopoese , Proliferação de Células , Montagem e Desmontagem da Cromatina , Proteínas de Ligação a DNA/metabolismo , Humanos , Células K562 , Megacariócitos/efeitos dos fármacos , Megacariócitos/metabolismo , Acetato de Tetradecanoilforbol/farmacologia
2.
Mol Syst Biol ; 15(8): e9059, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31464368

RESUMO

Haapaniemi et al address the issues raised by Brown et al and discuss several differences between the analyses performed by the two groups.


Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Edição de Genes , Sistemas CRISPR-Cas , Dano ao DNA , Proteína Supressora de Tumor p53
3.
Nat Med ; 24(7): 927-930, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29892067

RESUMO

Here, we report that genome editing by CRISPR-Cas9 induces a p53-mediated DNA damage response and cell cycle arrest in immortalized human retinal pigment epithelial cells, leading to a selection against cells with a functional p53 pathway. Inhibition of p53 prevents the damage response and increases the rate of homologous recombination from a donor template. These results suggest that p53 inhibition may improve the efficiency of genome editing of untransformed cells and that p53 function should be monitored when developing cell-based therapies utilizing CRISPR-Cas9.


Assuntos
Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , Dano ao DNA , Edição de Genes , Proteína Supressora de Tumor p53/metabolismo , Ciclo Celular , Linhagem Celular Tumoral , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Células HEK293 , Humanos , Ribonucleoproteínas/metabolismo
4.
EMBO Rep ; 17(5): 753-68, 2016 05.
Artigo em Inglês | MEDLINE | ID: mdl-26902262

RESUMO

Retrotransposons, the ancestors of retroviruses, have the potential for gene disruption and genomic takeover if not kept in check. Paradoxically, although host cells repress these elements by multiple mechanisms, they are transcribed and are even activated under stress conditions. Here, we describe a new mechanism of retrotransposon regulation through transcription start site (TSS) selection by altered nucleosome occupancy. We show that Fun30 chromatin remodelers cooperate to maintain a high level of nucleosome occupancy at retrotransposon-flanking long terminal repeat (LTR) elements. This enforces the use of a downstream TSS and the production of a truncated RNA incapable of reverse transcription and retrotransposition. However, in stressed cells, nucleosome occupancy at LTR elements is reduced, and the TSS shifts to allow for productive transcription. We propose that controlled retrotransposon transcription from a nonproductive TSS allows for rapid stress-induced activation, while preventing uncontrolled transposon activity in the genome.


Assuntos
Regulação da Expressão Gênica , Retroelementos , Sítio de Iniciação de Transcrição , Sequência de Bases , Catálise , Cromatina/genética , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina , Modelos Biológicos , Mutação , Nucleossomos , Fenótipo , Estresse Fisiológico , Sequências Repetidas Terminais , Ativação Transcricional
5.
PLoS Genet ; 11(3): e1005101, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25798942

RESUMO

In eukaryotic cells, local chromatin structure and chromatin organization in the nucleus both influence transcriptional regulation. At the local level, the Fun30 chromatin remodeler Fft3 is essential for maintaining proper chromatin structure at centromeres and subtelomeres in fission yeast. Using genome-wide mapping and live cell imaging, we show that this role is linked to controlling nuclear organization of its targets. In fft3∆ cells, subtelomeres lose their association with the LEM domain protein Man1 at the nuclear periphery and move to the interior of the nucleus. Furthermore, genes in these domains are upregulated and active chromatin marks increase. Fft3 is also enriched at retrotransposon-derived long terminal repeat (LTR) elements and at tRNA genes. In cells lacking Fft3, these sites lose their peripheral positioning and show reduced nucleosome occupancy. We propose that Fft3 has a global role in mediating association between specific chromatin domains and the nuclear envelope.


