RESUMEN
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 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
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
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
To identify the mechanisms by which multiple signaling pathways coordinately affect gene expression, we investigated regulation of the S. cerevisiae INO1 gene. Full activation of INO1 transcription occurs in the absence of inositol and requires the Snf1 protein kinase in addition to other signaling molecules and transcription factors. Here, we present evidence that the Sit4 protein phosphatase negatively regulates INO1 transcription. A mutation in SIT4 was uncovered as a suppressor of the inositol auxotrophy of snf1Delta strains. We found that sit4 mutant strains exhibit an Spt(-) phenotype, suggesting a more general role for Sit4 in transcription. In fact, like the gene-specific regulators of INO1 transcription, Opi1, Ino2, and Ino4, both Snf1 and Sit4 regulate binding of TBP to the INO1 promoter, as determined by chromatin immunoprecipitation analysis. Experiments involving double-mutant strains indicate that the negative effect of Sit4 on INO1 transcription is unlikely to occur through dephosphorylation of histone H3 or Opi1. Sit4 is a known component of the target of rapamycin (TOR) signaling pathway, and treatment of cells with rapamycin reduces INO1 activation. However, analysis of rapamycin-treated cells suggests that Sit4 represses INO1 transcription through multiple mechanisms, only one of which may involve inhibition of TOR signaling.
Asunto(s)
Regulación Fúngica de la Expresión Génica , Mio-Inositol-1-Fosfato Sintasa/genética , Fosfoproteínas Fosfatasas/genética , Fosfoproteínas Fosfatasas/fisiología , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/fisiología , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/fisiología , Proteína de Unión a TATA-Box/metabolismo , Alelos , Inmunoprecipitación de Cromatina , Clonación Molecular , Codón sin Sentido , Cartilla de ADN/química , Genotipo , Histonas/metabolismo , Immunoblotting , Operón Lac , Modelos Genéticos , Mutación , Fenotipo , Fosforilación , Plásmidos/metabolismo , Unión Proteica , Proteínas Quinasas/farmacología , Proteína Fosfatasa 2 , Proteínas Represoras/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transducción de Señal , Sirolimus/farmacología , Serina-Treonina Quinasas TOR , Transcripción Genética , beta-Galactosidasa/metabolismoRESUMEN
The Saccharomyces cerevisiae Snf1 kinase plays a critical role in recalibrating cellular metabolism in response to glucose depletion. Hundreds of genes show changes in expression levels when the SNF1 gene is deleted. However, cells can adapt to the absence of a specific gene when grown in long term culture. Here we apply a chemical genetic method to rapidly and selectively inactivate a modified Snf1 kinase using a pyrazolopyrimidine inhibitor. By allowing cells to adjust to a change in carbon source prior to inhibition of the Snf1 kinase activity, we identified a set of genes whose expression increased when Snf1 was inhibited. Prominent in this set are genes that are activated by Gcn4, a transcriptional activator of amino acid biosynthetic genes. Deletion of Snf1 increased Gcn4 protein levels without affecting its mRNA levels. The increased Gcn4 protein levels required the Gcn2 kinase and Gcn20, regulators of GCN4 translation. These data indicate that Snf1 functions upstream of Gcn20 to regulate control of GCN4 translation in S. cerevisiae.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Regulación Fúngica de la Expresión Génica/fisiología , Biosíntesis de Proteínas/fisiología , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/metabolismo , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico , Proteínas de Unión al ADN/genética , Eliminación de Gen , Regulación Fúngica de la Expresión Génica/efectos de los fármacos , Biosíntesis de Proteínas/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/genética , Pirimidinas/farmacología , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Factores de Transcripción/genética , Triazoles/farmacologíaRESUMEN
Phosphorylation of the Saccharomyces cerevisiae Snf1 kinase activation loop is determined by the integration of two reaction rates: the rate of phosphorylation by upstream kinases and the rate of dephosphorylation by Glc7. The activities of the Snf1-activating kinases do not appear to be glucose-regulated, since immune complex kinase assays with each of the three Snf1-activating kinases show similar levels of activity when prepared from cells grown in either high or low glucose. In contrast, the dephosphorylation of the Snf1 activation loop was strongly regulated by glucose. When de novo phosphorylation of Snf1 was inhibited, phosphorylation of the Snf1 activation loop was found to be stable in low glucose but rapidly lost upon the addition of glucose. A greater than 10-fold difference in the rates of Snf1 activation loop dephosphorylation was detected. However, the activity of the Glc7-Reg1 phosphatase may not itself be directly regulated by glucose, since the Glc7-Reg1 enzyme was active in low glucose toward another substrate, the transcription factor Mig1. Glucose-mediated regulation of Snf1 activation loop dephosphorylation is controlled by changes in the ability of the Snf1 activation loop to act as a substrate for Glc7.