RESUMEN
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 transcriptional coactivators Mediator and two histone acetyltransferase (HAT) complexes, NuA4 and SAGA, play global roles in transcriptional activation. Here we explore the relative contributions of these factors to RNA polymerase II association at specific genes and gene classes by rapid nuclear depletion of key complex subunits. We show that the NuA4 HAT Esa1 differentially affects certain groups of genes, whereas the SAGA HAT Gcn5 has a weaker but more uniform effect. Relative dependence on Esa1 and Tra1, a shared component of NuA4 and SAGA, distinguishes two large groups of coregulated growth-promoting genes. In contrast, we show that the activity of Mediator is particularly important at a separate, small set of highly transcribed TATA-box-containing genes. Our analysis indicates that at least three distinct combinations of coactivator deployment are used to generate moderate or high transcription levels and suggests that each may be associated with distinct forms of regulation.
Asunto(s)
Regulación Fúngica de la Expresión Génica , Histona Acetiltransferasas/fisiología , Complejo Mediador/fisiología , Proteínas de Saccharomyces cerevisiae/fisiología , Saccharomyces cerevisiae/genética , Activación Transcripcional , Acetilación , Histonas/metabolismo , Complejo Mediador/metabolismo , Estrés Oxidativo/genética , Regiones Promotoras Genéticas , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteína de Unión a TATA-Box/metabolismo , Transcripción GenéticaRESUMEN
Accessible chromatin is important for RNA polymerase II recruitment and transcription initiation at eukaryotic promoters. We investigated the mechanistic links between promoter DNA sequence, nucleosome positioning, and transcription. Our results indicate that positioning of the transcription start site-associated +1 nucleosome in yeast is critical for efficient TBP binding and is driven by two key factors, the essential chromatin remodeler RSC and a small set of ubiquitous general regulatory factors (GRFs). Our findings indicate that the strength and directionality of RSC action on promoter nucleosomes depends on the arrangement and proximity of two specific DNA motifs. This, together with the effect on nucleosome position observed in double depletion experiments, suggests that, despite their widespread co-localization, RSC and GRFs predominantly act through independent signals to generate accessible chromatin. Our results provide mechanistic insight into how the promoter DNA sequence instructs trans-acting factors to control nucleosome architecture and stimulate transcription initiation.
Asunto(s)
Ensamble y Desensamble de Cromatina , Nucleosomas/metabolismo , ARN Polimerasa II/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Transcripción Genética , Nucleosomas/genética , ARN Polimerasa II/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Improvements in deep sequencing, together with methods to rapidly deplete essential transcription factors (TFs) and chromatin remodelers, have recently led to a more detailed picture of promoter nucleosome architecture in yeast and its relationship to transcriptional regulation. These studies revealed that â¼40% of all budding yeast protein-coding genes possess a unique promoter structure, where we propose that an unusually unstable nucleosome forms immediately upstream of the transcription start site (TSS). This "fragile" nucleosome (FN) promoter architecture relies on the combined action of the essential RSC (Remodels Structure of Chromatin) nucleosome remodeler and pioneer transcription factors (PTFs). FNs are associated with genes whose expression is high, coupled to cell growth, and characterized by low cell-to-cell variability (noise), suggesting that they may promote these features. Recent studies in metazoans suggest that the presence of dynamic nucleosomes upstream of the TSS at highly expressed genes may be conserved throughout evolution.
Asunto(s)
Nucleosomas/genética , Regiones Promotoras Genéticas , Factores de Transcripción/genética , Animales , Ensamble y Desensamble de Cromatina/genética , Fragilidad Cromosómica/genética , Proteínas de Unión al ADN/genética , Genes Fúngicos , Humanos , Modelos Genéticos , Plantas/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Schizosaccharomyces/genéticaRESUMEN
In this issue of Molecular Cell, Chereji et al. (2017) present new data on MNase-sensitive particles previously identified upstream of transcription start sites at many promoters in budding yeast, and they argue, based upon negative histone-ChIP results, that they are non-nucleosomal signals generated by transcription factors (TFs). We show instead, based upon functional experiments where the relevant TFs are rapidly depleted, that this explanation does not hold, and we argue instead that histone ChIP and chemical cleavage assays have a limited capacity to capture these highly dynamic, MNase-sensitive "fragile" nucleosomes.
Asunto(s)
Histonas/genética , Nucleosomas , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Sitio de Iniciación de la TranscripciónRESUMEN
Cell growth potential is determined by the rate of ribosome biogenesis, a complex process that requires massive and coordinated transcriptional output. In the yeast Saccharomyces cerevisiae, ribosome biogenesis is highly regulated at the transcriptional level. Although evidence for a system that coordinates ribosomal RNA (rRNA) and ribosomal protein gene (RPG) transcription has been described, the molecular mechanisms remain poorly understood. Here we show that an interaction between the RPG transcriptional activator Ifh1 and the rRNA processing factor Utp22 serves to coordinate RPG transcription with that of rRNA. We demonstrate that Ifh1 is rapidly released from RPG promoters by a Utp22-independent mechanism following growth inhibition, but that its long-term dissociation requires Utp22. We present evidence that RNA polymerase I activity inhibits the ability of Utp22 to titrate Ifh1 from RPG promoters and propose that a dynamic Ifh1-Utp22 interaction fine-tunes RPG expression to coordinate RPG and rRNA transcription.
