RESUMO
Histone chaperones are critical for controlling chromatin integrity during transcription, DNA replication, and DNA repair. Three conserved and essential chaperones, Spt6, Spn1/Iws1, and FACT, associate with elongating RNA polymerase II and interact with each other physically and/or functionally; however, there is little understanding of their individual functions or their relationships with each other. In this study, we selected for suppressors of a temperature-sensitive spt6 mutation that disrupts the Spt6-Spn1 physical interaction and that also causes both transcription and chromatin defects. This selection identified novel mutations in FACT. Surprisingly, suppression by FACT did not restore the Spt6-Spn1 interaction, based on coimmunoprecipitation, ChIP, and mass spectrometry experiments. Furthermore, suppression by FACT bypassed the complete loss of Spn1. Interestingly, the FACT suppressor mutations cluster along the FACT-nucleosome interface, suggesting that they alter FACT-nucleosome interactions. In agreement with this observation, we showed that the spt6 mutation that disrupts the Spt6-Spn1 interaction caused an elevated level of FACT association with chromatin, while the FACT suppressors reduced the level of FACT-chromatin association, thereby restoring a normal Spt6-FACT balance on chromatin. Taken together, these studies reveal previously unknown regulation between histone chaperones that is critical for their essential in vivo functions.
Assuntos
Cromatina/metabolismo , Regulação da Expressão Gênica/genética , Chaperonas de Histonas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Transcrição Gênica/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Grupo de Alta Mobilidade/genética , Proteínas de Grupo de Alta Mobilidade/metabolismo , Chaperonas de Histonas/genética , Mutação , Nucleossomos/genética , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Elongação da Transcrição/genética , Fatores de Elongação da Transcrição/metabolismoRESUMO
Transcription elongation requires elaborate coordination between the transcriptional machinery and chromatin regulatory factors to successfully produce RNA while preserving the epigenetic landscape. Recent structural and genomic studies have highlighted that suppressor of Ty 6 (Spt6), a conserved histone chaperone and transcription elongation factor, sits at the crux of the transcription elongation process. Other recent studies have revealed that Spt6 also promotes DNA replication and genome integrity. Here, we review recent studies of Spt6 that have provided new insights into the mechanisms by which Spt6 controls transcription and have revealed the breadth of Spt6 functions in eukaryotic cells.
Assuntos
Histonas , Humanos , Replicação do DNA/genética , Instabilidade Genômica/genética , Chaperonas de Histonas/genética , Chaperonas de Histonas/química , Chaperonas de Histonas/metabolismo , Histonas/genética , Histonas/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/genética , Transcrição Gênica , Fatores de Elongação da Transcrição/genética , Fatores de Elongação da Transcrição/química , Fatores de Elongação da Transcrição/metabolismo , AnimaisRESUMO
Spt6 is a conserved factor that controls transcription and chromatin structure across the genome. Although Spt6 is viewed as an elongation factor, spt6 mutations in Saccharomyces cerevisiae allow elevated levels of transcripts from within coding regions, suggesting that Spt6 also controls initiation. To address the requirements for Spt6 in transcription and chromatin structure, we have combined four genome-wide approaches. Our results demonstrate that Spt6 represses transcription initiation at thousands of intragenic promoters. We characterize these intragenic promoters and find sequence features conserved with genic promoters. Finally, we show that Spt6 also regulates transcription initiation at most genic promoters and propose a model of initiation site competition to account for this. Together, our results demonstrate that Spt6 controls the fidelity of transcription initiation throughout the genome.
