RESUMEN
Here, we present a detailed protocol to study the role of a human nuclear m6A RNA reader, YTHDC1, on chromatin-associated RNA targets. We describe steps for RNA extraction coupled to subnuclear fractionation to identify and study RNA-based regulations that take place in the chromatin-associated fraction. We then detail an RNA immunoprecipitation procedure adapted to identify chromatin-associated RNA targets. This protocol can be adapted to other human or mammalian chromatin-associated RNA binding proteins. For complete details on the use and execution of this protocol, please refer to Timcheva et al.1.
Asunto(s)
Adenina/análogos & derivados , Cromatina , ARN , Animales , Humanos , Cromatina/genética , ARN/genética , ARN Nuclear , ARN Nuclear Pequeño , MamíferosRESUMEN
Heat stress (HS) induces a cellular response leading to profound changes in gene expression. Here, we show that human YTHDC1, a reader of N6-methyladenosine (m6A) RNA modification, mostly associates to the chromatin fraction and that HS induces a redistribution of YTHDC1 across the genome, including to heat-induced heat shock protein (HSP) genes. YTHDC1 binding to m6A-modified HSP transcripts co-transcriptionally promotes expression of HSPs. In parallel, hundreds of the genes enriched in YTHDC1 during HS have their transcripts undergoing YTHDC1- and m6A-dependent intron retention. Later, YTHDC1 concentrates within nuclear stress bodies (nSBs) where it binds to m6A-modified SATIII non-coding RNAs, produced in an HSF1-dependent manner upon HS. These findings reveal that YTHDC1 plays a central role in a chromatin-associated m6A-based reprogramming of gene expression during HS. Furthermore, they support the model where the subsequent and temporary sequestration of YTHDC1 within nSBs calibrates the timing of this YTHDC1-dependent gene expression reprogramming.
Asunto(s)
Cromatina , Respuesta al Choque Térmico , Humanos , Respuesta al Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Expresión Génica , Factores de Empalme de ARN/metabolismo , Proteínas del Tejido Nervioso/metabolismoRESUMEN
To eliminate specific or aberrant transcripts, eukaryotes use nuclear RNA-targeting complexes that deliver them to the exosome for degradation. S. pombe MTREC, and its human counterpart PAXT, are key players in this mechanism but inner workings of these complexes are not understood in sufficient detail. Here, we present an NMR structure of an MTREC scaffold protein Red1 helix-turn-helix domain bound to the Iss10 N-terminus and show this interaction is required for proper cellular growth and meiotic mRNA degradation. We also report a crystal structure of a Red1-Ars2 complex explaining mutually exclusive interactions of hARS2 with various ED/EGEI/L motif-possessing RNA regulators, including hZFC3H1 of PAXT, hFLASH or hNCBP3. Finally, we show that both Red1 and hZFC3H1 homo-dimerize via their coiled-coil regions indicating that MTREC and PAXT likely function as dimers. Our results, combining structures of three Red1 interfaces with in vivo studies, provide mechanistic insights into conserved features of MTREC/PAXT architecture.
Asunto(s)
Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Proteínas Portadoras/metabolismo , Humanos , Meiosis , ARN/metabolismo , Estabilidad del ARN/genética , ARN Mensajero/metabolismo , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismoRESUMEN
In eukaryotes, the heat shock response is orchestrated by a transcription factor named Heat Shock Factor 1 (HSF1). HSF1 is mostly characterized for its role in activating the expression of a repertoire of protein-coding genes, including the heat shock protein (HSP) genes. Remarkably, a growing set of reports indicate that, upon heat shock, HSF1 also targets various non-coding regions of the genome. Focusing primarily on mammals, this review aims at reporting the identity of the non-coding genomic sites directly bound by HSF1, and at describing the molecular function of the long non-coding RNAs (lncRNAs) produced in response to HSF1 binding. The described non-coding genomic targets of HSF1 are pericentric Satellite DNA repeats, (sub)telomeric DNA repeats, Short Interspersed Nuclear Element (SINE) repeats, transcriptionally active enhancers and the NEAT1 gene. This diverse set of non-coding genomic sites, which already appears to be an integral part of the cellular response to stress, may only represent the first of many. Thus, the study of the evolutionary conserved heat stress response has the potential to emerge as a powerful cellular context to study lncRNAs, produced from repeated or unique DNA regions, with a regulatory function that is often well-documented but a mode of action that remains largely unknown.
