RESUMO
Epigenetically regulated heterochromatin domains govern essential cellular activities. A key feature of heterochromatin domains is the presence of hypoacetylated nucleosomes, which are methylated on lysine 9 of histone H3 (H3K9me). Here, we investigate the requirements for establishment, spreading and maintenance of heterochromatin using fission yeast centromeres as a paradigm. We show that establishment of heterochromatin on centromeric repeats is initiated at modular 'nucleation sites' by RNA interference (RNAi), ensuring the mitotic stability of centromere-bearing minichromosomes. We demonstrate that the histone deacetylases Sir2 and Clr3 and the chromodomain protein Swi6(HP1) are required for H3K9me spreading from nucleation sites, thus allowing formation of extended heterochromatin domains. We discovered that RNAi and Sir2 along with Swi6(HP1) operate in two independent pathways to maintain heterochromatin. Finally, we demonstrate that tethering of Sir2 is pivotal to the maintenance of heterochromatin at an ectopic locus in the absence of RNAi. These analyses reveal that Sir2, together with RNAi, are sufficient to ensure heterochromatin integrity and provide evidence for sequential establishment, spreading and maintenance steps in the assembly of centromeric heterochromatin.
Assuntos
Centrômero/genética , Montagem e Desmontagem da Cromatina/genética , Heterocromatina/metabolismo , Interferência de RNA/fisiologia , Proteínas de Schizosaccharomyces pombe/fisiologia , Centrômero/metabolismo , Epigênese Genética/genética , Epigênese Genética/fisiologia , Heterocromatina/genética , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Histonas/química , Histonas/metabolismo , Metiltransferases/genética , Metiltransferases/metabolismo , Modelos Biológicos , Organismos Geneticamente Modificados , Processamento de Proteína Pós-Traducional , Elementos Reguladores de Transcrição/genética , Schizosaccharomyces/genética , Schizosaccharomyces/fisiologia , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismoRESUMO
Non-coding transcription can trigger histone post-translational modifications forming specialized chromatin. In fission yeast, heterochromatin formation requires RNAi and the histone H3K9 methyltransferase complex CLRC, composed of Clr4, Raf1, Raf2, Cul4, and Rik1. CLRC mediates H3K9 methylation and siRNA production; it also displays E3-ubiquitin ligase activity in vitro. DCAFs act as substrate receptors for E3 ligases and may couple ubiquitination with histone methylation. Here, structural alignment and mutation of signature WDxR motifs in Raf1 indicate that it is a DCAF for CLRC. We demonstrate that Raf1 promotes H3K9 methylation and siRNA amplification via two distinct, separable functions. The association of the DCAF Raf1 with Cul4-Rik1 is critical for H3K9 methylation, but dispensable for processing of centromeric transcripts into siRNAs. Thus the association of a DCAF, Raf1, with its adaptor, Rik1, is required for histone methylation and to allow RNAi to signal to chromatin.
Assuntos
Proteínas Cromossômicas não Histona/genética , Proteínas de Ligação a DNA/genética , Histonas/genética , Proteínas Proto-Oncogênicas c-raf/genética , RNA Interferente Pequeno/genética , Proteínas de Schizosaccharomyces pombe/genética , Schizosaccharomyces/genética , Proteínas Cdc20 , Proteínas de Ciclo Celular/genética , Montagem e Desmontagem da Cromatina , Heterocromatina/genética , Histona-Lisina N-Metiltransferase/genética , Humanos , Metilação , Metiltransferases/genética , Complexos Multiproteicos/genética , Mutação , Processamento de Proteína Pós-Traducional , Schizosaccharomyces/metabolismo , Homologia Estrutural de Proteína , Ubiquitina-Proteína Ligases/genética , UbiquitinaçãoRESUMO
Argonaute (Ago) proteins mediate silencing of nucleic acid targets by small RNAs. In fission yeast, Ago1, Tas3 and Chp1 assemble into a RITS complex, which silences transcription near centromeres. Here we describe a repetitive motif within Tas3, termed the 'Argonaute hook', that is conserved from yeast to humans and binds Ago proteins through their PIWI domains in vitro and in vivo. Site-directed mutation of key residues in the motif disrupts Ago binding and heterochromatic silencing in vivo. Unexpectedly, a PIWI domain pocket that binds the 5' end of the short interfering RNA guide strand is required for direct binding of the Ago hook. Moreover, wild-type but not mutant Ago hook peptides derepress microRNA-mediated translational silencing of a target messenger RNA. Proteins containing the conserved Ago hook may thus be important regulatory components of effector complexes in RNA interference.