Assuntos
Chaperonas de Histonas/genética , Chaperonas de Histonas/fisiologia , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/fisiologia , Iniciação da Transcrição Genética/fisiologia , Fatores de Elongação da Transcrição/genética , Fatores de Elongação da Transcrição/fisiologia , Cromatina/fisiologia , Regulação Fúngica da Expressão Gênica/genética , Chaperonas de Histonas/metabolismo , Histonas/fisiologia , Proteínas Nucleares , Nucleossomos , Fatores de Alongamento de Peptídeos/fisiologia , Regiões Promotoras Genéticas/genética , RNA Polimerase II , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Proteínas de Schizosaccharomyces pombe/fisiologia , Fatores de Transcrição/fisiologia , Sítio de Iniciação de Transcrição/fisiologia , Transcrição Gênica/genética , Fatores de Elongação da Transcrição/metabolismoRESUMO
Spt5 is an essential and conserved factor that functions in transcription and co-transcriptional processes. However, many aspects of the requirement for Spt5 in transcription are poorly understood. We have analyzed the consequences of Spt5 depletion in Schizosaccharomyces pombe using four genome-wide approaches. Our results demonstrate that Spt5 is crucial for a normal rate of RNA synthesis and distribution of RNAPII over transcription units. In the absence of Spt5, RNAPII localization changes dramatically, with reduced levels and a relative accumulation over the first â¼500 bp, suggesting that Spt5 is required for transcription past a barrier. Spt5 depletion also results in widespread antisense transcription initiating within this barrier region. Deletions of this region alter the distribution of RNAPII on the sense strand, suggesting that the barrier observed after Spt5 depletion is normally a site at which Spt5 stimulates elongation. Our results reveal a global requirement for Spt5 in transcription elongation.
Assuntos
Proteínas Cromossômicas não Histona/metabolismo , RNA Antissenso/biossíntese , RNA Fúngico/biossíntese , RNA Mensageiro/biossíntese , Saccharomyces cerevisiae/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Elongação da Transcrição Genética , Fatores de Elongação da Transcrição/metabolismo , Proteínas Cromossômicas não Histona/genética , Biologia Computacional , Bases de Dados Genéticas , Regulação Fúngica da Expressão Gênica , Genoma Fúngico , Genótipo , Mutação , Fenótipo , Regiões Promotoras Genéticas , RNA Polimerase II/metabolismo , Splicing de RNA , RNA Antissenso/genética , RNA Fúngico/genética , RNA Mensageiro/genética , Saccharomyces cerevisiae/genética , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética , Fatores de Tempo , Fatores de Elongação da Transcrição/genéticaRESUMO
The histone chaperone Spt6 is involved in promoting elongation of RNA polymerase II (RNAPII), maintaining chromatin structure, regulating cotranscriptional histone modifications, and controlling mRNA processing. These diverse functions of Spt6 are partly mediated through its interactions with RNAPII and other factors in the transcription elongation complex. In this study, we used mass spectrometry to characterize the differences in RNAPII-interacting factors between wildtype cells and those depleted for Spt6, leading to the identification of proteins that depend on Spt6 for their interaction with RNAPII. The altered association of some of these factors could be attributed to changes in steady-state protein levels. However, Abd1, the mRNA cap methyltransferase, had decreased association with RNAPII after Spt6 depletion despite unchanged Abd1 protein levels, showing a requirement for Spt6 in mediating the Abd1-RNAPII interaction. Genome-wide studies showed that Spt6 is required for maintaining the level of Abd1 over transcribed regions, as well as the level of Spt5, another protein known to recruit Abd1 to chromatin. Abd1 levels were particularly decreased at the 5' ends of genes after Spt6 depletion, suggesting a greater need for Spt6 in Abd1 recruitment over these regions. Together, our results show that Spt6 is important in regulating the composition of the transcription elongation complex and reveal a previously unknown function for Spt6 in the recruitment of Abd1.