Asunto(s)
ARN Largo no Codificante , Animales , ADN , Factores de Transcripción del Choque Térmico/genética , Factores de Transcripción del Choque Térmico/metabolismo , Respuesta al Choque Térmico/genética , Mamíferos/genética , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Factores de Transcripción/metabolismoRESUMEN
Taking advantage of the evolutionary conserved nature of ATAD2, we report here a series of parallel functional studies in human, mouse, and Schizosaccharomyces pombe to investigate ATAD2's conserved functions. In S. pombe, the deletion of ATAD2 ortholog, abo1, leads to a dramatic decrease in cell growth, with the appearance of suppressor clones recovering normal growth. The identification of the corresponding suppressor mutations revealed a strong genetic interaction between Abo1 and the histone chaperone HIRA. In human cancer cell lines and in mouse embryonic stem cells, we observed that the KO of ATAD2 leads to an accumulation of HIRA. A ChIP-seq mapping of nucleosome-bound HIRA and FACT in Atad2 KO mouse ES cells demonstrated that both chaperones are trapped on nucleosomes at the transcription start sites of active genes, resulting in the abnormal presence of a chaperone-bound nucleosome on the TSS-associated nucleosome-free regions. Overall, these data highlight an important layer of regulation of chromatin dynamics ensuring the turnover of histone-bound chaperones.
Asunto(s)
ATPasas Asociadas con Actividades Celulares Diversas/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Unión al ADN/metabolismo , Chaperonas de Histonas/metabolismo , Células Madre Embrionarias de Ratones/metabolismo , Nucleosomas/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Transducción de Señal/genética , Factores de Transcripción/metabolismo , ATPasas Asociadas con Actividades Celulares Diversas/genética , Animales , Proliferación Celular/genética , Proteínas de Unión al ADN/genética , Eliminación de Gen , Técnicas de Inactivación de Genes , Genotipo , Células HeLa , Células Hep G2 , Humanos , Ratones , Microorganismos Modificados Genéticamente , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética , TransfecciónRESUMEN
The heat shock factor 1 (HSF1)-dependent transcriptional activation of human pericentric heterochromatin in heat-shocked cells is the most striking example of transcriptional activation of heterochromatin. Until now, pericentric heterochromatin of chromosome 9 has been identified as the primary target of HSF1, in both normal and tumor heat-shocked cells. Transcriptional awakening of this large genomic region results in the nuclear accumulation of satellite III (SATIII) noncoding RNAs (ncRNAs) and the formation in cis of specific structures known as nuclear stress bodies (nSBs). Here, we show that, in four different male cell lines, including primary human fibroblasts and amniocytes, pericentric heterochromatin of chromosome Y can also serve as a unique primary site of HSF1-dependent heterochromatin transcriptional activation, production of SATIII ncRNA, and nucleation of nuclear stress bodies (nSBs) upon heat shock. Our observation suggests that the chromosomal origin of SATIII transcripts in cells submitted to heat shock is not a determinant factor as such, but that transcription of SATIII repetitive units or the SATIII ncRNA molecules is the critical element of HSF1-dependent transcription activation of constitutive heterochromatin.
Asunto(s)
Cromosomas Humanos Y/genética , ADN Satélite/genética , Fibroblastos/metabolismo , Factores de Transcripción del Choque Térmico/metabolismo , Heterocromatina/genética , Factores de Empalme Serina-Arginina/metabolismo , Estrés Fisiológico , Femenino , Fibroblastos/citología , Factores de Transcripción del Choque Térmico/genética , Respuesta al Choque Térmico , Humanos , Masculino , Factores de Empalme Serina-Arginina/genética , Transcripción GenéticaRESUMEN
Long non-coding RNAs (lncRNAs) regulating gene expression at the chromatin level are widespread among eukaryotes. However, their functions and the mechanisms by which they act are not fully understood. Here, we identify new fission yeast regulatory lncRNAs that are targeted, at their site of transcription, by the YTH domain of the RNA-binding protein Mmi1 and degraded by the nuclear exosome. We uncover that one of them, nam1, regulates entry into sexual differentiation. Importantly, we demonstrate that Mmi1 binding to this lncRNA not only triggers its degradation but also mediates its transcription termination, thus preventing lncRNA transcription from invading and repressing the downstream gene encoding a mitogen-activated protein kinase kinase kinase (MAPKKK) essential to sexual differentiation. In addition, we show that Mmi1-mediated termination of lncRNA transcription also takes place at pericentromeric regions where it contributes to heterochromatin gene silencing together with RNA interference (RNAi). These findings reveal an important role for selective termination of lncRNA transcription in both euchromatic and heterochromatic lncRNA-based gene silencing processes.
