RESUMO
Transcription of non-protein-coding DNA (ncDNA) and its noncoding RNA (ncRNA) products are beginning to emerge as key regulators of gene expression. We previously identified a regulatory system in Saccharomyces cerevisiae whereby transcription of intergenic ncDNA (SRG1) represses transcription of an adjacent protein-coding gene (SER3) through transcription interference. We now provide evidence that SRG1 transcription causes repression of SER3 by directing a high level of nucleosomes over SRG1, which overlaps the SER3 promoter. Repression by SRG1 transcription is dependent on the Spt6 and Spt16 transcription elongation factors. Significantly, spt6 and spt16 mutations reduce nucleosome levels over the SER3 promoter without reducing intergenic SRG1 transcription, strongly suggesting that nucleosome levels, not transcription levels, cause SER3 repression. Finally, we show that spt6 and spt16 mutations allow transcription factor access to the SER3 promoter. Our results raise the possibility that transcription of ncDNA may contribute to nucleosome positioning on a genome-wide scale where, in some cases, it negatively impacts protein-DNA interactions.
Assuntos
DNA Intergênico/metabolismo , Regulação Fúngica da Expressão Gênica , Nucleossomos/metabolismo , Saccharomyces cerevisiae/metabolismo , Transcrição Gênica , DNA Intergênico/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Grupo de Alta Mobilidade/metabolismo , Chaperonas de Histonas , Mutação/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosfoglicerato Desidrogenase/metabolismo , Regiões Promotoras Genéticas , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Temperatura , Fatores de Elongação da Transcrição/genética , Fatores de Elongação da Transcrição/metabolismoRESUMO
Previous studies have shown that repression of the Saccharomyces cerevisiae SER3 gene is dependent on transcription of SRG1 from noncoding DNA initiating within the intergenic region 5' of SER3 and extending across the SER3 promoter region. By a mechanism dependent on the activities of the Swi/Snf chromatin remodeling factor, the HMG-like factor Spt2, and the Spt6 and Spt16 histone chaperones, SRG1 transcription deposits nucleosomes over the SER3 promoter to prevent transcription factors from binding and activating SER3. In this study, we uncover a role for the Paf1 transcription elongation complex in SER3 repression. We find that SER3 repression is primarily dependent on the Paf1 and Ctr9 subunits of this complex, with minor contributions by the Rtf1, Cdc73, and Leo1 subunits. We show that the Paf1 complex localizes to the SRG1 transcribed region under conditions that repress SER3, consistent with it having a direct role in mediating SRG1 transcription-dependent SER3 repression. Importantly, we show that the defect in SER3 repression in strains lacking Paf1 subunits is not a result of reduced SRG1 transcription or reduced levels of known Paf1 complex-dependent histone modifications. Rather, we find that strains lacking subunits of the Paf1 complex exhibit reduced nucleosome occupancy and reduced recruitment of Spt16 and, to a lesser extent, Spt6 at the SER3 promoter. Taken together, our results suggest that Paf1 and Ctr9 repress SER3 by maintaining SRG1 transcription-dependent nucleosome occupancy.
Assuntos
DNA Intergênico/genética , Regulação para Baixo , Proteínas Nucleares/metabolismo , Nucleossomos/metabolismo , Fosfoglicerato Desidrogenase/genética , Regiões Promotoras Genéticas , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Transcrição Gênica , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , DNA Intergênico/metabolismo , Regulação Fúngica da Expressão Gênica , Proteínas Nucleares/genética , Nucleossomos/genética , Fosfoglicerato Desidrogenase/metabolismo , Ligação Proteica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Fatores de Elongação da Transcrição/genética , Fatores de Elongação da Transcrição/metabolismoRESUMO
Transcription by RNA polymerase II in Saccharomyces cerevisiae and in humans is widespread, even in genomic regions that do not encode proteins. The purpose of such intergenic transcription is largely unknown, although it can be regulatory. We have discovered a role for one case of intergenic transcription by studying the S. cerevisiae SER3 gene. Our previous results demonstrated that transcription of SER3 is tightly repressed during growth in rich medium. We now show that the regulatory region of this gene is highly transcribed under these conditions and produces a non-protein-coding RNA (SRG1). Expression of the SRG1 RNA is required for repression of SER3. Additional experiments have demonstrated that repression occurs by a transcription-interference mechanism in which SRG1 transcription across the SER3 promoter interferes with the binding of activators. This work identifies a previously unknown class of transcriptional regulatory genes.
