RESUMO
In eukaryotes, heterochromatin is generally located at the nuclear periphery. This study investigates the biological significance of perinuclear positioning for heterochromatin maintenance and gene silencing. We identify the nuclear rim protein Amo1NUPL2 as a factor required for the propagation of heterochromatin at endogenous and ectopic sites in the fission yeast genome. Amo1 associates with the Rix1PELP1-containing RNA processing complex RIXC and with the histone chaperone complex FACT. RIXC, which binds to heterochromatin protein Swi6HP1 across silenced chromosomal domains and to surrounding boundary elements, connects heterochromatin with Amo1 at the nuclear periphery. In turn, the Amo1-enriched subdomain is critical for Swi6 association with FACT that precludes histone turnover to promote gene silencing and preserve epigenetic stability of heterochromatin. In addition to uncovering conserved factors required for perinuclear positioning of heterochromatin, these analyses elucidate a mechanism by which a peripheral subdomain enforces stable gene repression and maintains heterochromatin in a heritable manner.
Assuntos
Epigênese Genética/genética , Heterocromatina/genética , Heterocromatina/metabolismo , Proteínas de Ciclo Celular/metabolismo , Núcleo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Repressão Epigenética/genética , Inativação Gênica , Hereditariedade , Histonas/genética , Histonas/metabolismo , Metilação , Proteínas Nucleares/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismoRESUMO
The regulation of protein-coding and noncoding RNAs is linked to nuclear processes, including chromatin modifications and gene silencing. However, the mechanisms that distinguish RNAs and mediate their functions are poorly understood. We describe a nuclear RNA-processing network in fission yeast with a core module comprising the Mtr4-like protein, Mtl1, and the zinc-finger protein, Red1. The Mtl1-Red1 core promotes degradation of mRNAs and noncoding RNAs and associates with different proteins to assemble heterochromatin via distinct mechanisms. Mtl1 also forms Red1-independent interactions with evolutionarily conserved proteins named Nrl1 and Ctr1, which associate with splicing factors. Whereas Nrl1 targets transcripts with cryptic introns to form heterochromatin at developmental genes and retrotransposons, Ctr1 functions in processing intron-containing telomerase RNA. Together with our discovery of widespread cryptic introns, including in noncoding RNAs, these findings reveal unique cellular strategies for recognizing regulatory RNAs and coordinating their functions in response to developmental and environmental cues.
Assuntos
RNA Helicases DEAD-box/metabolismo , Processamento Pós-Transcricional do RNA , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Telômero/metabolismo , Animais , Caenorhabditis elegans/metabolismo , Proteínas de Transporte/metabolismo , Montagem e Desmontagem da Cromatina , Heterocromatina/metabolismo , ÍntronsRESUMO
Heterochromatin, defined by histone H3 lysine 9 methylation (H3K9me), spreads across large domains and can be epigenetically inherited in a self-propagating manner. Heterochromatin propagation depends upon a read-write mechanism, where the Clr4/Suv39h methyltransferase binds to preexisting trimethylated H3K9 (H3K9me3) and further deposits H3K9me. How the parental methylated histone template is preserved during DNA replication is not well understood. Here, we demonstrate using Schizosaccharomyces pombe that heterochromatic regions are specialized replication domains demarcated by their surrounding boundary elements. DNA replication throughout these domains is distinguished by an abundance of replisome components and is coordinated by Swi6/HP1. Although mutations in the replicative helicase subunit Mcm2 that affect histone binding impede the maintenance of a heterochromatin domain at an artificially targeted ectopic site, they have only a modest impact on heterochromatin propagation via the read-write mechanism at an endogenous site. Instead, our findings suggest a crucial role for the replication factor Mcl1 in retaining parental histones and promoting heterochromatin propagation via a mechanism involving the histone chaperone FACT. Engagement of FACT with heterochromatin requires boundary elements, which position the heterochromatic domain at the nuclear peripheral subdomain enriched for heterochromatin factors. Our findings highlight the importance of replisome components and boundary elements in creating a specialized environment for the retention of parental methylated histones, which facilitates epigenetic inheritance of heterochromatin.