Asunto(s)
Regulación Fúngica de la Expresión Génica , ARN Ribosómico/genética , Proteínas Ribosómicas/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Transactivadores/genética , Biogénesis de Organelos , Regiones Promotoras Genéticas , Unión Proteica , ARN Polimerasa I/genética , ARN Polimerasa I/metabolismo , ARN Ribosómico/biosíntesis , Proteínas Ribosómicas/biosíntesis , Proteínas Ribosómicas/metabolismo , Ribosomas/genética , Ribosomas/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transactivadores/metabolismo , Transcripción GenéticaRESUMEN
Previous studies indicate that eukaryotic promoters display a stereotypical chromatin landscape characterized by a well-positioned +1 nucleosome near the transcription start site and an upstream -1 nucleosome that together demarcate a nucleosome-free (or -depleted) region. Here we present evidence that there are two distinct types of promoters distinguished by the resistance of the -1 nucleosome to micrococcal nuclease digestion. These different architectures are characterized by two sequence motifs that are broadly deployed at one set of promoters where a nuclease-sensitive ("fragile") nucleosome forms, but concentrated in a narrower, nucleosome-free region at all other promoters. The RSC nucleosome remodeler acts through the motifs to establish stable +1 and -1 nucleosome positions, while binding of a small set of general regulatory (pioneer) factors at fragile nucleosome promoters plays a key role in their destabilization. We propose that the fragile nucleosome promoter architecture is adapted for regulation of highly expressed, growth-related genes.
Asunto(s)
Regulación Fúngica de la Expresión Génica/fisiología , Nucleosomas/metabolismo , Regiones Promotoras Genéticas/fisiología , Proteínas de Saccharomyces cerevisiae/biosíntesis , Saccharomyces cerevisiae/metabolismo , Nucleosomas/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
In yeast, ribosome production is controlled transcriptionally by tight coregulation of the 138 ribosomal protein genes (RPGs). RPG promoters display limited sequence homology, and the molecular basis for their coregulation remains largely unknown. Here we identify two prevalent RPG promoter types, both characterized by upstream binding of the general transcription factor (TF) Rap1 followed by the RPG-specific Fhl1/Ifh1 pair, with one type also binding the HMG-B protein Hmo1. We show that the regulatory properties of the two promoter types are remarkably similar, suggesting that they are determined to a large extent by Rap1 and the Fhl1/Ifh1 pair. Rapid depletion experiments allowed us to define a hierarchy of TF binding in which Rap1 acts as a pioneer factor required for binding of all other TFs. We also uncovered unexpected features underlying recruitment of Fhl1, whose forkhead DNA-binding domain is not required for binding at most promoters, and Hmo1, whose binding is supported by repeated motifs. Finally, we describe unusually micrococcal nuclease (MNase)-sensitive nucleosomes at all RPG promoters, located between the canonical +1 and -1 nucleosomes, which coincide with sites of Fhl1/Ifh1 and Hmo1 binding. We speculate that these "fragile" nucleosomes play an important role in regulating RPG transcriptional output.
Asunto(s)
Regulación Fúngica de la Expresión Génica , Nucleosomas/metabolismo , Regiones Promotoras Genéticas/genética , Proteínas Ribosómicas/genética , Saccharomyces cerevisiae/genética , Secuencias de Aminoácidos , Unión Proteica , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Complejo Shelterina , Proteínas de Unión a Telómeros/genética , Proteínas de Unión a Telómeros/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
A series of recent studies demonstrated an unexpectedly high frequency of intronic RNA polymerase (pol) III transcription units spread throughout the human genome. The investigation of a subset of these transcripts revealed their tissue/cell-specific transcription together with the involvement in relevant physiopathological pathways. Despite this evidence, these transcripts did not seem to have murine orthologs, based on their nucleotide sequence, resulting in a limitation of the experimental approaches aimed to study their function. In this work, we have extended our investigation to the murine genome identifying 121 pairs of mouse/human transcripts displaying syntenic subchromosomal localization. The analysis in silico of this set of putative noncoding (nc)RNAs suggest their association with alternative splicing as suggested by recent experimental evidence. The investigation of one of these pairs taken as experimental model in mouse hippocampal neurons provided evidence of a human/mouse functional homology that does not depend on underlying sequence conservation. In this light, the collection of transcriptional units here reported can be considered as a novel source for the identification and the study of novel regulatory elements involved in relevant biological processes.