Assuntos
Montagem e Desmontagem da Cromatina/genética , Cromatina/genética , Proteínas Cromossômicas não Histona/genética , Proteínas de Schizosaccharomyces pombe/genética , Telômero/genética , Transcrição Gênica , Núcleo Celular/genética , Proteínas Cromossômicas não Histona/biossíntese , Regulação Fúngica da Expressão Gênica , Elementos Isolantes/genética , Proteínas de Membrana/biossíntese , Proteínas de Membrana/genética , Proteínas Nucleares/biossíntese , Proteínas Nucleares/genética , Nucleossomos/genética , RNA de Transferência/genética , Schizosaccharomyces , Proteínas de Schizosaccharomyces pombe/biossíntese , Sequências Repetidas Terminais/genética
6.
EMBO J ; 31(23): 4388-403, 2012 Nov 28.
Artigo em Inglês | MEDLINE | ID: mdl-23103765

RESUMO

Nucleosome positioning governs access to eukaryotic genomes. Many genes show a stereotypic organisation at their 5'end: a nucleosome free region just upstream of the transcription start site (TSS) followed by a regular nucleosomal array over the coding region. The determinants for this pattern are unclear, but nucleosome remodelers are likely critical. Here we study the role of remodelers in global nucleosome positioning in S. pombe and the corresponding changes in expression. We find a striking evolutionary shift in remodeler usage between budding and fission yeast. The S. pombe RSC complex does not seem to be involved in nucleosome positioning, despite its prominent role in S. cerevisiae. While S. pombe lacks ISWI-type remodelers, it has two CHD1-type ATPases, Hrp1 and Hrp3. We demonstrate nucleosome spacing activity for Hrp1 and Hrp3 in vitro, and that together they are essential for linking regular genic arrays to most TSSs in vivo. Impaired arrays in the absence of either or both remodelers may lead to increased cryptic antisense transcription, but overall gene expression levels are only mildly affected.


Assuntos
Adenosina Trifosfatases/fisiologia , DNA Helicases/fisiologia , Proteínas de Ligação a DNA/fisiologia , Regulação Fúngica da Expressão Gênica , Nucleossomos/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/metabolismo , Proteínas de Schizosaccharomyces pombe/fisiologia , Schizosaccharomyces/metabolismo , Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , DNA Helicases/química , Proteínas de Ligação a DNA/química , Dactinomicina/farmacologia , Deleção de Genes , Histonas/química , Modelos Biológicos , Mutação , Oligonucleotídeos Antissenso/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/química , Transcrição Gênica , Transcriptoma
7.
Transcription ; 2(2): 66-70, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21468231

RESUMO

In eukaryotes transcription is complicated by the DNA being packed in nucleosomes and by supercoils induced by opening of the DNA double helix during elongation. Here we discuss our recent genome-wide work regarding topoisomerases and their role in chromatin remodeling during the transcription cycle and we report a novel function for topoisomerases in transcription termination.

8.
EMBO J ; 29(13): 2126-34, 2010 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-20526281

RESUMO

DNA topoisomerases regulate the topological state of the DNA double helix and are key enzymes in the processes of DNA replication, transcription and genome stability. Using the fission yeast model Schizosaccharomyces pombe, we investigate genome wide how DNA topoisomerases I and II affect chromatin dynamics and gene expression in vivo. We show that topoisomerase I activity is directly required for efficient nucleosome disassembly at gene promoter regions. Lack of topoisomerase activity results in increased nucleosome occupancy, perturbed histone modifications and reduced transcription from these promoters. Strong correlative evidence suggests that topoisomerase I cooperates with the ATP-dependent chromatin remodeller Hrp1 in nucleosome disassembly. Our study links topoisomerase activity to the maintenance of open chromatin and regulating transcription in vivo.


Assuntos
Cromatina/metabolismo , DNA Topoisomerases Tipo I/metabolismo , Regulação Fúngica da Expressão Gênica , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , DNA Helicases/metabolismo , DNA Topoisomerases Tipo II/metabolismo , Histonas/metabolismo , Nucleossomos/metabolismo , Regiões Promotoras Genéticas , RNA Polimerase II/metabolismo , Schizosaccharomyces/genética , Transcrição Gênica
9.
Exp Cell Res ; 316(8): 1316-23, 2010 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-20211173

RESUMO

Eukaryotic DNA is packaged around octamers of histone proteins into nucleosomes, the basic unit of chromatin. In addition to enabling meters of DNA to fit within the confines of a nucleus, the structure of chromatin has functional implications for cell identity. Covalent chemical modifications to the DNA and to histones, histone variants, ATP-dependent chromatin remodelers, small noncoding RNAs and the level of chromatin compaction all contribute to chromosomal structure and to the activity or silencing of genes. These chromatin-level alterations are defined as epigenetic when they are heritable from mother to daughter cell. The great diversity of epigenomes that can arise from a single genome permits a single, totipotent cell to generate the hundreds of distinct cell types found in humans. Two recent studies in mouse and in fly have highlighted the importance of Chd1 chromatin remodelers for maintaining an open, active chromatin state. Based on evidence from fission yeast as a model system, we speculate that Chd1 remodelers are involved in the disassembly of nucleosomes at promoter regions, thus promoting active transcription and open chromatin. It is likely that these nucleosomes are specifically marked for disassembly by the histone variant H2A.Z.


