RESUMO
Partitioning of chromosomes into euchromatic and heterochromatic domains requires mechanisms that specify boundaries. The S. pombe JmjC family protein Epe1 prevents the ectopic spread of heterochromatin and is itself concentrated at boundaries. Paradoxically, Epe1 is recruited to heterochromatin by HP1 silencing factors that are distributed throughout heterochromatin. We demonstrate here that the selective enrichment of Epe1 at boundaries requires its regulation by the conserved Cul4-Ddb1(Cdt)² ubiquitin ligase, which directly recognizes Epe1 and promotes its polyubiquitylation and degradation. Strikingly, in cells lacking the ligase, Epe1 persists in the body of heterochromatin thereby inducing a defect in gene silencing. Epe1 is the sole target of the Cul4-Ddb1(Cdt)² complex whose destruction is necessary for the preservation of heterochromatin. This mechanism acts parallel with phosphorylation of HP1/Swi6 by CK2 to restrict Epe1. We conclude that the ubiquitin-dependent sculpting of the chromosomal distribution of an antisilencing factor is critical for heterochromatin boundaries to form correctly.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Ligação a DNA/metabolismo , Heterocromatina/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Complexos Ubiquitina-Proteína Ligase/metabolismo , Proteínas Nucleares/metabolismo , Schizosaccharomyces/enzimologia , Schizosaccharomyces/genética , Transdução de SinaisRESUMO
Long noncoding RNAs (lncRNAs) can trigger repressive chromatin, but how they recruit silencing factors remains unclear. In Schizosaccharomyces pombe, heterochromatin assembly on transcribed noncoding pericentromeric repeats requires both RNAi and RNAi-independent mechanisms. In Saccharomyces cerevisiae, which lacks a repressive chromatin mark (H3K9me [methylated Lys9 on histone H3]), unstable ncRNAs are recognized by the RNA-binding protein Nrd1. We show that the S. pombe ortholog Seb1 is associated with pericentromeric lncRNAs. Individual mutation of dcr1+ (Dicer) or seb1+ results in equivalent partial reductions of pericentromeric H3K9me levels, but a double mutation eliminates this mark. Seb1 functions independently of RNAi by recruiting the NuRD (nucleosome remodeling and deacetylase)-related chromatin-modifying complex SHREC (Snf2-HDAC [histone deacetylase] repressor complex).
Assuntos
Regulação Fúngica da Expressão Gênica , Inativação Gênica/fisiologia , Heterocromatina/metabolismo , Proteínas Repressoras/metabolismo , Ribonucleoproteínas/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Interferência de RNA , RNA Longo não Codificante/metabolismo , Proteínas Repressoras/genética , Ribonucleoproteínas/genética , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Transporte Vesicular/metabolismoRESUMO
HP1 proteins are central to the assembly and spread of heterochromatin containing histone H3K9 methylation. The chromodomain (CD) of HP1 proteins specifically recognizes the methyl mark on H3 peptides, but the same extent of specificity is not observed within chromatin. The chromoshadow domain of HP1 proteins promotes homodimerization, but this alone cannot explain heterochromatin spread. Using the S. pombe HP1 protein, Swi6, we show that recognition of H3K9-methylated chromatin in vitro relies on an interface between two CDs. This interaction causes Swi6 to tetramerize on a nucleosome, generating two vacant CD sticky ends. On nucleosomal arrays, methyl mark recognition is highly sensitive to internucleosomal distance, suggesting that the CD sticky ends bridge nearby methylated nucleosomes. Strengthening the CD-CD interaction enhances silencing and heterochromatin spread in vivo. Our findings suggest that recognition of methylated nucleosomes and HP1 spread on chromatin are structurally coupled and imply that methylation and nucleosome arrangement synergistically regulate HP1 function.
Assuntos
Montagem e Desmontagem da Cromatina , Proteínas Cromossômicas não Histona/metabolismo , Heterocromatina/metabolismo , Nucleossomos/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Proteínas Cromossômicas não Histona/fisiologia , Histonas/metabolismo , Metilação , Multimerização Proteica , Proteínas de Schizosaccharomyces pombe/fisiologia , Especificidade por SubstratoRESUMO
The elongation phase of transcription by RNA polymerase is highly regulated and modulated. Both general and operon-specific elongation factors determine the local rate and extent of transcription to coordinate the appearance of transcript with its use as a messenger or functional ribonucleoprotein or regulatory element, as well as to provide operon-specific gene regulation.
Assuntos
Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Fatores de Elongação da Transcrição/metabolismo , Bactérias/genética , Bacteriófago lambda/metabolismo , Sequência de Bases , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Modelos Biológicos , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , Transcrição Gênica , Proteínas Virais/metabolismoRESUMO
Centromeric silencing and heterochromatin formation in Schizosaccharomyces pombe require the RNA interference (RNAi) machinery. Three factors that mediate this mechanism have been identified: 1) the RNA-dependent RNA polymerase complex RdRC, 2) the Argonaute-containing RITS (RNA-induced initiation of transcriptional silencing) complex, and 3) the endoribonuclease Dicer ortholog Dcr1. S. pombe mutants lacking a new factor described here, Ers1, are completely defective in RNAi-dependent silencing of centromeric regions but, importantly, not in RNAi-independent silencing at the mat3M or tel2R loci. ers1Delta cells likewise fail to convert centromeric pre-small interfering RNA transcripts into small interfering RNAs, are defective in histone H3 Lys(9) methylation, and are unable to recruit the RITS complex to centromeric sequences. Surprisingly, Ers1 lacks obvious orthologs outside of the genus Schizosaccharomyces. Within this group, it is diverging rapidly, raising the possibility that it is coevolving with target RNA elements.
Assuntos
Proteínas Fúngicas/fisiologia , Heterocromatina/genética , Interferência de RNA , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/fisiologia , Sequência de Aminoácidos , Centrômero/metabolismo , Cromossomos Fúngicos , Proteínas Fúngicas/genética , Modelos Genéticos , Dados de Sequência Molecular , RNA Interferente Pequeno/metabolismo , Complexo de Inativação Induzido por RNA/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Schizosaccharomyces , Homologia de Sequência de AminoácidosRESUMO
The universal bacterial transcription elongation factor NusA mediates elongation activities of RNA polymerase. By itself, NusA induces transcription pausing and facilitates intrinsic termination, but NusA also is a cofactor of antiterminators that antagonize pausing and prevent termination. We show that NusA is required for lambda-related phage 82 antiterminator Q(82) to construct a stable complex in which RNA-based termination mechanisms have restricted access to the emerging transcript; this result suggests a locale for both Q(82) and NusA near the beta flap domain of RNA polymerase. Furthermore, as NusA is not required for the antipausing activity of Q(82) in vitro, we distinguish two distinct activities of antiterminators, namely antipausing and RNA occlusion, and discuss their roles in Q(82) function.