Asunto(s)
Silenciador del Gen , Heterocromatina/genética , ARN Largo no Codificante/genética , Regulación Fúngica de la Expresión Génica , Schizosaccharomyces/genética , Schizosaccharomyces/fisiología , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismoRESUMEN
The heat shock response is characterized by the transcriptional activation of both hsp genes and noncoding and repeated satellite III DNA sequences located at pericentric heterochromatin. Both events are under the control of Heat Shock Factor I (HSF1). Here we show that under heat shock, HSF1 recruits major cellular acetyltransferases, GCN5, TIP60 and p300 to pericentric heterochromatin leading to a targeted hyperacetylation of pericentric chromatin. Redistribution of histone acetylation toward pericentric region in turn directs the recruitment of Bromodomain and Extra-Terminal (BET) proteins BRD2, BRD3, BRD4, which are required for satellite III transcription by RNAP II. Altogether we uncover here a critical role for HSF1 in stressed cells relying on the restricted use of histone acetylation signaling over pericentric heterochromatin (HC).
Asunto(s)
Respuesta al Choque Térmico , Heterocromatina/genética , Transducción de Señal/genética , Activación Transcripcional , Animales , Células COS , Proteínas de Ciclo Celular , Chlorocebus aethiops , Células HeLa , Factores de Transcripción del Choque Térmico/genética , Factores de Transcripción del Choque Térmico/metabolismo , Heterocromatina/metabolismo , Histona Acetiltransferasas/metabolismo , Histonas/metabolismo , Humanos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , ARN Polimerasa II/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
In response to metabolic or environmental stress, cells activate powerful defense mechanisms to prevent the formation and accumulation of toxic protein aggregates. The main orchestrator of this cellular response is HSF1 (heat shock factor 1), a transcription factor involved in the up-regulation of protein-coding genes with protective roles. It has become very clear that HSF1 has a broader function than initially expected. Indeed, our previous work demonstrated that, upon stress, HSF1 activates the transcription of a non-coding RNA, named Satellite III, at pericentromeric heterochromatin. Here, we observe that the function of HSF1 extends to telomeres and identify subtelomeric DNA as a new genomic target of HSF1. We show that the binding of HSF1 to subtelomeric regions plays an essential role in the upregulation of non-coding TElomeric Repeat containing RNA (TERRA) transcription upon heat shock. Importantly, our data show that telomere integrity is impacted by heat shock and that telomeric DNA damages are markedly enhanced in HSF1 deficient cells. Altogether, our findings reveal a new direct and essential function of HSF1 in the transcriptional activation of TERRA and in telomere protection upon stress.
Asunto(s)
Factores de Transcripción del Choque Térmico/fisiología , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Telómero/metabolismo , Células HeLa , Respuesta al Choque Térmico , Humanos , Estabilidad del ARN , Homeostasis del Telómero , Transcripción Genética , Activación TranscripcionalRESUMEN
ATAD2, a remarkably conserved, yet poorly characterized factor is found upregulated and associated with poor prognosis in a variety of independent cancers in human. Studies conducted on the yeast Saccharomyces cerevisiae ATAD2 homologue, Yta7, are now indicating that the members of this family may primarily be regulators of chromatin dynamics and that their action on gene expression could only be one facet of their general activity. In this review, we present an overview of the literature on Yta7 and discuss the possibility of translating these findings into other organisms to further define the involvement of ATAD2 and other members of its family in regulating chromatin structure and function both in normal and pathological situations.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Cromatina/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , ATPasas Asociadas con Actividades Celulares Diversas , Regulación de la Expresión Génica , Genoma Fúngico , Genoma Humano , Chaperonas de Histonas/metabolismo , Humanos , Filogenia , Saccharomyces cerevisiae/genética , Activación TranscripcionalRESUMEN
Germ cell differentiation, the cellular process by which a diploid progenitor cell produces by meiotic divisions haploid cells, is conserved from the unicellular yeasts to mammals. Over the recent years, yeast germ cell differentiation process has proven to be a powerful biological system to identify and study several long noncoding RNAs (lncRNAs) that play a central role in regulating cellular differentiation by acting directly on chromatin. Remarkably, in the well-studied budding yeast Saccharomyces cerevisiae and fission yeast Schizosaccharomyces pombe, the lncRNA-based chromatin regulations of germ cell differentiation are quite different. In this review, we present an overview of these regulations by focusing on the mechanisms and their respective functions both in S. cerevisiae and in S. pombe. Part of these lncRNA-based chromatin regulations may be conserved in other eukaryotes and play critical roles either in the context of germ cell differentiation or, more generally, in the development of multicellular organisms.