Assuntos
Chaperonas de Histonas/metabolismo , Metiltransferases/metabolismo , Elementos de Resposta , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Transcrição Gênica , Fatores de Elongação da Transcrição/metabolismo , Cromatina/genética , Cromatina/metabolismo , Chaperonas de Histonas/genética , Espectrometria de Massas , Metiltransferases/genética , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética , Fatores de Elongação da Transcrição/genéticaRESUMO
FACT (FAcilitates Chromatin Transcription) has long been considered to be a transcription elongation factor whose ability to destabilize nucleosomes promotes RNAPII progression on chromatin templates. However, this is just one function of this histone chaperone, as FACT also functions in DNA replication. While broadly conserved among eukaryotes and essential for viability in many organisms, dependence on FACT varies widely, with some differentiated cells proliferating normally in its absence. It is therefore unclear what the core functions of FACT are, whether they differ in different circumstances, and what makes FACT essential in some situations but not others. Here, we review recent advances and propose a unifying model for FACT activity. By analogy to DNA repair, we propose that the ability of FACT to both destabilize and assemble nucleosomes allows it to monitor and restore nucleosome integrity as part of a system of chromatin repair, in which disruptions in the packaging of DNA are sensed and returned to their normal state. The requirement for FACT then depends on the level of chromatin disruption occurring in the cell, and the cell's ability to tolerate packaging defects. The role of FACT in transcription would then be just one facet of a broader system for maintaining chromatin integrity.
Assuntos
Montagem e Desmontagem da Cromatina , Reparo do DNA , Proteínas de Ligação a DNA/genética , DNA/genética , Proteínas de Grupo de Alta Mobilidade/genética , Chaperonas de Histonas/genética , Nucleossomos/genética , RNA Polimerase II/genética , Fatores de Elongação da Transcrição/genética , Sítios de Ligação , DNA/química , DNA/metabolismo , Dano ao DNA , Replicação do DNA , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Proteínas de Grupo de Alta Mobilidade/química , Proteínas de Grupo de Alta Mobilidade/metabolismo , Chaperonas de Histonas/química , Chaperonas de Histonas/metabolismo , Humanos , Modelos Moleculares , Nucleossomos/química , Nucleossomos/metabolismo , Especificidade de Órgãos , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , RNA Polimerase II/química , RNA Polimerase II/metabolismo , Transcrição Gênica , Fatores de Elongação da Transcrição/química , Fatores de Elongação da Transcrição/metabolismoRESUMO
Spn1/Iws1 is a conserved protein involved in transcription and chromatin dynamics, yet its general in vivo requirement for these functions is unknown. Using a Spn1 depletion system in Saccharomyces cerevisiae, we demonstrate that Spn1 broadly influences several aspects of gene expression on a genome-wide scale. We show that Spn1 is globally required for normal mRNA levels and for normal splicing of ribosomal protein transcripts. Furthermore, Spn1 maintains the localization of H3K36 and H3K4 methylation across the genome and is required for normal histone levels at highly expressed genes. Finally, we show that the association of Spn1 with the transcription machinery is strongly dependent on its binding partner, Spt6, while the association of Spt6 and Set2 with transcribed regions is partially dependent on Spn1. Taken together, our results show that Spn1 affects multiple aspects of gene expression and provide additional evidence that it functions as a histone chaperone in vivo.
Assuntos
Chaperonas de Histonas/genética , Metiltransferases/genética , Proteínas de Saccharomyces cerevisiae/genética , Transcrição Gênica/genética , Fatores de Elongação da Transcrição/genética , Processamento Alternativo/genética , Regulação Fúngica da Expressão Gênica/genética , Código das Histonas/genética , Saccharomyces cerevisiae/genéticaRESUMO
The transcription elongation factor Spt6 and the H3K36 methyltransferase Set2 are both required for H3K36 methylation and transcriptional fidelity in Saccharomyces cerevisiae. However, the nature of the requirement for Spt6 has remained elusive. By selecting for suppressors of a transcriptional defect in an spt6 mutant, we have isolated several highly clustered, dominant SET2 mutations (SET2sup mutations) in a region encoding a proposed autoinhibitory domain. SET2sup mutations suppress the H3K36 methylation defect in the spt6 mutant, as well as in other mutants that impair H3K36 methylation. We also show that SET2sup mutations overcome the requirement for certain Set2 domains for H3K36 methylation. In vivo, SET2sup mutants have elevated levels of H3K36 methylation and the purified Set2sup mutant protein has greater enzymatic activityin vitro. ChIP-seq studies demonstrate that the H3K36 methylation defect in the spt6 mutant, as well as its suppression by a SET2sup mutation, occurs at a step following the recruitment of Set2 to chromatin. Other experiments show that a similar genetic relationship between Spt6 and Set2 exists in Schizosaccharomyces pombe. Taken together, our results suggest a conserved mechanism by which the Set2 autoinhibitory domain requires multiple Set2 interactions to ensure that H3K36 methylation occurs specifically on actively transcribed chromatin.