Assuntos
Montagem e Desmontagem da Cromatina/fisiologia , Cromatina/fisiologia , DNA Helicases/fisiologia , Proteínas de Ligação a DNA/fisiologia , Epigênese Genética , Animais , Humanos
10.
Mol Cell Biol ; 30(3): 657-74, 2010 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19933844

RESUMO

Ino80 is an ATP-dependent nucleosome-remodeling enzyme involved in transcription, replication, and the DNA damage response. Here, we characterize the fission yeast Ino80 and find that it is essential for cell viability. We show that the Ino80 complex from fission yeast mediates ATP-dependent nucleosome remodeling in vitro. The purification of the Ino80-associated complex identified a highly conserved complex and the presence of a novel zinc finger protein with similarities to the mammalian transcriptional regulator Yin Yang 1 (YY1) and other members of the GLI-Krüppel family of proteins. Deletion of this Iec1 protein or the Ino80 complex subunit arp8, ies6, or ies2 causes defects in DNA damage repair, the response to replication stress, and nucleotide metabolism. We show that Iec1 is important for the correct expression of genes involved in nucleotide metabolism, including the ribonucleotide reductase subunit cdc22 and phosphate- and adenine-responsive genes. We find that Ino80 is recruited to a large number of promoter regions on phosphate starvation, including those of phosphate- and adenine-responsive genes that depend on Iec1 for correct expression. Iec1 is required for the binding of Ino80 to target genes and subsequent histone loss at the promoter and throughout the body of these genes on phosphate starvation. This suggests that the Iec1-Ino80 complex promotes transcription through nucleosome eviction.


Assuntos
Nucleossomos/metabolismo , Nucleotídeos/metabolismo , Fosfatos/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Fatores de Transcrição/metabolismo , Dedos de Zinco , Adenina/metabolismo , Sequência de Aminoácidos , Proteínas de Ciclo Celular/metabolismo , Dano ao DNA , Regulação Fúngica da Expressão Gênica , Análise em Microsséries , Dados de Sequência Molecular , Proteínas de Schizosaccharomyces pombe/genética , Fatores de Transcrição/genética
11.
PLoS Genet ; 4(8): e1000145, 2008 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-18670629

RESUMO

At the imprinted Rasgrf1 locus in mouse, a cis-acting sequence controls DNA methylation at a differentially methylated domain (DMD). While characterizing epigenetic marks over the DMD, we observed that DNA and H3K27 trimethylation are mutually exclusive, with DNA and H3K27 methylation limited to the paternal and maternal sequences, respectively. The mutual exclusion arises because one mark prevents placement of the other. We demonstrated this in five ways: using 5-azacytidine treatments and mutations at the endogenous locus that disrupt DNA methylation; using a transgenic model in which the maternal DMD inappropriately acquired DNA methylation; and by analyzing materials from cells and embryos lacking SUZ12 and YY1. SUZ12 is part of the PRC2 complex, which is needed for placing H3K27me3, and YY1 recruits PRC2 to sites of action. Results from each experimental system consistently demonstrated antagonism between H3K27me3 and DNA methylation. When DNA methylation was lost, H3K27me3 encroached into sites where it had not been before; inappropriate acquisition of DNA methylation excluded normal placement of H3K27me3, and loss of factors needed for H3K27 methylation enabled DNA methylation to appear where it had been excluded. These data reveal the previously unknown antagonism between H3K27 and DNA methylation and identify a means by which epigenetic states may change during disease and development.


Assuntos
Metilação de DNA , Impressão Genômica , Histonas/metabolismo , Lisina/metabolismo , ras-GRF1/genética , Alelos , Animais , Células Cultivadas , Feminino , Masculino , Metilação , Camundongos , Camundongos Endogâmicos , Modelos Genéticos , Especificidade da Espécie , ras-GRF1/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...