RESUMEN
Scientists have long been fascinated by the complexity of eukaryotic transcription and the large numbers of proteins involved at each step in the process. In this issue of Cell, Schilbach et al. bring us one important step closer to the goal of a complete understanding of transcription at atomic resolution.
Asunto(s)
Eucariontes , Células EucariotasRESUMEN
Transcription elongation is a key regulatory step in gene expression that is controlled by many proteins, and mechanisms, ranging from RNA Polymerase II pausing to cotranscriptional histone modifications.
Asunto(s)
Eucariontes/metabolismo , Elongación de la Transcripción Genética , Animales , Ensamble y Desensamble de Cromatina , Histonas/metabolismo , Humanos , ARN Polimerasa II/metabolismoRESUMEN
The RNA polymerase II transcription cycle is often divided into three major stages: initiation, elongation, and termination. Research over the last decade has blurred these divisions and emphasized the tightly regulated transitions that occur as RNA polymerase II synthesizes a transcript from start to finish. Transcription termination, the process that marks the end of transcription elongation, is regulated by proteins that interact with the polymerase, nascent transcript, and/or chromatin template. The failure to terminate transcription can cause accumulation of aberrant transcripts and interfere with transcription at downstream genes. Here, we review the mechanism, regulation, and physiological impact of a termination pathway that targets small noncoding transcripts produced by RNA polymerase II. We emphasize the Nrd1-Nab3-Sen1 pathway in yeast, in which the process has been extensively studied. The importance of understanding small RNA termination pathways is underscored by the need to control noncoding transcription in eukaryotic genomes.
Asunto(s)
ARN Polimerasa II/metabolismo , ARN Pequeño no Traducido/metabolismo , Terminación de la Transcripción Genética , Animales , Fenómenos Fisiológicos Celulares , Humanos , Procesamiento Postranscripcional del ARN , Saccharomyces cerevisiae/genéticaRESUMEN
Histone modifications coupled to transcription elongation play important roles in regulating the accuracy and efficiency of gene expression. The monoubiquitylation of a conserved lysine in H2B (K123 in Saccharomyces cerevisiae; K120 in humans) occurs cotranscriptionally and is required for initiating a histone modification cascade on active genes. H2BK123 ubiquitylation (H2BK123ub) requires the RNA polymerase II (RNAPII)-associated Paf1 transcription elongation complex (Paf1C). Through its histone modification domain (HMD), the Rtf1 subunit of Paf1C directly interacts with the ubiquitin conjugase Rad6, leading to the stimulation of H2BK123ub in vivo and in vitro. To understand the molecular mechanisms that target Rad6 to its histone substrate, we identified the site of interaction for the HMD on Rad6. Using in vitro cross-linking followed by mass spectrometry, we localized the primary contact surface for the HMD to the highly conserved N-terminal helix of Rad6. Using a combination of genetic, biochemical, and in vivo protein cross-linking experiments, we characterized separation-of-function mutations in S. cerevisiae RAD6 that greatly impair the Rad6-HMD interaction and H2BK123 ubiquitylation but not other Rad6 functions. By employing RNA-sequencing as a sensitive approach for comparing mutant phenotypes, we show that mutating either side of the proposed Rad6-HMD interface yields strikingly similar transcriptome profiles that extensively overlap with those of a mutant that lacks the site of ubiquitylation in H2B. Our results fit a model in which a specific interface between a transcription elongation factor and a ubiquitin conjugase guides substrate selection toward a highly conserved chromatin target during active gene expression.