Assuntos
Regulação Fúngica da Expressão Gênica , Chaperonas de Histonas/genética , Histonas/genética , Metiltransferases/genética , Processamento de Proteína Pós-Traducional , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Schizosaccharomyces/genética , Fatores de Elongação da Transcrição/genética , Sequência de Aminoácidos , Animais , Baculoviridae/genética , Baculoviridae/metabolismo , Cromatina/química , Cromatina/metabolismo , Clonagem Molecular , Sequência Conservada , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Chaperonas de Histonas/metabolismo , Histonas/metabolismo , Metilação , Metiltransferases/metabolismo , Mutação , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Schizosaccharomyces/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Células Sf9 , Spodoptera , Transcrição Gênica , Fatores de Elongação da Transcrição/metabolismoRESUMO
The SAGA complex is a conserved, multifunctional co-activator that has broad roles in eukaryotic transcription. Previous studies suggested that Tra1, the largest SAGA component, is required either for SAGA assembly or for SAGA recruitment by DNA-bound transcriptional activators. In contrast to Saccharomyces cerevisiae and mouse, a tra1Δ mutant is viable in Schizosaccharomyces pombe, allowing us to test these issues in vivo. We find that, in a tra1Δ mutant, SAGA assembles and is recruited to some, but not all, promoters. Consistent with these findings, Tra1 regulates the expression of only a subset of SAGA-dependent genes. We previously reported that the SAGA subunits Gcn5 and Spt8 have opposing regulatory roles during S. pombe sexual differentiation. We show here that, like Gcn5, Tra1 represses this pathway, although by a distinct mechanism. Thus, our study reveals that Tra1 has specific regulatory roles, rather than global functions, within SAGA.
Assuntos
Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Acetiltransferases/genética , Acetiltransferases/metabolismo , Biomarcadores/metabolismo , Western Blotting , Sobrevivência Celular , Imunoprecipitação da Cromatina , Cromatografia de Afinidade , Perfilação da Expressão Gênica , Mutação/genética , Análise de Sequência com Séries de Oligonucleotídeos , Fenótipo , Regiões Promotoras Genéticas/genética , Ligação Proteica , RNA Mensageiro/genética , Sequências Reguladoras de Ácido Nucleico , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Schizosaccharomyces/genética , Schizosaccharomyces/crescimento & desenvolvimento , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Binding of elongation factor Spt6 to Iws1 provides an effective means for coupling eukaryotic mRNA synthesis, chromatin remodelling and mRNA export. We show that an N-terminal region of Spt6 (Spt6N) is responsible for interaction with Iws1. The crystallographic structures of Encephalitozoon cuniculi Iws1 and the Iws1/Spt6N complex reveal two conserved binding subdomains in Iws1. The first subdomain (one HEAT repeat; HEAT subdomain) is a putative phosphoprotein-binding site most likely involved in an Spt6-independent function of Iws1. The second subdomain (two ARM repeats; ARM subdomain) specifically recognizes a bipartite N-terminal region of Spt6. Mutations that alter this region of Spt6 cause severe phenotypes in vivo. Importantly, the ARM subdomain of Iws1 is conserved in several transcription factors, including TFIIS, Elongin A and Med26. We show that the homologous region in yeast TFIIS enables this factor to interact with SAGA and the Mediator subunits Spt8 and Med13, suggesting the molecular basis for TFIIS recruitment at promoters. Taken together, our results provide new structural information about the Iws1/Spt6 complex and reveal a novel interaction domain used for the formation of transcription networks.