Assuntos
Desidrogenases de Carboidrato/genética , DNA Intergênico/genética , Regulação Fúngica da Expressão Gênica , Genes Fúngicos/genética , RNA não Traduzido/genética , Saccharomyces cerevisiae/genética , Transcrição Gênica/genética , Sequência de Bases , Regulação para Baixo/genética , Fosfoglicerato Desidrogenase , Regiões Promotoras Genéticas/genética , RNA Fúngico/biossíntese , RNA Fúngico/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Saccharomyces cerevisiae/citologiaAssuntos
RNA não Traduzido/genética , Transcrição Gênica , Histonas/metabolismo , Lisina/metabolismo , Metilação , Regiões Promotoras Genéticas , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Members of the Swi/Snf family of chromatin-remodeling complexes play critical roles in transcriptional control. Recent studies have made significant advances in our understanding of the fundamental aspects of Swi/Snf complexes, including the roles of specific subunits, the repression of transcription, and the mechanism of remodeling. In addition, new findings also indicate an important role for the Swi/Snf-related complex, RSC, in controlling gene expression.
Assuntos
Cromatina/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Fatores de Transcrição/metabolismo , Animais , Regulação da Expressão Gênica/fisiologia , Humanos , Nucleossomos/metabolismo , Transcrição GênicaRESUMO
BACKGROUND: Nucleosomes have an important role in modulating access of DNA by regulatory factors. The role specific histone residues have in this process has been shown to be an important mechanism of transcription regulation. Previously, we identified eight amino acids in histones H3 and H4 that are required for nucleosome occupancy over highly transcribed regions of the genome. RESULTS: We investigate the mechanism through which three of these previously identified histone H3 amino acids regulate nucleosome architecture. We find that histone H3 K122, Q120, and R49 are required for Spt2, Spt6, and Spt16 occupancies at genomic locations where transcription rates are high, but not over regions of low transcription rates. Furthermore, substitution at one residue, K122, located on the dyad axis of the nucleosome, results in improper reassembly and disassembly of nucleosomes, likely accounting for the transcription rate-dependent regulation by these mutant histones. CONCLUSIONS: These data show that when specific amino acids of histone proteins are substituted, Spt2, Spt6, and Spt16 occupancies are reduced and nucleosome dynamics are altered. Therefore, these data support a mechanism for histone chaperone binding where these factors interact with histone proteins to promote their activities during transcription.
RESUMO
Transcription of nonprotein-coding DNA is widespread in eukaryotes and plays important regulatory roles for many genes, including genes that are misregulated in cancer cells. Its pervasiveness presents the potential for a wealth of diverse regulatory roles for noncoding transcription. We previously showed that the act of transcribing noncoding DNA (ncDNA) across the promoter of the protein-coding SER3 gene in Saccharomyces cerevisiae positions nucleosomes over the upstream activating sequences, leading to strong repression of SER3 transcription. To explore the possibility of other regulatory roles for ncDNA transcription, we selected six candidate S. cerevisiae genes that express ncRNAs over their promoters and analyzed the regulation of one of these genes, ECM3, in detail. Because noncoding transcription can lead to changes in the local chromatin landscape that impinge on the expression of nearby coding genes, we surveyed the effects of various chromatin regulators on the expression of ECM3 These analyses identified roles for the Paf1 complex in positively regulating ECM3 transcription through methylation of histone H3 at lysine 4 (K4) and for Paf1 in controlling the pattern of intergenic transcription at this locus. By deleting a putative promoter for the noncoding transcription unit that lies upstream of ECM3, we provide evidence for a positive correlation between intergenic transcription and ECM3 expression. Our results are consistent with a model in which cotranscriptional methylation of histone H3 K4, mediated by the Paf1 complex and noncoding transcription, leads to activation of ECM3 transcription.
Assuntos
Adenosina Trifosfatases/genética , Regulação Fúngica da Expressão Gênica/genética , Proteínas Nucleares/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Cromatina/genética , Proteínas de Ligação a DNA/genética , Histonas/genética , Lisina/genética , Metilação , Nucleossomos , Regiões Promotoras Genéticas , RNA não Traduzido/genética , Transcrição GênicaRESUMO
The highly conserved FACT (Facilitates Chromatin Transactions) complex performs essential functions in eukaryotic cells through the reorganization of nucleosomes. During transcription, FACT reorganizes nucleosomes to allow passage of RNA Polymerase II and then assists in restoring these nucleosomes after RNA Polymerase II has passed. We have previously shown, consistent with this function, that Spt16 facilitates repression of the Saccharomyces cerevisiae SER3 gene by maintaining nucleosome occupancy over the promoter of this gene as a consequence of intergenic transcription of SRG1 noncoding DNA. In this study, we report the results of a genetic screen to identify mutations in SPT16 that derepress SER3. Twenty-five spt16 mutant alleles were found to derepress SER3 without causing significant reductions in either SRG1 RNA levels or Spt16 protein levels. Additional phenotypic assays indicate that these mutants have general transcription defects related to altered chromatin structure. Our analyses of a subset of these spt16 mutants reveal defects in SRG1 transcription-coupled nucleosome occupancy over the SER3 promoter. We provide evidence that these mutants broadly impair transcription-coupled nucleosome occupancy at highly transcribed genes but not at lowly transcribed genes. Finally, we show that one consequence shared by these mutations is the reduced binding of mutant Spt16 proteins across SRG1 and other highly transcribed genes. Taken together, our results highlight an important role for Spt16 in orchestrating transcription-coupled nucleosome assembly at highly transcribed regions of the genome, possibly by facilitating the association of Spt16 during this process.