Asunto(s)
Histonas , Proteínas Nucleares , Proteínas de Saccharomyces cerevisiae , Proteína de Unión a TATA-Box , Enzimas Ubiquitina-Conjugadoras , gamma-Glutamil Hidrolasa , Histonas/metabolismo , Proteínas Nucleares/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Ubiquitina/metabolismo , Enzimas Ubiquitina-Conjugadoras/genética , Enzimas Ubiquitina-Conjugadoras/metabolismo , Ubiquitinación , Proteína de Unión a TATA-Box/genética , Proteína de Unión a TATA-Box/metabolismoRESUMEN
The Paf1 complex (Paf1C) is a conserved transcription elongation factor that regulates transcription elongation efficiency, facilitates co-transcriptional histone modifications, and impacts molecular processes linked to RNA synthesis, such as polyA site selection. Coupling of the activities of Paf1C to transcription elongation requires its association with RNA polymerase II (Pol II). Mutational studies in yeast identified Paf1C subunits Cdc73 and Rtf1 as important mediators of Paf1C association with Pol II on active genes. While the interaction between Rtf1 and the general elongation factor Spt5 is relatively well-understood, the interactions involving Cdc73 have not been fully elucidated. Using a site-specific protein cross-linking strategy in yeast cells, we identified direct interactions between Cdc73 and two components of the Pol II elongation complex, the elongation factor Spt6 and the largest subunit of Pol II. Both of these interactions require the tandem SH2 domain of Spt6. We also show that Cdc73 and Spt6 can interact in vitro and that rapid depletion of Spt6 dissociates Paf1 from chromatin, altering patterns of Paf1C-dependent histone modifications genome-wide. These results reveal interactions between Cdc73 and the Pol II elongation complex and identify Spt6 as a key factor contributing to the occupancy of Paf1C at active genes in Saccharomyces cerevisiae.
Asunto(s)
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Proteínas Nucleares/metabolismo , Factores de Elongación de Péptidos/metabolismo , ARN Polimerasa II/genética , ARN Polimerasa II/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/metabolismo , Transcripción Genética , Factores de Elongación Transcripcional/genética , Factores de Elongación Transcripcional/metabolismoRESUMEN
The five-subunit yeast Paf1 complex (Paf1C) regulates all stages of transcription and is critical for the monoubiquitylation of histone H2B (H2Bub), a modification that broadly influences chromatin structure and eukaryotic transcription. Here, we show that the histone modification domain (HMD) of Paf1C subunit Rtf1 directly interacts with the ubiquitin conjugase Rad6 and stimulates H2Bub independently of transcription. We present the crystal structure of the Rtf1 HMD and use site-specific, in vivo crosslinking to identify a conserved Rad6 interaction surface. Utilizing ChIP-exo analysis, we define the localization patterns of the H2Bub machinery at high resolution and demonstrate the importance of Paf1C in targeting the Rtf1 HMD, and thereby H2Bub, to its appropriate genomic locations. Finally, we observe HMD-dependent stimulation of H2Bub in a transcription-free, reconstituted in vitro system. Taken together, our results argue for an active role for Paf1C in promoting H2Bub and ensuring its proper localization in vivo.
Asunto(s)
Regulación Fúngica de la Expresión Génica , Histonas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteína de Unión a TATA-Box/metabolismo , Enzimas Ubiquitina-Conjugadoras/metabolismo , Secuencias de Aminoácidos , Sitios de Unión , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Reactivos de Enlaces Cruzados/química , Cristalografía por Rayos X , Formaldehído/química , Histonas/química , Histonas/genética , Modelos Moleculares , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Dominios y Motivos de Interacción de Proteínas , ARN Polimerasa II/genética , ARN Polimerasa II/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteína de Unión a TATA-Box/química , Proteína de Unión a TATA-Box/genética , Transcripción Genética , Factores de Elongación Transcripcional/genética , Factores de Elongación Transcripcional/metabolismo , Enzimas Ubiquitina-Conjugadoras/química , Enzimas Ubiquitina-Conjugadoras/genética , UbiquitinaciónRESUMEN
The conserved, multifunctional Polymerase-Associated Factor 1 complex (Paf1C) regulates all stages of the RNA polymerase (Pol) II transcription cycle. In this review, we examine a diverse set of recent studies from various organisms that build on foundational studies in budding yeast. These studies identify new roles for Paf1C in the control of gene expression and the regulation of chromatin structure. In exploring these advances, we find that various functions of Paf1C, such as the regulation of promoter-proximal pausing and development in higher eukaryotes, are complex and context dependent. As more becomes known about the role of Paf1C in human disease, interest in the molecular mechanisms underpinning Paf1C function will continue to increase.