Assuntos
Encephalitozoon cuniculi/química , Proteínas Fúngicas/química , Domínios e Motivos de Interação entre Proteínas , Fatores de Transcrição/química , Fatores de Elongação da Transcrição/química , Sequência de Aminoácidos , Sítios de Ligação , Cristalografia por Raios X , Elonguina , Encephalitozoon cuniculi/genética , Encephalitozoon cuniculi/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Fenótipo , Mutação Puntual , Estrutura Terciária de Proteína , Alinhamento de Sequência , Transcrição Gênica , Fatores de Elongação da Transcrição/genética , Fatores de Elongação da Transcrição/metabolismoRESUMO
The Schizosaccharomyces pombe inv1(+) gene encodes invertase, the enzyme required for hydrolysis of sucrose and raffinose. Transcription of inv1(+) is regulated by glucose levels, with transcription tightly repressed in high glucose and strongly induced in low glucose. To understand this regulation, we have analyzed the inv1(+) cis-regulatory region and the requirement for the trans-acting coactivators SAGA and Swi/Snf. Surprisingly, deletion of the entire 1-kilobase intergenic region between the inv1(+) TATA element and the upstream open reading frame SPCC191.10 does not significantly alter regulation of inv1(+) transcription. However, a longer deletion that extends through SPCC191.10 abolishes inv1(+) induction in low glucose. Additional analysis demonstrates that there are multiple, redundant regulatory regions spread over 1.5 kb 5' of inv1(+), including within SPCC191.10, that can confer glucose-mediated transcriptional regulation to inv1(+). Furthermore, SPCC191.10 can regulate inv1(+) transcription in an orientation-independent fashion and from a distance as great as 3 kb. With respect to trans-acting factors, both SAGA and Swi/Snf are recruited to SPCC191.10 and to other locations in the large inv1(+) regulatory region in a glucose-dependent fashion, and both are required for inv1(+) derepression. Taken together, these results demonstrate that inv1(+) regulation in S. pombe occurs via the use of multiple regulatory elements and that activation can occur over a great distance, even from elements within other open reading frames.
Assuntos
Regulação Fúngica da Expressão Gênica , Elementos Reguladores de Transcrição , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/genética , Transativadores/metabolismo , beta-Frutofuranosidase/genética , Regiões 3' não Traduzidas , Regiões 5' não Traduzidas , Deleção de Genes , Glucose/metabolismo , RNA Mensageiro/biossíntese , Schizosaccharomyces/metabolismo , TATA BoxRESUMO
Histone chaperones are an important class of proteins that regulate chromatin accessibility for DNA-templated processes. Spt6 is a conserved histone chaperone and key regulator of transcription and chromatin structure. However, its functions outside of these roles have been little explored. In this work, we demonstrate a requirement for S. cerevisiae Spt6 in DNA replication and, more broadly, as a regulator of genome stability. Depletion or mutation of Spt6 impairs DNA replication in vivo. Additionally, spt6 mutants are sensitive to DNA replication stress-inducing agents. Interestingly, this sensitivity is independent of the association of Spt6 with RNA polymerase II (RNAPII), suggesting that spt6 mutants have a transcription-independent impairment of DNA replication. Specifically, genomic studies reveal that spt6 mutants have decreased loading of the MCM replicative helicase at replication origins, suggesting that Spt6 promotes origin licensing. Our results identify Spt6 as a regulator of genome stability, at least in part through a role in DNA replication.