Assuntos
Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Histonas/metabolismo , Heterocromatina/genética , Heterocromatina/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Montagem e Desmontagem da Cromatina , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Epigênese GenéticaRESUMO
Heterochromatin can be epigenetically inherited in cis, leading to stable gene silencing. However, the mechanisms underlying heterochromatin inheritance remain unclear. Here, we identify Fft3, a fission yeast homolog of the mammalian SMARCAD1 SNF2 chromatin remodeler, as a factor uniquely required for heterochromatin inheritance, rather than for de novo assembly. Importantly, we find that Fft3 suppresses turnover of histones at heterochromatic loci to facilitate epigenetic transmission of heterochromatin in cycling cells. Moreover, Fft3 also precludes nucleosome turnover at several euchromatic loci to prevent R-loop formation, ensuring proper replication progression. Our analyses show that overexpression of Clr4/Suv39h, which is also required for efficient replication through these loci, suppresses phenotypes associated with the loss of Fft3. This work uncovers a conserved factor critical for epigenetic inheritance of heterochromatin and describes a mechanism in which suppression of nucleosome turnover prevents formation of structural barriers that impede replication at fragile regions in the genome.
Assuntos
Montagem e Desmontagem da Cromatina , Proteínas Cromossômicas não Histona/metabolismo , Replicação do DNA , DNA Fúngico/biossíntese , Epigênese Genética , Hereditariedade , Heterocromatina/metabolismo , Nucleossomos/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/genética , DNA Fúngico/genética , Genótipo , Heterocromatina/genética , Histona-Lisina N-Metiltransferase , Histonas/metabolismo , Metiltransferases/genética , Metiltransferases/metabolismo , Mutação , Nucleossomos/genética , Fenótipo , Schizosaccharomyces/genética , Schizosaccharomyces/crescimento & desenvolvimento , Proteínas de Schizosaccharomyces pombe/genética , Fatores de TempoRESUMO
Facultative heterochromatin regulates gene expression, but its assembly is poorly understood. Previously, we identified facultative heterochromatin islands in the fission yeast genome and found that RNA elimination machinery promotes island assembly at meiotic genes. Here, we report that Taz1, a component of the telomere protection complex Shelterin, is required to assemble heterochromatin islands at regions corresponding to late replication origins that are sites of double-strand break formation during meiosis. The loss of Taz1 or other Shelterin subunits, including Ccq1 that interacts with Clr4/Suv39h, abolishes heterochromatin at late origins and causes derepression of associated genes. Moreover, the late-origin regulator Rif1 affects heterochromatin at Taz1-dependent islands and subtelomeric regions. We explore the connection between facultative heterochromatin and replication control and show that heterochromatin machinery affects replication timing. These analyses reveal the role of Shelterin in facultative heterochromatin assembly at late origins, which has important implications for genome stability and gene regulation.
