RESUMO
Heterochromatin comprises tightly compacted repetitive regions of eukaryotic chromosomes. The inheritance of heterochromatin through mitosis requires RNA interference (RNAi), which guides histone modification during the DNA replication phase of the cell cycle. Here we show that the alternating arrangement of origins of replication and non-coding RNA in pericentromeric heterochromatin results in competition between transcription and replication in Schizosaccharomyces pombe. Co-transcriptional RNAi releases RNA polymerase II (Pol II), allowing completion of DNA replication by the leading strand DNA polymerase, and associated histone modifying enzymes that spread heterochromatin with the replication fork. In the absence of RNAi, stalled forks are repaired by homologous recombination without histone modification.
Assuntos
Replicação do DNA/fisiologia , Inativação Gênica , Heterocromatina/genética , Heterocromatina/metabolismo , Interferência de RNA , RNA Polimerase II/metabolismo , Schizosaccharomyces/genética , Centrômero/genética , Centrômero/metabolismo , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Dano ao DNA , DNA Polimerase Dirigida por DNA/metabolismo , Histonas/metabolismo , Recombinação Homóloga , Modelos Genéticos , Dados de Sequência Molecular , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Origem de Replicação , Fase S , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Transcrição GênicaRESUMO
Centromere-binding protein B (CENP-B) is a widely conserved DNA binding factor associated with heterochromatin and centromeric satellite repeats. In fission yeast, CENP-B homologues have been shown to silence long terminal repeat (LTR) retrotransposons by recruiting histone deacetylases. However, CENP-B factors also have unexplained roles in DNA replication. Here we show that a molecular function of CENP-B is to promote replication-fork progression through the LTR. Mutants have increased genomic instability caused by replication-fork blockage that depends on the DNA binding factor switch-activating protein 1 (Sap1), which is directly recruited by the LTR. The loss of Sap1-dependent barrier activity allows the unhindered progression of the replication fork, but results in rearrangements deleterious to the retrotransposon. We conclude that retrotransposons influence replication polarity through recruitment of Sap1 and transposition near replication-fork blocks, whereas CENP-B counteracts this activity and promotes fork stability. Our results may account for the role of LTR in fragile sites, and for the association of CENP-B with pericentromeric heterochromatin and tandem satellite repeats.
Assuntos
Proteína B de Centrômero/metabolismo , Replicação do DNA/genética , Genoma Fúngico/genética , Instabilidade Genômica/genética , Retroelementos/genética , Schizosaccharomyces/genética , Sequências Repetidas Terminais/genética , Proteína B de Centrômero/deficiência , Proteína B de Centrômero/genética , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Sequência Conservada/genética , Dano ao DNA/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Recombinação Genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismoRESUMO
Fission yeast is an important model for epigenetic studies due to the ease with which genetic mutants can be isolated. However, it can be difficult to complement epigenetic phenotypes with genomic libraries in order to identify the genes responsible. This is because epigenetic phenotypes are typically unstable, and can prohibit complementation if silencing cannot be reestablished. Here we have resequenced the fission yeast genome following mutagenesis to readily identify a novel mutant involved in heterochromatic silencing. Candidate genes were identified as functional single base changes linked to the mutation, which were then reconstituted in a wild-type strain to recapitulate the mutant phenotype. By this procedure we identified a weak allele of ubc4, which encodes an essential E2 ubiquitin ligase, as responsible for the swi*603 mutant phenotype. In combination with a large collection of mutants and suppressor plasmids, next-generation genomic resequencing promises to dramatically enhance the power of yeast genetics, permitting the isolation of subtle alleles of essential genes, alleles with quantitative effects, and enhancers and suppressors of heterochromatic silencing.