Asunto(s)
Diferenciación Celular/genética , Cromatina/genética , Células Germinativas/crecimiento & desarrollo , ARN Largo no Codificante/genética , Regulación del Desarrollo de la Expresión Génica , Silenciador del Gen , Células Germinativas/citología , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Schizosaccharomyces/genética , Schizosaccharomyces/crecimiento & desarrollo , Esporas Fúngicas/genética , Esporas Fúngicas/crecimiento & desarrolloRESUMEN
Regularly positioned nucleosomes are a common feature of 5' ends of most eukaryotic genes. A series of three studies, Shim et al (2012) and Pointner et al (2012) in this issue of The EMBO Journal and Hennig et al (2012) in EMBO Reports, now show that in the fission yeast Schizosaccharomyces pombe this intragenic nucleosome positioning mostly requires two ATP-dependent remodellers of the CHD family, Hrp1 and Hrp3. Moreover, they suggest that Hrp1- and Hrp3-dependent nucleosome spacing contributes to the silencing of cryptic antisense transcription.
Asunto(s)
Adenosina Trifosfatasas/fisiología , Adenosina Trifosfato/química , ADN Helicasas/fisiología , Proteínas de Unión al ADN/fisiología , Eucromatina/química , Regulación Fúngica de la Expresión Génica , Heterocromatina/química , Nucleosomas/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiología , Saccharomyces cerevisiae/metabolismo , Proteínas de Schizosaccharomyces pombe/fisiología , Schizosaccharomyces/metabolismoRESUMEN
The selective elimination system blocks the accumulation of meiosis-specific mRNAs during the mitotic cell cycle in fission yeast. These mRNAs harbour a region, the determinant of selective removal (DSR), which is recognized by a YTH-family RNA-binding protein, Mmi1. Mmi1 directs target transcripts to destruction in association with nuclear exosomes. Hence, the interaction between DSR and Mmi1 is crucial to discriminate mitosis from meiosis. Here, we show that Mmi1 interacts with repeats of the hexanucleotide U(U/C)AAAC that are enriched in the DSR. Disruption of this 'DSR core motif' in a target mRNA inhibits its elimination. Tandem repeats of the motif can function as an artificial DSR. Mmi1 binds to it in vitro. Thus, a core motif cluster is responsible for the DSR activity. Furthermore, certain variant hexanucleotide motifs can augment the function of the DSR core motif. Notably, meiRNA, which composes the nuclear Mei2 dot required to suppress Mmi1 activity during meiosis, carries numerous copies of the core/augmenting motifs on its tail and is indeed degraded by the Mmi1/exosome system, indicating its likely role as decoy bait for Mmi1.
Asunto(s)
ARN de Hongos/genética , ARN de Hongos/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Secuencia de Bases , Exosomas/metabolismo , Silenciador del Gen , Genes Fúngicos , Meiosis/genética , Mutagénesis , ARN de Hongos/química , Schizosaccharomyces/citología , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Secuencias Repetidas en Tándem , Factores de Escisión y Poliadenilación de ARNm/genética , Factores de Escisión y Poliadenilación de ARNm/metabolismoRESUMEN
RNA interference (RNAi) silences gene expression by acting both at the transcriptional and post-transcriptional levels in a broad range of eukaryotes. In the fission yeast Schizosaccharomyces pombe the RNA-Induced Transcriptional Silencing (RITS) RNAi complex mediates heterochromatin formation at non-coding and repetitive DNA. However, the targeting and role of RITS at other genomic regions, including protein-coding genes, remain unknown. Here we show that RITS localizes to specific meiotic genes and mRNAs. Remarkably, RITS is guided to these meiotic targets by the RNA-binding protein Mmi1 and its associated RNA surveillance machinery that together degrade selective meiotic mRNAs during vegetative growth. Upon sexual differentiation, RITS localization to the meiotic genes and mRNAs is lost. Large-scale identification of Mmi1 RNA targets reveals that RITS subunit Chp1 associates with the vast majority of them. In addition, loss of RNAi affects the effective repression of sexual differentiation mediated by the Mmi1 RNA surveillance machinery. These findings uncover a new mechanism for recruiting RNAi to specific meiotic genes and suggest that RNAi participates in the control of sexual differentiation in fission yeast.