Asunto(s)
Regulación de la Expresión Génica , Proteínas Nucleares/metabolismo , Humanos , Factores de TranscripciónRESUMEN
The nucleosome core regulates DNA-templated processes through the highly conserved nucleosome acidic patch. While structural and biochemical studies have shown that the acidic patch controls chromatin factor binding and activity, few studies have elucidated its functions in vivo. We employed site-specific crosslinking to identify proteins that directly bind the acidic patch in Saccharomyces cerevisiae and demonstrated crosslinking of histone H2A to Paf1 complex subunit Rtf1 and FACT subunit Spt16. Rtf1 bound to nucleosomes through its histone modification domain, supporting its role as a cofactor in H2B K123 ubiquitylation. An acidic patch mutant showed defects in nucleosome positioning and occupancy genome-wide. Our results provide new information on the chromatin engagement of two central players in transcription elongation and emphasize the importance of the nucleosome core as a hub for proteins that regulate chromatin during transcription.
Asunto(s)
ADN de Hongos/genética , Proteínas de Unión al ADN/genética , Regulación Fúngica de la Expresión Génica , Genoma Fúngico , Proteínas del Grupo de Alta Movilidad/genética , Proteínas Nucleares/genética , Nucleosomas/ultraestructura , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Factores de Elongación Transcripcional/genética , Sitios de Unión , ADN de Hongos/química , ADN de Hongos/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Proteínas del Grupo de Alta Movilidad/química , Proteínas del Grupo de Alta Movilidad/metabolismo , Histonas/química , Histonas/genética , Histonas/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Conformación de Ácido Nucleico , Nucleosomas/metabolismo , Unión Proteica , Conformación Proteica , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteína de Unión a TATA-Box/química , Proteína de Unión a TATA-Box/genética , Proteína de Unión a TATA-Box/metabolismo , Transcripción Genética , Factores de Elongación Transcripcional/química , Factores de Elongación Transcripcional/metabolismo , UbiquitinaciónRESUMEN
Eukaryotes regulate gene expression and other nuclear processes through the posttranslational modification of histones. In S. cerevisiae, the mono-ubiquitylation of histone H2B on lysine 123 (H2B K123ub) affects nucleosome stability, broadly influences gene expression and other DNA-templated processes, and is a prerequisite for additional conserved histone modifications that are associated with active transcription, namely the methylation of lysine residues in H3. While the enzymes that promote these chromatin marks are known, regions of the nucleosome required for the recruitment of these enzymes are undefined. To identify histone residues required for H2B K123ub, we exploited a functional interaction between the ubiquitin-protein ligase, Rkr1/Ltn1, and H2B K123ub in S. cerevisiae. Specifically, we performed a synthetic lethal screen with cells lacking RKR1 and a comprehensive library of H2A and H2B residue substitutions, and identified H2A residues that are required for H2B K123ub. Many of these residues map to the nucleosome acidic patch. The substitutions in the acidic patch confer varying histone modification defects downstream of H2B K123ub, indicating that this region contributes differentially to multiple histone modifications. Interestingly, substitutions in the acidic patch result in decreased recruitment of H2B K123ub machinery to active genes and defects in transcription elongation and termination. Together, our findings reveal a role for the nucleosome acidic patch in recruitment of histone modification machinery and maintenance of transcriptional integrity.