Assuntos
Genoma Fúngico/genética , Mutação , Schizosaccharomyces/genética , Análise de Sequência de DNA/métodos , Substituição de Aminoácidos , Mapeamento Cromossômico/métodos , Cromossomos Fúngicos/genética , Epigênese Genética , Proteínas Fúngicas/genética , Genes Essenciais , Fenótipo , Polimorfismo de Nucleotídeo Único , Enzimas de Conjugação de Ubiquitina/genéticaRESUMO
Noncoding RNA has long been proposed to control gene expression via sequence-specific interactions with regulatory regions. Here, we review the role of noncoding RNA in heterochromatic silencing and in the silencing of transposable elements (TEs), unpaired DNA in meiosis, and developmentally excised DNA. The role of cotranscriptional processing by RNA interference and by other mechanisms is discussed, as well as parallels with RNA silencing in imprinting, paramutation, polycomb silencing, and X inactivation. Interactions with regulatory sequences may well occur, but at the RNA rather than at the DNA level.
Assuntos
Epigênese Genética/genética , Regulação da Expressão Gênica/genética , Inativação Gênica/fisiologia , RNA não Traduzido/genética , Animais , Elementos de DNA Transponíveis/genética , Humanos , Proteínas do Grupo Polycomb , Interferência de RNA/fisiologia , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Inativação do Cromossomo X/genéticaRESUMO
Small interfering RNA (siRNA) guides dimethylation of histone H3 lysine-9 (H3K9me2) via the Argonaute and RNA-dependent RNA polymerase complexes, as well as base-pairing with either RNA or DNA. We show that Argonaute requires the conserved aspartate-aspartate-histidine motif for heterochromatic silencing and for ribonuclease H-like cleavage (slicing) of target messages complementary to siRNA. In the fission yeast Schizosaccharomyces pombe, heterochromatic repeats are transcribed by polymerase II. We show that H3K9me2 spreads into silent reporter genes when they are embedded within these transcripts and that spreading requires read-through transcription, as well as slicing by Argonaute. Thus, siRNA guides histone modification by basepairing interactions with RNA.
Assuntos
Heterocromatina/metabolismo , Interferência de RNA , RNA Fúngico/metabolismo , RNA Interferente Pequeno/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/genética , Motivos de Aminoácidos , Proteínas Argonautas , Pareamento de Bases , Genes Reporter , Heterocromatina/genética , Histonas/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA , Proteínas Recombinantes de Fusão/metabolismo , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/genética , Transcrição GênicaRESUMO
The ability to create fully functional human chromosome vectors represents a potentially exciting gene-delivery system for the correction of human genetic disorders with several advantages over viral delivery systems. However, for the full potential of chromosome-based gene-delivery vectors to be realized, several key obstacles must be overcome. Methods must be developed to insert therapeutic genes reliably and efficiently and to enable the stable transfer of the resulting chromosomal vectors to different therapeutic cell types. Research to achieve these outcomes continues to encounter major challenges; however recent developments have reiterated the potential of chromosome-based vectors for therapeutic gene delivery. Here we review the different strategies under development and discuss the advantages and problems associated with each.
Assuntos
Cromossomos Humanos/genética , Engenharia Genética/métodos , Terapia Genética/métodos , Vetores Genéticos/administração & dosagem , Animais , Linhagem Celular Transformada , Técnicas de Transferência de Genes , Humanos , Microinjeções , TransfecçãoRESUMO
We have expressed an EGFP-CENP-A fusion protein in human cells in order to quantitate the level of CENP-A incorporated into normal and variant human centromeres. The results revealed a 3.2-fold difference in the level of CENP-A incorporation into alpha-satellite repeat DNA-based centromeres, with the Y centromere showing the lowest level of all normal human chromosomes. Identification of individual chromosomes revealed a statistically significant, though not absolute, correlation between chromosome size and CENP-A incorporation. Analysis of three independent neocentromeres revealed a significantly reduced level of CENP-A compared to normal centromeres. Truncation of a neocentric marker chromosome to produce a minichromosome further reduced CENP-A levels, indicating a remodelling of centromeric chromatin. These results suggest a role for increased CENP-A incorporation in the faithful segregation of larger chromosomes and support a model of centromere evolution in which neocentromeres represent ancestral centromeres that, through adaptive evolution, acquire satellite repeats to facilitate the incorporation of higher numbers of CENP-A containing nucleosomes, thereby facilitating the assembly of larger kinetochore structures.