Asunto(s)
Regulación Fúngica de la Expresión Génica , Genes Fúngicos , Complejo Silenciador Inducido por ARN/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Factores de Escisión y Poliadenilación de ARNm/metabolismo , Modelos Biológicos , Unión Proteica , ARN de Hongos/metabolismoRESUMEN
Small interfering RNAs (siRNAs) act through RNA interference (RNAi) pathways to silence gene expression either at the transcriptional or post-transcriptional level. Here, we review mechanisms and functions of siRNA-mediated silencing pathways that promote chromatin modifications in the fission yeast Schizosaccharomyces pombe, plants and animals. In fission yeast, siRNAs are involved in heterochromatin formation and key aspects of the underlying siRNA-dependent pathway have been uncovered. Two RNAi complexes, the RNA-Induced Transcriptional Silencing complex (RITS), which contains a siRNA bound to an Argonaute protein, and the RNA-Directed RNA polymerase Complex (RDRC) are critical components of the pathway. In addition, this pathway implicates non-coding nascent transcripts synthesized by RNA polymerase II (RNApII) and the RNApII itself. In Arabidopsis thaliana, the RNA-directed DNA methylation (RdDM) pathway appears to share a similar set of proteins and enzymatic activities, suggesting that, beyond certain aspects that are specific to each pathway, part of the siRNA-mediated epigenetic silencing mechanisms are conserved between fission yeast and plants. Moreover, in both organisms the pathways target repetitive DNA sequences. This conservation of mechanisms and genomic targets might actually extend to animals as recent investigations revealed the existence of endogenous siRNA-based pathways directed against repetitive DNA sequences in flies and mammals.
Asunto(s)
Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Complejo Silenciador Inducido por ARN/metabolismo , Animales , Cromatina/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Modelos Biológicos , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , ARN Interferente Pequeño/genética , Complejo Silenciador Inducido por ARN/genética , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Transducción de SeñalRESUMEN
LSD1 represses and activates transcription by demethylating histone H3K4me and H3K9me, respectively. Genetic ablation of the S. pombe homologs, splsd1 and splsd2, resulted in slow growth and lethality, respectively, underscoring their physiological importance. spLsd1 and spLsd2 form a stable protein complex, which exhibits demethylase activity toward methylated H3K9 in vitro. Both proteins were associated with the heterochromatin boundary regions and euchromatic gene promoters. Loss of spLsd1 resulted in increased H3K9 methylation accompanied by reduced euchromatic gene transcription and heterochromatin propagation. Removal of the H3K9 methylase Clr4 partially suppressed the slow growth phenotype of splsd1Delta. Conversely, catalytically inactivating point mutations in the splsd1 and splsd2 genes partially mimicked the growth and heterochromatin propagation phenotypes. Taken together, these findings suggest the importance of both enzymatic and nonenzymatic roles of spLsd1 in regulating heterochromatin propagation and euchromatic transcription and also suggest that misregulation of spLsd1/2 is likely to impact the epigenetic state of the cell.
Asunto(s)
Eucromatina/genética , Regulación Fúngica de la Expresión Génica , Genoma Fúngico , Heterocromatina/metabolismo , Oxidorreductasas N-Desmetilantes/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/genética , Transcripción Genética , Secuencia de Aminoácidos , Supervivencia Celular , Metilación de ADN , Heterocromatina/genética , Heterocromatina/fisiología , Histonas/metabolismo , Datos de Secuencia Molecular , Oxidorreductasas N-Desmetilantes/genética , Oxidorreductasas N-Desmetilantes/fisiología , Schizosaccharomyces/enzimología , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/fisiología , Homología de Secuencia de AminoácidoRESUMEN
In the fission yeast Schizosaccharomyces pombe, formation of pericentromeric heterochromatin involves RNA interference (RNAi). Recent data indicate that two RNAi complexes, RITS (RNA-induced transcriptional silencing complex) and RDRC (RNA-directed RNA polymerase complex), their respective enzymatic activity, and RNA polymerase II are essential for RNAi-mediated heterochromatin formation. At the site where heterochromatin formation takes place, RNA polymerase II synthesizes an RNA that would serve as an RNA platform to recruit in a siRNA-dependent manner RITS and RDRC, and thereby initiate heterochromatin assembly. Once recruited, RITS and RDRC seem to also contribute to the processing of the RNA platform. Therefore, RNAi-driven heterochromatin assembly appears to take place through a dynamic process of RNA synthesis, RNA-dependant recruitment of RNAi complexes and RNA degradation that all occur in cis.