Asunto(s)
Histonas/metabolismo , Saccharomyces cerevisiae/genética , Elongación de la Transcripción Genética , Ubiquitinación , Endopeptidasas/genética , Regulación Fúngica de la Expresión Génica , Técnicas de Inactivación de Genes , Histonas/química , Metilación , Nucleosomas/metabolismo , Dominios y Motivos de Interacción de Proteínas , ARN Polimerasa II/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Polymerase associated factor 1 complex (Paf1C) broadly influences gene expression by regulating chromatin structure and the recruitment of RNA-processing factors during transcription elongation. The Plus3 domain of the Rtf1 subunit mediates Paf1C recruitment to genes by binding a repeating domain within the elongation factor Spt5 (suppressor of Ty). Here we provide a molecular description of this interaction by reporting the structure of human Rtf1 Plus3 in complex with a phosphorylated Spt5 repeat. We find that Spt5 binding is mediated by an extended surface containing phosphothreonine recognition and hydrophobic interfaces that interact with residues outside the Spt5 motif. Changes within these interfaces diminish binding of Spt5 in vitro and chromatin localization of Rtf1 in vivo. The structure reveals the basis for recognition of the repeat motif of Spt5, a key player in the recruitment of gene regulatory factors to RNA polymerase II.
Asunto(s)
Cromatina/genética , Proteínas Nucleares/metabolismo , Transcripción Genética , Factores de Elongación Transcripcional/metabolismo , Secuencias de Aminoácidos/genética , Secuencia de Aminoácidos , Sitios de Unión/genética , Western Blotting , Cromatina/metabolismo , Inmunoprecipitación de Cromatina , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Proteínas Nucleares/química , Proteínas Nucleares/genética , Fosforilación , Unión Proteica , Estructura Terciaria de Proteína , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Homología de Secuencia de Aminoácido , Factores de Transcripción/química , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Factores de Elongación Transcripcional/química , Factores de Elongación Transcripcional/genéticaRESUMEN
Histone modifications regulate transcription by RNA polymerase II and maintain a balance between active and repressed chromatin states. The conserved Paf1 complex (Paf1C) promotes specific histone modifications during transcription elongation, but the mechanisms by which it facilitates these marks are undefined. We previously identified a 90-amino acid region within the Rtf1 subunit of Paf1C that is necessary for Paf1C-dependent histone modifications in Saccharomyces cerevisiae. Here we show that this histone modification domain (HMD), when expressed as the only source of Rtf1, can promote H3 K4 and K79 methylation and H2B K123 ubiquitylation in yeast. The HMD can restore histone modifications in rtf1Δ cells whether or not it is directed to DNA by a fusion to a DNA binding domain. The HMD can facilitate histone modifications independently of other Paf1C subunits and does not bypass the requirement for Rad6-Bre1. The isolated HMD localizes to chromatin, and this interaction requires residues important for histone modification. When expressed outside the context of full-length Rtf1, the HMD associates with and causes Paf1C-dependent histone modifications to appear at transcriptionally inactive loci, suggesting that its function has become deregulated. Finally, the Rtf1 HMDs from other species can function in yeast. Our findings suggest a direct and conserved role for Paf1C in coupling histone modifications to transcription elongation.
Asunto(s)
Histonas/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteína de Unión a TATA-Box/química , Proteína de Unión a TATA-Box/metabolismo , Cromatina/genética , Cromatina/metabolismo , Secuencia Conservada , Evolución Molecular , Regulación Fúngica de la Expresión Génica/fisiología , Histonas/genética , Proteínas Nucleares/genética , Nucleosomas/genética , Nucleosomas/metabolismo , ARN Polimerasa II/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteína de Unión a TATA-Box/genética , Transcripción Genética/fisiología , Ubiquitinación/fisiologíaRESUMEN
The Paf1 complex was originally identified over fifteen years ago in budding yeast through its physical association with RNA polymerase II. The Paf1 complex is now known to be conserved throughout eukaryotes and is well studied for promoting RNA polymerase II transcription elongation and transcription-coupled histone modifications. Through these critical regulatory functions, the Paf1 complex participates in numerous cellular processes such as gene expression and silencing, RNA maturation, DNA repair, cell cycle progression and prevention of disease states in higher eukaryotes. In this review, we describe the historic and current research involving the eukaryotic Paf1 complex to explain the cellular roles that underlie its conservation and functional importance. This article is part of a Special Issue entitled: RNA polymerase II Transcript Elongation.