Assuntos
Montagem e Desmontagem da Cromatina , Cromossomos Fúngicos , DNA Fúngico/metabolismo , Regulação Fúngica da Expressão Gênica , Heterocromatina/metabolismo , Origem de Replicação , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Proteínas de Ligação a Telômeros/metabolismo , Sítios de Ligação , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Metilação de DNA , DNA Fúngico/genética , Inativação Gênica , Heterocromatina/genética , Histona-Lisina N-Metiltransferase , Histonas/metabolismo , Metiltransferases/genética , Metiltransferases/metabolismo , Ligação Proteica , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Ligação a Telômeros/genética , Fatores de TempoRESUMO
Uniparental disomy (UPD), in which an individual contains a pair of homologous chromosomes originating from only one parent, is a frequent phenomenon that is linked to congenital disorders and various cancers. UPD is thought to result mostly from pre- or post-zygotic chromosome missegregation. However, the factors that drive UPD remain unknown. Here we use the fission yeast Schizosaccharomyces pombe as a model to investigate UPD, and show that defects in the RNA interference (RNAi) machinery or in the YTH domain-containing RNA elimination factor Mmi1 cause high levels of UPD in vegetative diploid cells. This phenomenon is not due to defects in heterochromatin assembly at centromeres. Notably, in cells lacking RNAi components or Mmi1, UPD is associated with the untimely expression of gametogenic genes. Deletion of the upregulated gene encoding the meiotic cohesin Rec8 or the cyclin Crs1 suppresses UPD in both RNAi and mmi1 mutants. Moreover, overexpression of Rec8 is sufficient to trigger UPD in wild-type cells. Rec8 expressed in vegetative cells localizes to chromosomal arms and to the centromere core, where it is required for localization of the cohesin subunit Psc3. The centromeric localization of Rec8 and Psc3 promotes UPD by uniquely affecting chromosome segregation, causing a reductional segregation of one homologue. Together, these findings establish the untimely vegetative expression of gametogenic genes as a causative factor of UPD, and provide a solid foundation for understanding this phenomenon, which is linked to diverse human diseases.
Assuntos
Regulação Fúngica da Expressão Gênica , Células Germinativas/metabolismo , Modelos Biológicos , Mutação , Schizosaccharomyces/citologia , Schizosaccharomyces/genética , Dissomia Uniparental/genética , Centrômero/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Segregação de Cromossomos/genética , Ciclinas/deficiência , Ciclinas/genética , Diploide , Heterocromatina/metabolismo , Humanos , Meiose/genética , Fosfoproteínas/deficiência , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Interferência de RNA , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Fatores de Tempo , Dissomia Uniparental/patologia , Fatores de Poliadenilação e Clivagem de mRNA/deficiência , Fatores de Poliadenilação e Clivagem de mRNA/genética , Fatores de Poliadenilação e Clivagem de mRNA/metabolismoRESUMO
Heterochromatin impacts various nuclear processes by providing a recruiting platform for diverse chromosomal proteins. In fission yeast, HP1 proteins Chp2 and Swi6, which bind to methylated histone H3 lysine 9, associate with SHREC (Snf2/HDAC repressor complex) and Clr6 histone deacetylases (HDACs) involved in heterochromatic silencing. However, heterochromatic silencing machinery is not fully defined. We describe a histone chaperone complex containing Asf1 and HIRA that spreads across silenced domains via its association with Swi6 to enforce transcriptional silencing. Asf1 functions in concert with a Clr6 HDAC complex to silence heterochromatic repeats, and it suppresses antisense transcription by promoting histone deacetylation. Furthermore, we demonstrate that Asf1 and SHREC facilitate nucleosome occupancy at heterochromatic regions but TFIIIC transcription factor binding sites within boundary elements are refractory to these factors. These analyses uncover a role for Asf1 in global histone deacetylation and suggest that HP1-associated histone chaperone promotes nucleosome occupancy to assemble repressive heterochromatin.