Asunto(s)
Regulación Fúngica de la Expresión Génica/genética , Heterocromatina/genética , Interferencia de ARN , ARN de Hongos/genética , Schizosaccharomyces/genética , Núcleo Celular/metabolismo , Endorribonucleasas/fisiología , Modelos Genéticos , Nucleosomas/ultraestructura , ARN Polimerasa II/genética , Estabilidad del ARN , ARN Bicatenario/biosíntesis , ARN Bicatenario/genética , ARN de Hongos/biosíntesis , ARN Polimerasa Dependiente del ARN/genética , Complejo Silenciador Inducido por ARN/genética , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/fisiologíaRESUMEN
In the fission yeast Schizosaccharomyces pombe, the RNA-Induced Transcriptional Silencing (RITS) complex has been proposed to target the chromosome via siRNA-dependent base-pairing interactions to initiate heterochromatin formation. Here we show that tethering of the RITS subunit, Tas3, to the RNA transcript of the normally active ura4+ gene silences ura4+ expression. This silencing depends on a functional RNAi pathway, requires the heterochromatin proteins, Swi6/HP1, Clr4/Suv39h, and Sir2, and is accompanied by the generation of ura4+ siRNAs, histone H3-lysine 9 methylation, and Swi6 binding. Furthermore, the ability of the newly generated ura4+ siRNAs to silence a second ura4+ allele in trans is strongly inhibited by the conserved siRNA nuclease, Eri1. Surprisingly, silencing of tethered ura4+, or ura4+ inserted within centromeric heterochromatin, or some of the endogenous centromeric repeat promoters, is not associated with changes in RNA polymerase II occupancy. These findings support a model in which targeting of nascent transcripts by RITS mediates chromatin modifications and suggest that cotranscriptional processing events play a primary role in the silencing mechanism.
Asunto(s)
Heterocromatina/genética , Interferencia de ARN/fisiología , Precursores del ARN/genética , ARN Interferente Pequeño/genética , Complejo Silenciador Inducido por ARN/genética , Schizosaccharomyces/genética , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Metilación de ADN , Exorribonucleasas/genética , Exorribonucleasas/metabolismo , Regulación Fúngica de la Expresión Génica/genética , N-Metiltransferasa de Histona-Lisina , Histonas/metabolismo , Metiltransferasas/genética , Metiltransferasas/metabolismo , Proteínas de Unión al ARN/genética , Elementos Reguladores de la Transcripción/genética , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Sirtuinas/genética , Sirtuinas/metabolismoRESUMEN
HP1 is well known as a key silencing protein. However, in the June 9 issue of Molecular Cell, report that Swi6/HP1 recruits an antisilencing protein, Epe1, to facilitate transcription, leading to a model in which Swi6/HP1 is used as a platform to recruit both silencing and antisilencing activities.
Asunto(s)
Proteínas Cromosómicas no Histona/metabolismo , Silenciador del Gen/fisiología , Heterocromatina/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Transcripción Genética/genética , Animales , Homólogo de la Proteína Chromobox 5 , Proteínas Cromosómicas no Histona/genética , Heterocromatina/genética , Humanos , Proteínas Nucleares/genética , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genéticaRESUMEN
Heterochromatin is an epigenetically heritable and conserved feature of eukaryotic chromosomes with important roles in chromosome segregation, genome stability, and gene regulation. The formation of heterochromatin involves an ordered array of chromatin changes, including histone deacetylation, histone H3-lysine 9 methylation, and recruitment of histone binding proteins such as Swi6/HP1. Recent discoveries have uncovered a role for the RNA interference (RNAi) pathway in heterochromatin assembly in the fission yeast Schizosaccharomyces pombe and other eukaryotes. Purification of two RNAi complexes, RITS and RDRC, from fission yeast has provided further insight into the mechanism of RNAi-mediated heterochromatin assembly. These discoveries have given rise to a model in which small interfering RNA molecules act as specificity factors that initiate epigenetic chromatin modifications and double strand RNA synthesis at specific chromosome regions.