Asunto(s)
Enfermedad/genética , Eucariontes/genética , Histonas/metabolismo , Proteínas Nucleares/fisiología , Transcripción Genética/genética , Animales , Secuencia Conservada , Humanos , Modelos Biológicos , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Modificación Traduccional de las Proteínas/genética , Modificación Traduccional de las Proteínas/fisiología , Subunidades de Proteína/química , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Subunidades de Proteína/fisiología , Factores de Transcripción , Transcripción Genética/fisiologíaRESUMEN
Paf1C is a highly conserved protein complex with critical functions during eukaryotic transcription. Previous studies have shown that Paf1C is multi-functional, controlling specific aspects of transcription, ranging from RNAPII processivity to histone modifications. However, it is unclear how specific Paf1C subunits directly impact transcription and coupled processes. We have compared conditional depletion to steady-state deletion for each Paf1C subunit to determine the direct and indirect contributions to gene expression in Saccharomyces cerevisiae. Using nascent transcript sequencing, RNAPII profiling, and modeling of transcription elongation dynamics, we have demonstrated direct effects of Paf1C subunits on RNAPII processivity and elongation rate and indirect effects on transcript splicing and repression of antisense transcripts. Further, our results suggest that the direct transcriptional effects of Paf1C cannot be readily assigned to any particular histone modification. This work comprehensively analyzes both the immediate and extended roles of each Paf1C subunit in transcription elongation and transcript regulation.
RESUMEN
Nucleosomes are major determinants of eukaryotic genome organization and regulation. Many studies, incorporating a diversity of experimental approaches, have been focused on identifying and discerning the contributions of histone post-translational modifications to DNA-centered processes. Among these, monoubiquitylation of H2B (H2Bub) on K120 in humans or K123 in budding yeast is a critical histone modification that has been implicated in a wide array of DNA transactions. H2B is co-transcriptionally ubiquitylated and deubiquitylated via the concerted action of an extensive network of proteins. In addition to altering the chemical and physical properties of the nucleosome, H2Bub is important for the proper control of gene expression and for the deposition of other histone modifications. In this review, we discuss the molecular mechanisms underlying the ubiquitylation cycle of H2B and how it connects to the regulation of transcription and chromatin structure.
Asunto(s)
Cromatina , Histonas , Nucleosomas , Transcripción Genética , Ubiquitinación , Histonas/metabolismo , Humanos , Cromatina/metabolismo , Nucleosomas/metabolismo , Procesamiento Proteico-Postraduccional , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , AnimalesRESUMEN
The conserved Paf1 complex localizes to the coding regions of genes and facilitates multiple processes during transcription elongation, including the regulation of histone modifications. However, the mechanisms that govern Paf1 complex recruitment to active genes are undefined. Here we describe a previously unrecognized domain within the Cdc73 subunit of the Paf1 complex, the Cdc73 C-domain, and demonstrate its importance for Paf1 complex occupancy on transcribed chromatin. Deletion of the C-domain causes phenotypes associated with elongation defects without an apparent loss of complex integrity. Simultaneous mutation of the C-domain and another subunit of the Paf1 complex, Rtf1, causes enhanced mutant phenotypes and loss of histone H3 lysine 36 trimethylation. The crystal structure of the C-domain reveals unexpected similarity to the Ras family of small GTPases. Instead of a deep nucleotide-binding pocket, the C-domain contains a large but comparatively flat surface of highly conserved residues, devoid of ligand. Deletion of the C-domain results in reduced chromatin association for multiple Paf1 complex subunits. We conclude that the Cdc73 C-domain probably constitutes a protein interaction surface that functions with Rtf1 in coupling the Paf1 complex to the RNA polymerase II elongation machinery.