Assuntos
Inativação Gênica , Histonas/metabolismo , Chaperonas Moleculares/fisiologia , Proteínas de Schizosaccharomyces pombe/fisiologia , Schizosaccharomyces/genética , Fatores de Transcrição/fisiologia , Acetilação , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/fisiologia , Montagem e Desmontagem da Cromatina , Proteínas Cromossômicas não Histona/metabolismo , Proteínas Cromossômicas não Histona/fisiologia , Epigênese Genética , Heterocromatina/metabolismo , Chaperonas Moleculares/metabolismo , Nucleossomos/metabolismo , RNA Antissenso/metabolismo , Recombinação Genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Chromatin regulatory proteins affect diverse developmental and environmental response pathways via their influence on nuclear processes such as the regulation of gene expression. Through a genome-wide genetic screen, we implicate a novel protein called X-chromosome-associated protein 5 (Xap5) in chromatin regulation. We show that Xap5 is a chromatin-associated protein acting in a similar manner as the histone variant H2A.Z to suppress expression of antisense and repeat element transcripts throughout the fission yeast genome. Xap5 is highly conserved across eukaryotes, and a plant homolog rescues xap5 mutant yeast. We propose that Xap5 likely functions as a chromatin regulator in diverse organisms.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Histonas/fisiologia , Proteínas de Schizosaccharomyces pombe/fisiologia , Schizosaccharomyces/genética , Elementos Antissenso (Genética) , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Genes Fúngicos , Teste de Complementação Genética , Ligação Proteica , RNA Fúngico/genética , RNA Fúngico/metabolismo , RNA Mensageiro/metabolismo , Sequências Repetitivas de Ácido Nucleico , Schizosaccharomyces/metabolismo , Transcrição Gênica , Regulação para CimaRESUMO
Eukaryotic transcriptomes are characterized by widespread transcription of noncoding and antisense RNAs, which is linked to key chromosomal processes, such as chromatin remodelling, gene regulation and heterochromatin assembly. However, these transcripts can be deleterious, and their accumulation is suppressed by several mechanisms including degradation by the nuclear exosome. The mechanisms by which cells differentiate coding RNAs from transcripts targeted for degradation are not clear. Here we show that the variant histone H2A.Z, which is loaded preferentially at the 5' ends of genes by the Swr1 complex containing a JmjC domain protein, mediates suppression of antisense transcripts in the fission yeast Schizosaccharomyces pombe genome. H2A.Z is partially redundant in this regard with the Clr4 (known as SUV39H in mammals)-containing heterochromatin silencing complex that is also distributed at euchromatic loci, and with RNA interference component Argonaute (Ago1). Loss of Clr4 or Ago1 alone has little effect on antisense transcript levels, but cells lacking either of these factors and H2A.Z show markedly increased levels of antisense RNAs that are normally degraded by the exosome. These analyses suggest that as well as performing other functions, H2A.Z is a component of a genome indexing mechanism that cooperates with heterochromatin and RNAi factors to suppress read-through antisense transcripts.
Assuntos
Regulação Fúngica da Expressão Gênica , Heterocromatina/metabolismo , Histonas/metabolismo , Interferência de RNA , RNA Antissenso/antagonistas & inibidores , RNA Antissenso/genética , Schizosaccharomyces/genética , Proteínas Argonautas , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Exossomos/metabolismo , Heterocromatina/genética , Histona-Lisina N-Metiltransferase , Histonas/deficiência , Histonas/genética , Metiltransferases/deficiência , Metiltransferases/genética , Metiltransferases/metabolismo , RNA Antissenso/biossíntese , RNA Fúngico/genética , RNA Fúngico/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismoRESUMO
Heterochromatin in eukaryotic genomes regulates diverse chromosomal processes including transcriptional silencing. However, in Schizosaccharomyces pombe RNA polymerase II (RNAPII) transcription of centromeric repeats is essential for RNA-interference-mediated heterochromatin assembly. Here we study heterochromatin dynamics during the cell cycle and its effect on RNAPII transcription. We describe a brief period during the S phase of the cell cycle in which RNAPII preferentially transcribes centromeric repeats. This period is enforced by heterochromatin, which restricts RNAPII accessibility at centromeric repeats for most of the cell cycle. RNAPII transcription during S phase is linked to loading of RNA interference and heterochromatin factors such as the Ago1 subunit of the RITS complex and the Clr4 methyltransferase complex subunit Rik1 (ref. 7). Moreover, Set2, an RNAPII-associated methyltransferase that methylates histone H3 lysine 36 at repeat loci during S phase, acts in a pathway parallel to Clr4 to promote heterochromatin assembly. We also show that phosphorylation of histone H3 serine 10 alters heterochromatin during mitosis, correlating with recruitment of condensin that affects silencing of centromeric repeats. Our analyses suggest at least two distinct modes of heterochromatin targeting to centromeric repeats, whereby RNAPII transcription of repeats and chromodomain proteins bound to methylated histone H3 lysine 9 mediate recruitment of silencing factors. Together, these processes probably facilitate heterochromatin maintenance through successive cell divisions.