Assuntos
Sequência de Aminoácidos , Proteínas de Transporte , Conformação Proteica , Proteínas de Schizosaccharomyces pombe , Proteínas Argonautas , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Núcleo Celular/metabolismo , Fator de Iniciação 2 em Eucariotos/genética , Fator de Iniciação 2 em Eucariotos/metabolismo , Inativação Gênica , Humanos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Ligação Proteica , Biossíntese de Proteínas , Interferência de RNA , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteínas de Ligação a RNA , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Alinhamento de Sequência , Técnicas do Sistema de Duplo-HíbridoRESUMO
The FACT complex is a conserved cofactor for RNA polymerase II elongation through nucleosomes. FACT bears histone chaperone activity and contributes to chromatin integrity. However, the molecular mechanisms behind FACT function remain elusive. Here we report biochemical, structural, and mutational analyses that identify the peptidase homology domain of the Schizosaccharomyces pombe FACT large subunit Spt16 (Spt16-N) as a binding module for histones H3 and H4. The 2.1-A crystal structure of Spt16-N reveals an aminopeptidase P fold whose enzymatic activity has been lost. Instead, the highly conserved fold directly binds histones H3-H4 through a tight interaction with their globular core domains, as well as with their N-terminal tails. Mutations within a conserved surface pocket in Spt16-N or posttranslational modification of the histone H4 tail reduce interaction in vitro, whereas the globular domains of H3-H4 and the H3 tail bind distinct Spt16-N surfaces. Our analysis suggests that the N-terminal domain of Spt16 may add to the known H2A-H2B chaperone activity of FACT by including a H3-H4 tail and H3-H4 core binding function mediated by the N terminus of Spt16. We suggest that these interactions may aid FACT-mediated nucleosome reorganization events.
Assuntos
Aminopeptidases/química , Histonas/metabolismo , Peptídeo Hidrolases/química , Peptídeo Hidrolases/metabolismo , Proteínas de Schizosaccharomyces pombe/química , Schizosaccharomyces/metabolismo , Aminopeptidases/metabolismo , Catálise , Ativação Enzimática , Histonas/química , Modelos Moleculares , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Schizosaccharomyces/enzimologia , Proteínas de Schizosaccharomyces pombe/metabolismoRESUMO
Centromeres exert vital cellular functions in mitosis and meiosis. A specialized histone and other chromatin-bound factors nucleate a dynamic protein assembly that is required for the proper segregation of sister chromatids. In several organisms, including the fission yeast, Schizosaccharomyces pombe, the RNAi pathway contributes to the formation of silent chromatin in pericentromeric regions. Little is known about how chromatin-remodeling factors contribute to heterochromatic integrity and centromere function. Here we show that the histone chaperone and remodeling complex FACT is required for centromeric-heterochromatin integrity and accurate chromosome segregation. We show that Spt16 and Pob3 are two subunits of the S. pombe FACT complex. Surprisingly, yeast strains deleted for pob3+ are viable and alleviate gene silencing at centromeric repeats and at the silent mating-type locus. Importantly, like heterochromatin and RNAi pathway mutants, Pob3 null strains exhibit lagging chromosomes on anaphase spindles. Whereas the processing of centromeric RNA transcripts into siRNAs is maintained in Pob3 mutants, Swi6-association with the centromere is reduced. Our studies provide the first experimental evidence for a role of the RNA polymerase II cofactor FACT in heterochromatin integrity and in centromere function.
Assuntos
Centrômero/metabolismo , Segregação de Cromossomos/fisiologia , Heterocromatina/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Regulação Fúngica da Expressão Gênica , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Interferência de RNA , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genéticaAssuntos
Proteínas de Ligação a DNA/metabolismo , NAD/metabolismo , Fosfoproteínas/metabolismo , Proteínas Repressoras/metabolismo , Transcrição Gênica/genética , Acetiltransferases/química , Acetiltransferases/metabolismo , Oxirredutases do Álcool , Animais , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ligação a DNA/genética , Histona Acetiltransferases , Histonas/metabolismo , Fosfoproteínas/genética , Proteínas Repressoras/genética , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Fatores de Transcrição de p300-CBPRESUMO
The methyltransferase component of type I DNA restriction and modification systems comprises three subunits, one DNA sequence specificity subunit and two DNA modification subunits. Limited proteolysis of the EcoKI methyltransferase shows that a 55-kDa N-terminal fragment of the 59-kDa modification subunit is resistant to degradation. We have purified this fragment and determined by mass spectrometry that proteolysis removes 43 or 44 amino acids from the C-terminus. The fragment fails to interact with the other subunits even though it still possesses secondary and tertiary structure and the ability to bind the S-adenosylmethionine cofactor. We conclude that the C-terminal region of the modification subunit of EcoKI is essential for the assembly of the EcoKI methyltransferase.
Assuntos
Subunidades Proteicas/química , DNA Metiltransferases Sítio Específica (Adenina-Específica)/biossíntese , Sequência de Aminoácidos , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/isolamento & purificação , Fragmentos de Peptídeos/metabolismo , Mapeamento de Peptídeos , Desnaturação Proteica , Dobramento de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Subunidades Proteicas/metabolismo , DNA Metiltransferases Sítio Específica (Adenina-Específica)/químicaRESUMO
Epigenetic mechanisms regulate genome structure and expression profiles in eukaryotes. RNA interference (RNAi) and other small RNA-based chromatin-modifying activities can act to reset the epigenetic landscape at defined chromatin domains. Centromeric heterochromatin assembly is a RNAi-dependent process in the fission yeast Schizosaccharomyces pombe, and provides a paradigm for detailed examination of such epigenetic processes. Here we review recent progress in understanding the mechanisms that underpin RNAi-mediated heterochromatin formation in S. pombe. We discuss recent analyses of the events that trigger RNAi and manipulations which uncouple RNAi and chromatin modification. Finally we provide an overview of similar molecular machineries across species where related small RNA pathways appear to drive the epigenetic reprogramming in germ cells and/or during early development in metazoans.