RESUMO
Transcription of ncDNA occurs throughout eukaryotic genomes, generating a wide array of ncRNAs. One large class of ncRNAs includes those transcribed over the promoter regions of nearby protein coding genes. Recent studies, primarily focusing on individual genes have uncovered multiple mechanisms by which promoter-associated transcriptional activity locally alters gene expression.
RESUMO
Our previous studies of Saccharomyces cerevisiae described a gene repression mechanism where the transcription of intergenic noncoding DNA (ncDNA) (SRG1) assembles nucleosomes across the promoter of the adjacent SER3 gene that interfere with the binding of transcription factors. To investigate the role of histones in this mechanism, we screened a comprehensive library of histone H3 and H4 mutants for those that derepress SER3. We identified mutations altering eight histone residues (H3 residues V46, R49, V117, Q120, and K122 and H4 residues R36, I46, and S47) that strongly increase SER3 expression without reducing the transcription of the intergenic SRG1 ncDNA. We detected reduced nucleosome occupancy across SRG1 in these mutants to degrees that correlate well with the level of SER3 derepression. The histone chromatin immunoprecipitation experiments on several other genes suggest that the loss of nucleosomes in these mutants is specific to highly transcribed regions. Interestingly, two of these histone mutants, H3 R49A and H3 V46A, reduce Set2-dependent methylation of lysine 36 of histone H3 and allow transcription initiation from cryptic intragenic promoters. Taken together, our data identify a new class of histone mutants that is defective for transcription-dependent nucleosome occupancy.
Assuntos
Histonas/genética , Mutação , Nucleossomos/genética , Proteínas de Saccharomyces cerevisiae/genética , Aminoácidos/química , Aminoácidos/genética , Aminoácidos/metabolismo , Northern Blotting , Western Blotting , DNA Intergênico/genética , DNA Intergênico/metabolismo , Regulação Fúngica da Expressão Gênica , Histonas/química , Histonas/metabolismo , Modelos Moleculares , Nucleossomos/metabolismo , Fosfoglicerato Desidrogenase/genética , Fosfoglicerato Desidrogenase/metabolismo , Regiões Promotoras Genéticas/genética , Conformação Proteica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Transcrição GênicaRESUMO
Recent studies have revealed that transcription of noncoding, intergenic DNA is abundant among eukaryotes. However, the functions of this transcription are poorly understood. We have previously shown that in Saccharomyces cerevisiae, expression of an intergenic transcript, SRG1, represses the transcription of the adjacent gene, SER3, by transcription interference. We now show that SRG1 transcription is regulated by serine, thereby conferring regulation of SER3, a serine biosynthetic gene. This regulation requires Cha4, a serine-dependent activator that binds to the SRG1 promoter and is required for SRG1 induction in the presence of serine. Furthermore, two coactivator complexes, SAGA and Swi/Snf, are also directly required for activation of SRG1 and transcription interference of SER3. Taken together, our results elucidate a physiological role for intergenic transcription in the regulation of SER3. Moreover, our results demonstrate a mechanism by which intergenic transcription allows activators to act indirectly as repressors.
Assuntos
DNA Intergênico/genética , Regulação Fúngica da Expressão Gênica/fisiologia , Saccharomyces cerevisiae/genética , Transcrição Gênica/fisiologia , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/fisiologia , Fosfoglicerato Desidrogenase/antagonistas & inibidores , Fosfoglicerato Desidrogenase/biossíntese , Fosfoglicerato Desidrogenase/genética , Regiões Promotoras Genéticas , Proteínas de Ligação a RNA/fisiologia , Proteínas Repressoras/fisiologia , Ribonucleoproteína Nuclear Pequena U1/fisiologia , Proteínas de Saccharomyces cerevisiae/antagonistas & inibidores , Proteínas de Saccharomyces cerevisiae/biossíntese , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiologia , Serina/fisiologia , Supressão Genética/genética , Transativadores/metabolismo , Fatores de Transcrição/fisiologiaRESUMO
Many studies have established that the Swi/Snf family of chromatin-remodeling complexes activate transcription. Recent reports have suggested the possibility that these complexes can also repress transcription. We now present chromatin immunoprecipitation evidence that the Swi/Snf complex of Saccharomyces cerevisiae directly represses transcription of the SER3 gene. Consistent with its role in nucleosome remodeling, Swi/Snf controls the chromatin structure of the SER3 promoter. However, in striking contrast to activation by Swi/Snf, which requires most Swi/Snf subunits, repression by Swi/Snf at SER3 is dependent primarily on one Swi/Snf component, Snf2. These results show distinct differences in the requirements for Swi/Snf components in transcriptional activation and repression.