Assuntos
Histonas , Proteínas de Saccharomyces cerevisiae , Humanos , Histonas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Chaperonas de Histonas/genética , Chaperonas de Histonas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Elongação da Transcrição/metabolismo , Cromatina/metabolismo , Replicação do DNA/genética , Instabilidade Genômica , Transcrição GênicaRESUMO
Proper histone levels are critical for transcription, chromosome segregation, and other chromatin-mediated processes(1-7). In Saccharomyces cerevisiae, the histones H2A and H2B are encoded by two gene pairs, named HTA1-HTB1 and HTA2-HTB2 (ref. 8). Previous studies have demonstrated that when HTA2-HTB2 is deleted, HTA1-HTB1 dosage compensates at the transcriptional level(4,9). Here we show that a different mechanism of dosage compensation, at the level of gene copy number, can occur when HTA1-HTB1 is deleted. In this case, HTA2-HTB2 amplifies via creation of a new, small, circular chromosome. This duplication, which contains 39 kb of chromosome II, includes HTA2-HTB2, the histone H3-H4 locus HHT1-HHF1, a centromere and origins of replication. Formation of the new chromosome occurs by recombination between two Ty1 retrotransposon elements that flank this region. Following meiosis, recombination between these two particular Ty1 elements occurs at a greatly elevated level in hta1-htb1Delta mutants, suggesting that a decreased level of histones H2A and H2B specifically stimulates this amplification of histone genes. Our results demonstrate another mechanism by which histone gene dosage is controlled to maintain genomic integrity.
Assuntos
Cromossomos Fúngicos/metabolismo , DNA Circular/genética , Amplificação de Genes/genética , Genes Fúngicos/genética , Histonas/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Cromossomos Fúngicos/genética , DNA Circular/metabolismo , Recombinação Genética/genéticaRESUMO
In Saccharomyces cerevisiae, transcriptional silencing occurs at three classes of genomic regions: near the telomeres, at the silent mating type loci, and within the ribosomal DNA (rDNA) repeats. In all three cases, silencing depends upon several factors, including specific types of histone modifications. In this work we have investigated the roles in silencing for Spt10 and Spt21, two proteins previously shown to control transcription of particular histone genes. Building on a recent study showing that Spt10 is required for telomeric silencing, our results show that in both spt10 and spt21 mutants, silencing is reduced near telomeres and at HMLα, while it is increased at the rDNA. Both spt10 and spt21 mutations cause modest effects on Sir protein recruitment and histone modifications at telomeric regions, and they cause significant changes in chromatin structure, as judged by its accessibility to dam methylase. These silencing and chromatin changes are not seen upon deletion of HTA2-HTB2, the primary histone locus regulated by Spt10 and Spt21. These results suggest that Spt10 and Spt21 control silencing in S. cerevisiae by altering chromatin structure through roles beyond the control of histone gene expression.
Assuntos
Histona Acetiltransferases/fisiologia , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/genética , Fatores de Transcrição/fisiologia , Cromatina/química , Cromatina/metabolismo , DNA Ribossômico/genética , Regulação para Baixo , Inativação Gênica , Histonas/metabolismo , Processamento de Proteína Pós-Traducional , Saccharomyces cerevisiae/metabolismo , Proteínas Reguladoras de Informação Silenciosa de Saccharomyces cerevisiae/metabolismo , Telômero/genética , Telômero/metabolismoRESUMO
Spn1/Iws1 is an essential eukaryotic transcription elongation factor that is conserved from yeast to humans as an integral member of the RNA polymerase II elongation complex. Several studies have shown that Spn1 functions as a histone chaperone to control transcription, RNA splicing, genome stability, and histone modifications. However, the precise role of Spn1 is not understood, and there is little understanding of why it is essential for viability. To address these issues, we have isolated 8 suppressor mutations that bypass the essential requirement for Spn1 in Saccharomyces cerevisiae. Unexpectedly, the suppressors identify several functionally distinct complexes and activities, including the histone chaperone FACT, the histone methyltransferase Set2, the Rpd3S histone deacetylase complex, the histone acetyltransferase Rtt109, the nucleosome remodeler Chd1, and a member of the SAGA coactivator complex, Sgf73. The identification of these distinct groups suggests that there are multiple ways in which Spn1 bypass can occur, including changes in histone acetylation and alterations in other histone chaperones. Thus, Spn1 may function to overcome repressive chromatin by multiple mechanisms during transcription. Our results suggest that bypassing a subset of these functions allows viability in the absence of Spn1.
