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Spt10 and Spt21 are required for transcriptional silencing in Saccharomyces cerevisiae.

ABSTRACT: 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 HML?, 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.

SUBMITTER: Chang JS

PROVIDER: S-EPMC3019801 | biostudies-other | 2011 Jan

REPOSITORIES: biostudies-other

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Spt10 and Spt21 are required for transcriptional silencing in Saccharomyces cerevisiae.

Chang Jennifer S JS Winston Fred F

Eukaryotic cell 20101105 1

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 show ...[more]

PMID: 21057056

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Project description:In Saccharomyces cerevisiae, heterochromatin is formed through interactions between site-specific DNA-binding factors, including the transcriptional activator Rap1, and Sir proteins. Despite a vast understanding of the establishment and maintenance of Sir-silenced chromatin, the mechanism of gene silencing by Sir proteins has remained a mystery. Utilizing high resolution chromatin immunoprecipitation, we found that Rap1, the native activator of the bi-directional HML α promoter, bound its recognition sequence in silenced chromatin and its binding was enhanced by the presence of Sir proteins. In contrast to prior results, various components of transcription machinery were not able to access HML α in the silenced state. These findings disproved the long-standing model of indiscriminate steric occlusion by Sir proteins and led to investigation of the transcriptional activator Rap1 in Sir-silenced chromatin. Using a highly sensitive assay that monitors loss-of-silencing events, we identified a novel role for promoter-bound Rap1 in the maintenance of silent chromatin through interactions with the Sir complex. We also found that promoter-bound Rap1 activated HML α when in an expressed state, and aided in the transition from transcription initiation to elongation. Highlighting the importance of epigenetic context in transcription factor function, these results point toward a model in which the duality of Rap1 function was mediated by local chromatin environment rather than binding-site availability.Significance statementThe coarse partitioning of the genome into regions of active euchromatin and repressed heterochromatin is an important, and conserved, level gene expression regulation in eukaryotes. Repressor Activator Protein (Rap1) is a transcription factor that promotes the activation of genes when recruited to promoters, and aids in the establishment of heterochromatin through interactions with silencer elements. Here, we investigate the role of Rap1 when bound to a promoter in silent chromatin and dissect the context-specific epigenetic cues that regulate the dual properties of this transcription factor. Together, our data highlight the importance of protein-protein interactions and local chromatin state on transcription factor function.

Project description:BACKGROUND: Propolis is a natural product of plant resins collected by honeybees (Apis mellifera) from various plant sources. Our previous studies indicated that propolis sensitivity is dependent on the mitochondrial function and that vacuolar acidification and autophagy are important for yeast cell death caused by propolis. Here, we extended our understanding of propolis-mediated cell death in the yeast Saccharomyces cerevisiae by applying systems biology tools to analyze the transcriptional profiling of cells exposed to propolis. METHODS: We have used transcriptional profiling of S. cerevisiae exposed to propolis. We validated our findings by using real-time PCR of selected genes. Systems biology tools (physical protein-protein interaction [PPPI] network) were applied to analyse the propolis-induced transcriptional bevavior, aiming to identify which pathways are modulated by propolis in S. cerevisiae and potentially influencing cell death. RESULTS: We were able to observe 1,339 genes modulated in at least one time point when compared to the reference time (propolis untreated samples) (t-test, p-value 0.01). Enrichment analysis performed by Gene Ontology (GO) Term finder tool showed enrichment for several biological categories among the genes up-regulated in the microarray hybridization such as transport and transmembrane transport and response to stress. Real-time RT-PCR analysis of selected genes showed by our microarray hybridization approach was capable of providing information about S. cerevisiae gene expression modulation with a considerably high level of confidence. Finally, a physical protein-protein (PPPI) network design and global topological analysis stressed the importance of these pathways in response of S. cerevisiae to propolis and were correlated with the transcriptional data obtained thorough the microarray analysis. CONCLUSIONS: In summary, our data indicate that propolis is largely affecting several pathways in the eukaryotic cell. However, the most prominent pathways are related to oxidative stress, mitochondrial electron transport chain, vacuolar acidification, regulation of macroautophagy associated with protein target to vacuole, cellular response to starvation, and negative regulation of transcription from RNA polymerase II promoter. Our work emphasizes again the importance of S. cerevisiae as a model system to understand at molecular level the mechanism whereby propolis causes cell death in this organism at the concentration herein tested. Our study is the first one that investigates systematically by using functional genomics how propolis influences and modulates the mRNA abundance of an organism and may stimulate further work on the propolis-mediated cell death mechanisms in fungi.

Dataset Information

Spt10 and Spt21 are required for transcriptional silencing in Saccharomyces cerevisiae.

Publications

Spt10 and Spt21 are required for transcriptional silencing in Saccharomyces cerevisiae.

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