RESUMEN
Heterochromatic gene silencing relies on combinatorial control by specific histone modifications, the occurrence of transcription, and/or RNA degradation. Once nucleated, heterochromatin propagates within defined chromosomal regions and is maintained throughout cell divisions to warrant proper genome expression and integrity. In the fission yeast Schizosaccharomyces pombe, the Ccr4-Not complex partakes in gene silencing, but its relative contribution to distinct heterochromatin domains and its role in nucleation versus spreading have remained elusive. Here, we unveil major functions for Ccr4-Not in silencing and heterochromatin spreading at the mating type locus and subtelomeres. Mutations of the catalytic subunits Caf1 or Mot2, involved in RNA deadenylation and protein ubiquitinylation, respectively, result in impaired propagation of H3K9me3 and massive accumulation of nucleation-distal heterochromatic transcripts. Both silencing and spreading defects are suppressed upon disruption of the heterochromatin antagonizing factor Epe1. Overall, our results position the Ccr4-Not complex as a critical, dual regulator of heterochromatic gene silencing and spreading.
Asunto(s)
Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Heterocromatina/genética , Heterocromatina/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Histonas/genética , Histonas/metabolismo , Silenciador del GenRESUMEN
Cellular homeostasis is maintained by surveillance mechanisms that intervene at virtually every step of gene expression. In the nucleus, the yeast chromatin remodeler Isw1 holds back maturing mRNA ribonucleoparticles to prevent their untimely export, but whether this activity operates beyond quality control of mRNA biogenesis to regulate gene expression is unknown. Here, we identify the mRNA encoding the central effector of the unfolded protein response (UPR) HAC1, as an Isw1 RNA target. The direct binding of Isw1 to the 3' untranslated region of HAC1 mRNA restricts its nuclear export and is required for accurate UPR abatement. Accordingly, ISW1 inactivation sensitizes cells to endoplasmic reticulum (ER) stress while its overexpression reduces UPR induction. Our results reveal an unsuspected mechanism, in which binding of ER-stress induced Isw1 to HAC1 mRNA limits its nuclear export, providing a feedback loop that fine-tunes UPR attenuation to guarantee homeostatic adaptation to ER stress.
Asunto(s)
Proteínas de Saccharomyces cerevisiae , Regiones no Traducidas 3' , Adenosina Trifosfatasas/metabolismo , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Cromatina/metabolismo , Proteínas de Unión al ADN/metabolismo , Retículo Endoplásmico/genética , Retículo Endoplásmico/metabolismo , Proteínas Represoras/metabolismo , Empalme del ARN , ARN Mensajero/genética , ARN Mensajero/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Respuesta de Proteína Desplegada/genética , Estrés del Retículo EndoplásmicoRESUMEN
Cellular homeostasis requires the coordination of several machineries concurrently engaged in the DNA. Wide-spread transcription can interfere with other processes, and transcription-replication conflicts (TRCs) threaten genome stability. The conserved Sen1 helicase not only terminates non-coding transcription but also interacts with the replisome and reportedly resolves genotoxic R-loops. Sen1 prevents genomic instability, but how this relates to its molecular functions remains unclear. We generated high-resolution, genome-wide maps of transcription-dependent conflicts and R-loops using a Sen1 mutant that has lost interaction with the replisome but is termination proficient. We show that, under physiological conditions, Sen1 removes RNA polymerase II at TRCs within genes and the rDNA and at sites of transcription-transcription conflicts, thus qualifying as a "key regulator of conflicts." We demonstrate that genomic stability is affected by Sen1 mutation only when in addition to its role at the replisome, the termination of non-coding transcription or R-loop removal are additionally compromised.
Asunto(s)
Proteínas de Saccharomyces cerevisiae , ADN Helicasas/genética , ADN Helicasas/metabolismo , Replicación del ADN/genética , Inestabilidad Genómica , ARN Helicasas/genética , ARN Helicasas/metabolismo , ARN Polimerasa II/genética , ARN Polimerasa II/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Transcripción GenéticaRESUMEN
Detecting protein-RNA interactions in vivo is essential for deciphering many important cellular pathways. Several methods have been described for this purpose, among which cross-linking analysis of cDNA, CRAC. This method relies on a first step of UV cross-linking of living yeast cells and several subsequent steps of purification of the protein-RNA complexes, some of which under denaturing condition. Without altering the general principle of the method, we have modified and improved the protocol, with the specific aim of sequencing the nascent RNA isolated from transcription complexes and generate high-resolution and directional transcription maps.