Assuntos
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Fatores de Elongação da Transcrição , Cromatina , Proteínas de Ligação a DNA/genética , Histona Acetiltransferases/genética , Chaperonas de Histonas/genética , Histona Desacetilases/genética , Histona Metiltransferases/genética , Histonas/genética , Nucleossomos , Fatores de Alongamento de Peptídeos/genética , RNA Polimerase II/genética , Proteínas de Ligação a RNA/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Supressão Genética , Fatores de Transcrição/genética , Transcrição Gênica , Fatores de Elongação da Transcrição/genéticaRESUMO
Src homology 2 (SH2) domains are mostly found in multicellular organisms where they recognize phosphotyrosine-containing signaling proteins. Spt6, a conserved transcription factor and putative histone chaperone, contains a C-terminal SH2 domain conserved from yeast to human. In mammals, this SH2 domain recognizes phosphoserines rather than phosphotyrosines and is essential for the recruitment of Spt6 by elongating RNA polymerase II (RNAPII), enabling Spt6 to participate in the coupling of transcription elongation, chromatin modulation, and mRNA export. We have determined the structure of the entire Spt6 C-terminal region from Antonospora locustae, revealing the presence of two highly conserved tandem SH2 domains rather than a single SH2 domain. Although the first SH2 domain has a canonical organization, the second SH2 domain is highly noncanonical and appears to be unique in the SH2 family. However, both SH2 domains have phosphate-binding determinants. Our biochemical and genetic data demonstrate that the complete tandem, but not the individual SH2 domains, are necessary and sufficient for the interaction of Spt6 with RNAPII and are important for Spt6 function in vivo. Furthermore, our data suggest that binding of RNAPII to the Spt6 tandem SH2 is more extensive than the mere recognition of a doubly phosphorylated C-terminal domain peptide by the tandem SH2. Taken together, our results show that Spt6 interaction with RNAPII via a novel arrangement of canonical and noncanonical SH2 domains is crucial for Spt6 function in vivo.
Assuntos
Proteínas Fúngicas/metabolismo , Microsporídios/metabolismo , RNA Polimerase II/metabolismo , Fatores de Transcrição/metabolismo , Domínios de Homologia de src/fisiologia , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Humanos , Microsporídios/genética , Fosforilação/fisiologia , Ligação Proteica/fisiologia , RNA Polimerase II/química , RNA Polimerase II/genética , Fatores de Transcrição/química , Fatores de Transcrição/genéticaRESUMO
Previous studies in Saccharomyces cerevisiae have demonstrated that cryptic promoters within coding regions activate transcription in particular mutants. We have performed a comprehensive analysis of cryptic transcription in order to identify factors that normally repress cryptic promoters, to determine the amount of cryptic transcription genome-wide, and to study the potential for expression of genetic information by cryptic transcription. Our results show that a large number of factors that control chromatin structure and transcription are required to repress cryptic transcription from at least 1,000 locations across the S. cerevisiae genome. Two results suggest that some cryptic transcripts are translated. First, as expected, many cryptic transcripts contain an ATG and an open reading frame of at least 100 codons. Second, several cryptic transcripts are translated into proteins. Furthermore, a subset of cryptic transcripts tested is transiently induced in wild-type cells following a nutritional shift, suggesting a possible physiological role in response to a change in growth conditions. Taken together, our results demonstrate that, during normal growth, the global integrity of gene expression is maintained by a wide range of factors and suggest that, under altered genetic or physiological conditions, the expression of alternative genetic information may occur.
Assuntos
Cromatina/metabolismo , Regulação Fúngica da Expressão Gênica , Fases de Leitura Aberta/fisiologia , Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica , Perfilação da Expressão Gênica , Genoma Fúngico , Dados de Sequência Molecular , Análise de Sequência com Séries de Oligonucleotídeos , Fases de Leitura Aberta/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/fisiologia , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/genéticaRESUMO
One of the major advances in our understanding of gene regulation in eukaryotes was the discovery of factors that regulate transcription by controlling chromatin structure. Prominent among these discoveries was the demonstration that Gcn5 is a histone acetyltransferase, establishing a direct connection between transcriptional activation and histone acetylation. This breakthrough was soon followed by the purification of a protein complex that contains Gcn5, the SAGA complex. In this article, we review the early genetic and biochemical experiments that led to the discovery of SAGA and the elucidation of its multiple activities.