Assuntos
Ciclo Celular/fisiologia , Centrômero/genética , Montagem e Desmontagem da Cromatina , Heterocromatina/metabolismo , Schizosaccharomyces/citologia , Schizosaccharomyces/genética , Transcrição Gênica , Proteínas Argonautas , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Inativação Gênica , Heterocromatina/genética , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/química , Histonas/metabolismo , Metilação , Metiltransferases/metabolismo , Fosforilação , RNA Polimerase II/metabolismo , Proteínas de Ligação a RNA , Fase S , Schizosaccharomyces/enzimologia , Proteínas de Schizosaccharomyces pombe/metabolismoRESUMO
RNA interference is a conserved mechanism by which double-stranded RNA is processed into short interfering RNAs (siRNAs) that can trigger both post-transcriptional and transcriptional gene silencing. In fission yeast, the RNA-induced initiation of transcriptional gene silencing (RITS) complex contains Dicer-generated siRNAs and is required for heterochromatic silencing. Here we show that RITS components, including Argonaute protein, bind to all known heterochromatic loci. At the mating-type region, RITS is recruited to the centromere-homologous repeat cenH in a Dicer-dependent manner, whereas the spreading of RITS across the entire 20-kb silenced domain, as well as its subsequent maintenance, requires heterochromatin machinery including Swi6 and occurs even in the absence of Dicer. Furthermore, our analyses suggest that RNA interference machinery operates in cis as a stable component of heterochromatic domains with RITS tethered to silenced loci by methylation of histone H3 at Lys9. This tethering promotes the processing of transcripts and generation of additional siRNAs for heterochromatin maintenance.
Assuntos
Inativação Gênica , Interferência de RNA , Schizosaccharomyces/genética , Animais , Proteínas Cromossômicas não Histona/genética , Cromossomos Fúngicos , Metilação de DNA , Proteínas Ligadas por GPI , Heterocromatina , Modelos Genéticos , RNA Interferente Pequeno , Receptores do Fator de Necrose Tumoral/genética , Membro 10c de Receptores do Fator de Necrose Tumoral , Proteínas de Schizosaccharomyces pombe/genética , Receptores Chamariz do Fator de Necrose TumoralRESUMO
Conserved chromosomal HP1 proteins capable of binding to histone H3 methylated at lysine 9 are believed to provide a dynamic platform for the recruitment and/or spreading of various regulatory proteins involved in diverse chromosomal processes. The fission yeast Schizosaccharomyces pombe HP1 family members Chp2 and Swi6 are important for heterochromatin assembly and transcriptional silencing, but their precise roles are not fully understood. Here, we show that Swi6 and Chp2 associate with histone deacetylase (HDAC) protein complexes containing class I HDAC Clr6 and class II HDAC Clr3 (a component of Snf2/HDAC repressor complex), which are critical for transcriptional silencing of centromeric repeats targeted by the heterochromatin machinery. Mapping of RNA polymerase (Pol) II distribution in single and double mutant backgrounds revealed that Swi6 and Chp2 proteins and their associated HDAC complexes have overlapping functions in limiting Pol II occupancy across pericentromeric heterochromatin domains. The purified Swi6 fraction also contains factors involved in various chromosomal processes such as chromatin remodeling and DNA replication. Also, Swi6 copurifies with Mis4 protein, a cohesin loading factor essential for sister chromatid cohesion, and with centromere-specific histone H3 variant CENP-A, which is incorporated into chromatin in a heterochromatin-dependent manner. These analyses suggest that among other functions, HP1 proteins associate with chromatin-modifying factors that in turn cooperate to assemble repressive chromatin; thus, precluding accessibility of underlying DNA sequences to transcriptional machinery.