Asunto(s)
Nucleótidos , ARN , Reactivos de Enlaces Cruzados/metabolismo , ADN Complementario/genética , ADN Complementario/metabolismo , ARN/metabolismoRESUMEN
mRNA homoeostasis is favoured by crosstalk between transcription and degradation machineries. Both the Ccr4-Not and the Xrn1-decaysome complexes have been described to influence transcription. While Ccr4-Not has been shown to directly stimulate transcription elongation, the information available on how Xrn1 influences transcription is scarce and contradictory. In this study we have addressed this issue by mapping RNA polymerase II (RNA pol II) at high resolution, using CRAC and BioGRO-seq techniques in Saccharomyces cerevisiae. We found significant effects of Xrn1 perturbation on RNA pol II profiles across the genome. RNA pol II profiles at 5' exhibited significant alterations that were compatible with decreased elongation rates in the absence of Xrn1. Nucleosome mapping detected altered chromatin configuration in the gene bodies. We also detected accumulation of RNA pol II shortly upstream of polyadenylation sites by CRAC, although not by BioGRO-seq, suggesting higher frequency of backtracking before pre-mRNA cleavage. This phenomenon was particularly linked to genes with poorly positioned nucleosomes at this position. Accumulation of RNA pol II at 3' was also detected in other mRNA decay mutants. According to these and other pieces of evidence, Xrn1 seems to influence transcription elongation at least in two ways: by directly favouring elongation rates and by a more general mechanism that connects mRNA decay to late elongation.
Asunto(s)
Cromatina/metabolismo , Exorribonucleasas/metabolismo , ARN Polimerasa II/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Elongación de la Transcripción Genética , Factores de Elongación Transcripcional/metabolismo , Cromatina/química , Cromatina/genética , Exorribonucleasas/genética , Regulación Fúngica de la Expresión Génica , Nucleosomas/genética , Nucleosomas/metabolismo , ARN Polimerasa II/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Factores de Elongación Transcripcional/genéticaRESUMEN
Precise nucleosome organization at eukaryotic promoters is thought to be generated by multiple chromatin remodeler (CR) enzymes and to affect transcription initiation. Using an integrated analysis of chromatin remodeler binding and nucleosome occupancy following rapid remodeler depletion, we investigated the interplay between these enzymes and their impact on transcription in yeast. We show that many promoters are affected by multiple CRs that operate in concert or in opposition to position the key transcription start site (TSS)-associated +1 nucleosome. We also show that nucleosome movement after CR inactivation usually results from the activity of another CR and that in the absence of any remodeling activity, +1 nucleosomes largely maintain their positions. Finally, we present functional assays suggesting that +1 nucleosome positioning often reflects a trade-off between maximizing RNA polymerase recruitment and minimizing transcription initiation at incorrect sites. Our results provide a detailed picture of fundamental mechanisms linking promoter nucleosome architecture to transcription initiation.
Asunto(s)
Ensamble y Desensamble de Cromatina/fisiología , Saccharomyces cerevisiae/genética , Sitio de Iniciación de la Transcripción , Iniciación de la Transcripción Genética/fisiología , Ensamble y Desensamble de Cromatina/genética , ADN de Hongos/genética , ADN Intergénico/genética , ADN Intergénico/metabolismo , Sustancias Macromoleculares/metabolismo , Nucleasa Microcócica/metabolismo , Nucleosomas/metabolismo , Saccharomyces cerevisiae/enzimología , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
The fidelity of transcription initiation is essential for accurate gene expression, but the determinants of start site selection are not fully understood. Rap1 and other general regulatory factors (GRFs) control the expression of many genes in yeast. We show that depletion of these factors induces widespread ectopic transcription initiation within promoters. This generates many novel non-coding RNAs and transcript isoforms with diverse stability, drastically altering the coding potential of the transcriptome. Ectopic transcription initiation strongly correlates with altered nucleosome positioning. We provide evidence that Rap1 can suppress ectopic initiation by a "place-holder" mechanism whereby it physically occludes inappropriate sites for pre-initiation complex formation. These results reveal an essential role for GRFs in the fidelity of transcription initiation and in the suppression of pervasive transcription, profoundly redefining current models for their function. They have important implications for the mechanism of transcription initiation and the control of gene expression.