Assuntos
Processamento de Proteína Pós-Traducional , Proteínas de Saccharomyces cerevisiae/metabolismo , Transativadores/metabolismo , Ativação Transcricional , Fatores de Transcrição de p300-CBP/metabolismo , Acetilação , Cromatina/metabolismo , Ensaios Enzimáticos , Histonas/metabolismo , Humanos , Processamento de Proteína Pós-Traducional/fisiologia , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/isolamento & purificação , Homologia de Sequência de Aminoácidos , Proteína de Ligação a TATA-Box/genética , Proteína de Ligação a TATA-Box/metabolismo , Transativadores/genética , Transativadores/isolamento & purificação , Ubiquitinação , Fatores de Transcrição de p300-CBP/genética , Fatores de Transcrição de p300-CBP/isolamento & purificaçãoRESUMO
Changes in oxygen levels cause widespread changes in gene expression in organisms ranging from bacteria to humans. In Saccharomyces cerevisiae, this response is mediated in part by Hap1, originally identified as a heme-dependent transcriptional activator that functions during aerobic growth. We show here that Hap1 also plays a significant and direct role under hypoxic conditions, not as an activator, but as a repressor. The repressive activity of Hap1 controls several genes, including three ERG genes required for ergosterol biosynthesis. Chromatin immunoprecipitation experiments showed that Hap1 binds to the ERG gene promoters, while additional experiments showed that the corepressor Tup1/Ssn6 is recruited by Hap1 and is also required for repression. Furthermore, mutational analysis demonstrated that conserved Hap1 binding sites in the ERG5 5' regulatory region are required for repression. The switch of Hap1 from acting as a hypoxic repressor to an aerobic activator is determined by heme, which is synthesized only in the presence of oxygen. The ability of Hap1 to function as a ligand-dependent repressor and activator is a property shared with mammalian nuclear hormone receptors and likely allows greater transcriptional control by Hap1 in response to changing oxygen levels.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Heme/metabolismo , Proteínas Repressoras/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Transativadores/metabolismo , Transcrição Gênica , Aerobiose/efeitos dos fármacos , Anaerobiose/efeitos dos fármacos , Sequência de Bases , Sítios de Ligação , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Citocromos c/genética , Citocromos c/metabolismo , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Genes Fúngicos , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Heme/farmacologia , Dados de Sequência Molecular , Proteínas Nucleares/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Oxirredutases/genética , Oxirredutases/metabolismo , Regiões Promotoras Genéticas/genética , Ligação Proteica/efeitos dos fármacos , Saccharomyces cerevisiae/efeitos dos fármacos , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição , Transcrição Gênica/efeitos dos fármacosRESUMO
Most fundamental aspects of transcription are conserved among eukaryotes. One striking difference between yeast Saccharomyces cerevisiae and metazoans, however, is the distance over which transcriptional activation occurs. In S. cerevisiae, upstream activation sequences (UASs) are generally located within a few hundred base pairs of a target gene, while in Drosophila and mammals, enhancers are often several kilobases away. To study the potential for long-distance activation in S. cerevisiae, we constructed and analyzed reporters in which the UAS-TATA distance varied. Our results show that UASs lose the ability to activate normal transcription as the UAS-TATA distance increases. Surprisingly, transcription does initiate, but proximally to the UAS, regardless of its location. To identify factors affecting long-distance activation, we screened for mutants allowing activation of a reporter when the UAS-TATA distance is 799 bp. These screens identified four loci, SIN4, SPT2, SPT10, and HTA1-HTB1, with sin4 mutations being the strongest. Our results strongly suggest that long-distance activation in S. cerevisiae is normally limited by Sin4 and other factors and that this constraint plays a role in ensuring UAS-core promoter specificity in the compact S. cerevisiae genome.