Assuntos
Centrômero/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Inativação Gênica , Heterocromatina/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Proteínas de Ciclo Celular/metabolismo , Centrômero/genética , Imunoprecipitação da Cromatina , Homólogo 5 da Proteína Cromobox , Proteínas Cromossômicas não Histona/genética , DNA Polimerase II/metabolismo , Histona Desacetilases/metabolismo , Proteínas Repressoras/metabolismo , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genéticaRESUMO
Heterochromatin assembly, involving histone H3 lysine-9 methylation (H3K9me), is nucleated at specific genomic sites but can self-propagate across extended domains and, indeed, generations. Self-propagation requires Clr4/Suv39h methyltransferase recruitment by pre-existing H3K9 tri-methylation (H3K9me3) to perpetuate H3K9me deposition and is dramatically affected by chromatin context. However, the mechanism priming self-propagation of heterochromatin remains undefined. We show that robust chromatin association of fission yeast class II histone deacetylase Clr3 is necessary and sufficient to support heterochromatin propagation in different chromosomal contexts. Efficient targeting of Clr3, which suppresses histone turnover and maintains H3K9me3, enables self-propagation of an ectopic heterochromatin domain via the Clr4/Suv39h read-write mechanism requiring methylated histones. The deacetylase activity of Clr3 is necessary and, when inactivated, heterochromatin propagation can be recapitulated by removing two major histone acetyltransferases. Our results show that histone deacetylation, a conserved heterochromatin feature, preserves H3K9me3 that transmits epigenetic memory for stable propagation of silenced chromatin domains through multiple generations.
Assuntos
Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Epigênese Genética , Heterocromatina/genética , Histona Acetiltransferases , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/genética , Histonas/metabolismo , Lisina/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismoRESUMO
Chromatin-remodeling complexes regulate access to nucleosomal DNA by mobilizing nucleosomes in an ATP-dependent manner. In this study, we find that chromatin remodeling by SWI/SNF and ISW2 involves DNA translocation inside nucleosomes two helical turns from the dyad axis at superhelical location-2. DNA translocation at this internal position does not require the propagation of a DNA twist from the site of translocation to the entry/exit sites for nucleosome movement. Nucleosomes are moved in 9- to 11- or approximately 50-base-pair increments by ISW2 or SWI/SNF, respectively, presumably through the formation of DNA loops on the nucleosome surface. Remodeling by ISW2 but not SWI/SNF requires DNA torsional strain near the site of translocation, which may work in conjunction with conformational changes of ISW2 to promote nucleosome movement on DNA. The difference in step size of nucleosome movement by SWI/SNF and ISW2 demonstrates how SWI/SNF may be more disruptive to nucleosome structure than ISW2.
Assuntos
Adenosina Trifosfatases/metabolismo , Montagem e Desmontagem da Cromatina , Proteínas Cromossômicas não Histona/metabolismo , DNA Fúngico/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Transporte Ativo do Núcleo Celular , Adenosina Trifosfatases/química , Proteínas Cromossômicas não Histona/química , DNA Fúngico/química , Cinética , Substâncias Macromoleculares , Modelos Moleculares , Conformação de Ácido Nucleico , Nucleossomos/metabolismo , Conformação Proteica , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Fatores de Transcrição/químicaRESUMO
Cell proliferation and differentiation require signalling pathways that enforce appropriate and timely gene expression. We find that Tor2, the catalytic subunit of the TORC1 complex in fission yeast, targets a conserved nuclear RNA elimination network, particularly the serine and proline-rich protein Pir1, to control gene expression through RNA decay and facultative heterochromatin assembly. Phosphorylation by Tor2 protects Pir1 from degradation by the ubiquitin-proteasome system involving the polyubiquitin Ubi4 stress-response protein and the Cul4-Ddb1 E3 ligase. This pathway suppresses widespread and untimely gene expression and is critical for sustaining cell proliferation. Moreover, we find that the dynamic nature of Tor2-mediated control of RNA elimination machinery defines gene expression patterns that coordinate fundamental chromosomal events during gametogenesis, such as meiotic double-strand-break formation and chromosome segregation. These findings have important implications for understanding how the TOR signalling pathway reprogrammes gene expression patterns and contributes to diseases such as cancer.