Asunto(s)
Regulación Fúngica de la Expresión Génica , ARN de Hongos/biosíntesis , ARN Mensajero/biosíntesis , ARN no Traducido/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Unión a Telómeros/metabolismo , Factores de Transcripción/metabolismo , Transcripción Genética , Sitios de Unión , Ensamble y Desensamble de Cromatina , Nucleosomas/genética , Nucleosomas/metabolismo , Regiones Promotoras Genéticas , Unión Proteica , ARN de Hongos/genética , ARN Mensajero/genética , ARN no Traducido/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Complejo Shelterina , Proteínas de Unión a Telómeros/genética , Factores de Transcripción/genética , Sitio de Iniciación de la Transcripción , Iniciación de la Transcripción GenéticaRESUMEN
Transcription termination delimits transcription units but also plays important roles in limiting pervasive transcription. We have previously shown that transcription termination occurs when elongating RNA polymerase II (RNAPII) collides with the DNA-bound general transcription factor Reb1. We demonstrate here that many different DNA-binding proteins can induce termination by a similar roadblock (RB) mechanism. We generated high-resolution transcription maps by the direct detection of RNAPII upon nuclear depletion of two essential RB factors or when the canonical termination pathways for coding and non-coding RNAs are defective. We show that RB termination occurs genomewide and functions independently of (and redundantly with) the main transcription termination pathways. We provide evidence that transcriptional readthrough at canonical terminators is a significant source of pervasive transcription, which is controlled to a large extent by RB termination. Finally, we demonstrate the occurrence of RB termination around centromeres and tRNA genes, which we suggest shields these regions from RNAPII to preserve their functional integrity.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , ARN Polimerasa II/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Terminación de la Transcripción Genética , Transcripción Genética , Proteínas de Unión al ADN/genética , Genoma Fúngico , ARN Polimerasa II/genética , ARN de Hongos , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
The Set1 family of histone H3 lysine 4 (H3K4) methyltransferases is highly conserved from yeast to human. Here we show that the Set1 complex (Set1C) directly binds RNA in vitro through the regions that comprise the double RNA recognition motifs (dRRM) and N-SET domain within Set1 and its subunit Spp1. To investigate the functional relevance of RNA binding, we performed UV RNA crosslinking (CRAC) for Set1 and RNA polymerase II in parallel with ChIP-seq experiments. Set1 binds nascent transcripts through its dRRM. RNA binding is important to define the appropriate topology of Set1C distribution along transcription units and correlates with the efficient deposition of the H3K4me3 mark. In addition, we uncovered that Set1 binds to different classes of RNAs to levels that largely exceed the levels of binding to the general population of transcripts, suggesting the Set1 persists on these RNAs after transcription. This class includes RNAs derived from SET1, Ty1 retrotransposons, specific transcription factors genes and snRNAs (small nuclear RNAs). We propose that Set1 modulates adaptive responses, as exemplified by the post-transcriptional inhibition of Ty1 retrotransposition.
RESUMEN
Chromatin dynamics play an essential role in regulating DNA transaction processes, but it is unclear whether transcription-associated chromatin modifications control the mRNA ribonucleoparticles (mRNPs) pipeline from synthesis to nuclear exit. Here, we identify the yeast ISW1 chromatin remodeling complex as an unanticipated mRNP nuclear export surveillance factor that retains export-incompetent transcripts near their transcription site. This tethering activity of ISW1 requires chromatin binding and is independent of nucleosome sliding activity or changes in RNA polymerase II processivity. Combination of in vivo UV-crosslinking and genome-wide RNA immunoprecipitation assays show that Isw1 and its cofactors interact directly with premature mRNPs. Our results highlight that the concerted action of Isw1 and the nuclear exosome ensures accurate surveillance mechanism that proofreads the efficiency of mRNA biogenesis.