Asunto(s)
Cromatina/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas ras/química , Secuencia de Aminoácidos , Secuencia Conservada , Histonas/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , Estructura Terciaria de Proteína , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Transporte de Proteínas , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/genética , Proteína de Unión a TATA-Box/metabolismo , Transcripción GenéticaRESUMEN
The conserved multifunctional Paf1 complex is important for the proper transcription of numerous genes, and yet the exact mechanisms by which it controls gene expression remain unclear. While previous studies indicate that the Paf1 complex is a positive regulator of transcription, the repression of many genes also requires the Paf1 complex. In this study we used ARG1 as a model gene to study transcriptional repression by the Paf1 complex in Saccharomyces cerevisiae. We found that several members of the Paf1 complex contribute to ARG1 repression and that the complex localizes to the ARG1 promoter and coding region in repressing conditions, which is consistent with a direct repressive function. Furthermore, Paf1 complex-dependent histone modifications are enriched at the ARG1 locus in repressing conditions, and histone H3 lysine 4 methylation contributes to ARG1 repression. Consistent with previous reports, histone H2B monoubiquitylation, the mark upstream of histone H3 lysine 4 methylation, is also important for ARG1 repression. To begin to identify the mechanistic basis for Paf1 complex-mediated repression of ARG1, we focused on the Rtf1 subunit of the complex. Through an analysis of RTF1 mutations that abrogate known Rtf1 activities, we found that Rtf1 mediates ARG1 repression primarily by facilitating histone modifications. Other members of the Paf1 complex, such as Paf1, appear to repress ARG1 through additional mechanisms. Together, our results suggest that Rtf1-dependent histone H2B ubiquitylation and H3 K4 methylation repress ARG1 expression and that histone modifications normally associated with active transcription can occur at repressed loci and contribute to transcriptional repression.
Asunto(s)
Arginasa/biosíntesis , Histonas/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Recombinantes/genética , Proteínas de Saccharomyces cerevisiae/biosíntesis , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae , Transcripción Genética , Sistemas de Transporte de Aminoácidos/genética , Sistemas de Transporte de Aminoácidos/metabolismo , Arginasa/genética , Regulación Fúngica de la Expresión Génica , Técnicas de Inactivación de Genes , Genes Reporteros , Metilación , Proteínas Nucleares/genética , Regiones Promotoras Genéticas , Procesamiento Proteico-Postraduccional , Proteínas Recombinantes/biosíntesis , Proteínas de Saccharomyces cerevisiae/genética , Proteína de Unión a TATA-Box/genética , Proteína de Unión a TATA-Box/metabolismo , UbiquitinaciónRESUMEN
The Bur1-Bur2 and Paf1 complexes function during transcription elongation and affect histone modifications. Here we describe new roles for Bur1-Bur2 and the Paf1 complex. We find that histone H3 K36 tri-methylation requires specific components of the Paf1 complex and that K36 tri-methylation is more strongly affected at the 5' ends of genes in paf1delta and bur2delta strains in parallel with increased acetylation of histones H3 and H4. Interestingly, the 5' increase in histone acetylation is independent of K36 methylation, and therefore is mechanistically distinct from the methylation-driven deacetylation that occurs at the 3' ends of genes. Finally, Bur1-Bur2 and the Paf1 complex have a second methylation-independent function, since bur2delta set2delta and paf1delta set2delta double mutants display enhanced histone acetylation at the 3' ends of genes and increased cryptic transcription initiation. These findings identify new functions for the Paf1 and Bur1-Bur2 complexes, provide evidence that histone modifications at the 5' and 3' ends of coding regions are regulated by distinct mechanisms, and reveal that the Bur1-Bur2 and Paf1 complexes repress cryptic transcription through a Set2-independent pathway.
Asunto(s)
Quinasas Ciclina-Dependientes/metabolismo , Ciclinas/metabolismo , Histonas/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Acetilación , Quinasas Ciclina-Dependientes/genética , Ciclinas/genética , Metiltransferasas/genética , Metiltransferasas/metabolismo , Mutación , Proteínas Nucleares/genética , Proteínas de Saccharomyces cerevisiae/genética , Transcripción GenéticaRESUMEN
The regulation of transcription by RNA polymerase II is closely intertwined with the regulation of chromatin structure. A host of proteins required for the disassembly, reassembly, and modification of nucleosomes interacts with Pol II to aid its movement and counteract its disruptive effects on chromatin. The highly conserved Polymerase Associated Factor 1 Complex, Paf1C, travels with Pol II and exerts control over transcription elongation and chromatin structure, while broadly impacting the transcriptome in both single cell and multicellular eukaryotes. Recent studies have yielded exciting new insights into the mechanisms by which Paf1C regulates transcription elongation, epigenetic modifications, and post-transcriptional steps in eukaryotic gene expression. Importantly, these functional studies are now supported by an extensive foundation of high-resolution structural information, providing intimate views of Paf1C and its integration into the larger Pol II elongation complex. As a global regulatory factor operating at the interface between chromatin and transcription, the impact of Paf1C is broad and its influence reverberates into other domains of nuclear regulation, including genome stability, telomere maintenance, and DNA replication.