Assuntos
Proliferação de Células , Montagem e Desmontagem da Cromatina , Regulação Fúngica da Expressão Gênica , Heterocromatina/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Processamento Pós-Transcricional do RNA , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/enzimologia , Heterocromatina/genética , Mitose , Fosfatidilinositol 3-Quinases/genética , Fosforilação , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Schizosaccharomyces/genética , Schizosaccharomyces/crescimento & desenvolvimento , Proteínas de Schizosaccharomyces pombe/genética , UbiquitinaçãoRESUMO
In eukaryotic genomes, heterochromatin is targeted by RNAi machinery and/or by pathways requiring RNA elimination and transcription termination factors. However, a direct connection between termination machinery and RNA polymerase II (RNAPII) transcriptional activity at heterochromatic loci has remained elusive. Here, we show that, in fission yeast, the conserved cleavage and polyadenylation factor (CPF) is a key component involved in RNAi-independent assembly of constitutive and facultative heterochromatin domains and that CPF is broadly required to silence genes regulated by Clr4SUV39H. Remarkably, CPF is recruited to non-canonical termination sites within the body of genes by the YTH family RNA-binding protein Mmi1 and is required for RNAPII transcription termination and facultative heterochromatin assembly. CPF loading by Mmi1 also promotes the selective termination of long non-coding RNAs that regulate gene expression in cis. These analyses delineate a mechanism in which CPF loaded onto non-canonical termination sites specifies targets of heterochromatin assembly and gene silencing.
Assuntos
Inativação Gênica , Heterocromatina/metabolismo , RNA Polimerase II/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/genética , Terminação da Transcrição Genética , Fatores de Poliadenilação e Clivagem de mRNA/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Montagem e Desmontagem da Cromatina/genética , Exorribonucleases/genética , Exorribonucleases/metabolismo , Regulação Fúngica da Expressão Gênica , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Meiose/genética , Fosfoproteínas Fosfatases/genética , Fosfoproteínas Fosfatases/metabolismo , Interferência de RNA , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Fatores de Poliadenilação e Clivagem de mRNA/genéticaRESUMO
A minimal amount of extranucleosomal DNA was required for nucleosome mobilization by ISW2 as shown by using a photochemical histone mapping approach to analyze nucleosome movement on a set of nucleosomes with varied lengths of extranucleosomal DNA. ISW2 was ineffective in repositioning or mobilizing nucleosomes with Assuntos
Adenosina Trifosfatases/genética
, Adenosina Trifosfatases/metabolismo
, DNA Fúngico/genética
, DNA Fúngico/metabolismo
, Proteínas de Ligação a DNA/genética
, Proteínas de Ligação a DNA/metabolismo
, Proteínas de Saccharomyces cerevisiae/genética
, Proteínas de Saccharomyces cerevisiae/metabolismo
, Saccharomyces cerevisiae/genética
, Saccharomyces cerevisiae/metabolismo
, Montagem e Desmontagem da Cromatina/genética
, Montagem e Desmontagem da Cromatina/fisiologia
, DNA Fúngico/química
, Nucleossomos/genética
, Nucleossomos/metabolismo
, Termodinâmica
RESUMO
The imitation switch (ISWI) complex from yeast containing the Isw2 and Itc1 proteins was shown to preferentially slide mononucleosomes with as little as 23 bp of linker DNA from the end to the center of DNA. The contacts of unique residues in the histone fold regions of H4, H2B, and H2A with DNA were determined with base pair resolution before and after chromatin remodeling by a site-specific photochemical cross-linking approach. The path of DNA and the conformation of the histone octamer in the nucleosome remodeled or slid by ISW2 were not altered, because after adjustment for the new translational position, the DNA contacts at specific sites in the histone octamer had not been changed. Maintenance of the canonical nucleosome structure after sliding was also demonstrated by DNA photoaffinity labeling of histone proteins at specific sites within the DNA template. In addition, nucleosomal DNA does not become more accessible during ISW2 remodeling, as assayed by restriction endonuclease cutting. ISW2 was also shown to have the novel capability of counteracting transcriptional activators by sliding nucleosomes through Gal4-VP16 bound initially to linker DNA and displacing the activator from DNA.