Asunto(s)
Factores de Transcripción/metabolismo , Núcleo Celular/genética , Núcleo Celular/metabolismo , Cromatina/genética , Cromatina/metabolismo , Células Eucariotas , Regulación de la Expresión Génica , Humanos , Proteínas Nucleares/metabolismo , Células Procariotas , Procesamiento Postranscripcional del ARN , Factores de Transcripción/química , Transcripción GenéticaRESUMEN
Maintenance of chromatin structure under the disruptive force of transcription requires cooperation among numerous regulatory factors. Histone post-translational modifications can regulate nucleosome stability and influence the disassembly and reassembly of nucleosomes during transcription elongation. The Paf1 transcription elongation complex, Paf1C, is required for several transcription-coupled histone modifications, including the mono-ubiquitylation of H2B. In Saccharomyces cerevisiae, amino acid substitutions in the Rtf1 subunit of Paf1C greatly diminish H2B ubiquitylation and cause transcription to initiate at a cryptic promoter within the coding region of the FLO8 gene, an indicator of chromatin disruption. In a genetic screen to identify factors that functionally interact with Paf1C, we identified mutations in HDA3, a gene encoding a subunit of the Hda1C histone deacetylase (HDAC), as suppressors of an rtf1 mutation. Absence of Hda1C also suppresses the cryptic initiation phenotype of other mutants defective in H2B ubiquitylation. The genetic interactions between Hda1C and the H2B ubiquitylation pathway appear specific: loss of Hda1C does not suppress the cryptic initiation phenotypes of other chromatin mutants and absence of other HDACs does not suppress the absence of H2B ubiquitylation. Providing further support for an appropriate balance of histone acetylation in regulating cryptic initiation, absence of the Sas3 histone acetyltransferase elevates cryptic initiation in rtf1 mutants. Our data suggest that the H2B ubiquitylation pathway and Hda1C coordinately regulate chromatin structure during transcription elongation and point to a potential role for a HDAC in supporting chromatin accessibility.
Asunto(s)
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Regulación Fúngica de la Expresión Génica , Histona Desacetilasas/genética , Histonas/genética , Histonas/metabolismo , Proteínas Nucleares/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Transcripción Genética , UbiquitinaciónRESUMEN
Numerous transcription accessory proteins cause alterations in chromatin structure that promote the progression of RNA polymerase II (Pol II) along open reading frames (ORFs). The Saccharomyces cerevisiae Paf1 complex colocalizes with actively transcribing Pol II and orchestrates modifications to the chromatin template during transcription elongation. To better understand the function of the Rtf1 subunit of the Paf1 complex, we created a series of sequential deletions along the length of the protein. Genetic and biochemical assays were performed on these mutants to identify residues required for the various activities of Rtf1. Our results establish that discrete nonoverlapping segments of Rtf1 are necessary for interaction with the ATP-dependent chromatin-remodeling protein Chd1, promoting covalent modification of histones H2B and H3, recruitment to active ORFs, and association with other Paf1 complex subunits. We observed transcription-related defects when regions of Rtf1 that mediate histone modification or association with active genes were deleted, but disruption of the physical association between Rtf1 and other Paf1 complex subunits caused only subtle mutant phenotypes. Together, our results indicate that Rtf1 influences transcription and chromatin structure through several independent functional domains and that Rtf1 may function independently of its association with other members of the Paf1 complex.