Assuntos
Adenosina Trifosfatases/metabolismo , DNA/química , Histonas/química , Nucleossomos/química , Fatores de Transcrição/metabolismo , Sequência de Bases , Cromatina/metabolismo , DNA/metabolismo , Relação Dose-Resposta a Droga , Escherichia coli , Histonas/metabolismo , Modelos Genéticos , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Nucleossomos/metabolismo , Plasmídeos/metabolismo , Ligação Proteica , Biossíntese de Proteínas , Conformação Proteica , Saccharomyces cerevisiae/metabolismo , Transativadores/metabolismoRESUMO
Facultative heterochromatin that changes during cellular differentiation coordinates regulated gene expression, but its assembly is poorly understood. Here, we describe facultative heterochromatin islands in fission yeast and show that their formation at meiotic genes requires factors that eliminate meiotic messenger RNAs (mRNAs) during vegetative growth. Blocking production of meiotic mRNA or loss of RNA elimination factors, including Mmi1 and Red1 proteins, abolishes heterochromatin islands. RNA elimination machinery is enriched at meiotic loci and interacts with Clr4/SUV39h, a methyltransferase involved in heterochromatin assembly. Heterochromatin islands disassemble in response to nutritional signals that induce sexual differentiation. This process involves the antisilencing factor Epe1, the loss of which causes dramatic increase in heterochromatic loci. Our analyses uncover unexpected regulatory roles for mRNA-processing factors that assemble dynamic heterochromatin to modulate gene expression.
Assuntos
Montagem e Desmontagem da Cromatina , Heterocromatina/metabolismo , Meiose/genética , RNA Fúngico/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Proteínas de Ciclo Celular/metabolismo , Imunoprecipitação da Cromatina , Complexo Dinactina , Regulação Fúngica da Expressão Gênica , Genes Fúngicos , Histona-Lisina N-Metiltransferase , Histonas/metabolismo , Metiltransferases/metabolismo , Proteínas dos Microtúbulos/genética , Proteínas dos Microtúbulos/metabolismo , Nitrogênio/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Interferência de RNA , RNA Fúngico/genética , RNA Mensageiro/genética , Schizosaccharomyces/genética , Schizosaccharomyces/crescimento & desenvolvimento , Proteínas de Schizosaccharomyces pombe/genética , Fatores de Poliadenilação e Clivagem de mRNA/metabolismoRESUMO
Pervasive transcription of eukaryotic genomes generates a plethora of noncoding RNAs. In fission yeast, the heterochromatin factor Clr4/Suv39 methyltransferase facilitates RNA interference (RNAi)-mediated processing of centromeric transcripts into small interfering RNAs (siRNAs). Clr4 also mediates degradation of antisense RNAs at euchromatic loci, but the underlying mechanism has remained elusive. We show that Clr4 and the RNAi effector RITS (RNA-induced transcriptional silencing) interact with Mlo3, a protein related to mRNA quality control and export factors. Loss of Clr4 impairs RITS interaction with Mlo3, which is required for centromeric siRNA production and antisense suppression. Mlo3 also interacts with the RNA surveillance factor TRAMP, which suppresses antisense RNAs targeted by Clr4 and RNAi. These findings link Clr4 to RNA quality control machinery and suggest a pathway for processing potentially deleterious RNAs through the coordinated actions of RNAi and other RNA processing activities.