Similar Datasets
Project description:The formation of heterochromatin at HML, HMR, and telomeres in Saccharomyces cerevisiae involves two main steps: the recruitment of Sir proteins to silencers and their spread throughout the silenced domain. We developed a method to study these two processes at single basepair resolution. Using a fusion protein between the heterochromatin protein Sir3 and the nonsite-specific bacterial adenine methyltransferase M.EcoGII, we mapped sites of Sir3-chromatin interactions genome-wide using long-read Nanopore sequencing to detect adenines methylated by the fusion protein and by ChIP-seq to map the distribution of Sir3-M.EcoGII. A silencing-deficient mutant of Sir3 lacking its Bromo-Adjacent Homology (BAH) domain, sir3-bah∆, was still recruited to HML, HMR, and telomeres. However, in the absence of the BAH domain, it was unable to spread away from those recruitment sites. Overexpression of Sir3 did not lead to further spreading at HML, HMR, and most telomeres. A few exceptional telomeres, like 6R, exhibited a small amount of Sir3 spreading, suggesting that boundaries at telomeres responded variably to Sir3-M.EcoGII overexpression. Finally, by using a temperature-sensitive allele of SIR3 fused to M.ECOGII, we tracked the positions first methylated after induction and found that repression of genes at HML and HMR began before Sir3 occupied the entire locus.
| S-EPMC8830885 | biostudies-literature
Project description:The formation of heterochromatin at HML, HMR, and telomeres in Saccharomyces cerevisiae involves two main steps: Recruitment of Sir proteins to silencers and their spread throughout the silenced domain. For the following datasets, we created a fusion protein between the heterochromatin protein Sir3 and the non-site-specific bacterial adenine methyltransferase M.EcoGII. We mapped sites of Sir3-chromatin interactions genome-wide using long-read Nanopore sequencing to detect adenines methylated by the fusion protein. We also used a temperature-sensitive allele of SIR3 (sir3-8) fused to M.ECOGII to induce m6A methylation over time. Time courses involved a switch from restrictive temperature (37°C) to permissive temperature (25°C).
2022-01-17 | GSE190136 | GEO
Project description:The formation of heterochromatin at HML, HMR, and telomeres in Saccharomyces cerevisiae involves two main steps: Recruitment of Sir proteins to silencers and their spread throughout the silenced domain. For the following datasets, we created a fusion protein between the heterochromatin protein Sir3 and the non-site-specific bacterial adenine methyltransferase M.EcoGII, with or without a 3xV5 epitope at the C-terminus. We performed ChIP-seq experiments (immunoprecipitated Sir3-M.EcoGII-3xV5) and MeDIP-seq experiments (immunoprecipitated m6A methylated DNA). We also used a temperature-sensitive allele of SIR3 (sir3-8) fused to M.ECOGII to induce m6A methylation for MeDIP-seq.
2021-11-24 | GSE189038 | GEO
Project description:Distinguishing between recruitment and spread of silent chromatin structures in Saccharomyces cerevisiae
| PRJNA785980 | ENA
Project description:Distinguishing between recruitment and spread of silent chromatin structures in Saccharomyces cerevisiae [I]
| PRJNA781240 | ENA
Project description:Distinguishing between recruitment and spread of silent chromatin structures in Saccharomyces cerevisiae [II]
| PRJNA785983 | ENA
Project description:Specialized chromatin domains repress transcription of genes within them and present a barrier to many DNA-protein interactions. Silent chromatin in the budding yeast Saccharomyces cerevisiae, akin to heterochromatin of metazoans and plants, inhibits transcription of PolII- and PolIII-transcribed genes, yet somehow grants access to proteins necessary for DNA transactions like replication and homologous recombination. In this study, we adapted a novel assay to detect even transient changes in the dynamics of transcriptional silencing at HML after it served as a template for homologous recombination. Homologous recombination specifically targeted to HML via double-strand-break formation at a homologous locus often led to transient loss of transcriptional silencing at HML Interestingly, many cells could template homology-directed repair at HML without an obligate loss of silencing, even in recombination events with extensive gene conversion tracts. In a population of cells that experienced silencing loss following recombination, transcription persisted for 2-3 hr after all double-strand breaks were repaired. mRNA levels from cells that experienced recombination-induced silencing loss did not approach the amount of mRNA seen in cells lacking transcriptional silencing. Thus, silencing loss at HML after homologous recombination was short-lived and limited.
| S-EPMC5844325 | biostudies-literature
Project description:Transcriptional silencing in Saccharomyces cerevisiae occurs at several genomic sites including the silent mating-type loci, telomeres, and the ribosomal DNA (rDNA) tandem array. Epigenetic silencing at each of these domains is characterized by the absence of nearly all histone modifications, including most prominently the lack of histone H4 lysine 16 acetylation. In all cases, silencing requires Sir2, a highly-conserved NAD(+)-dependent histone deacetylase. At locations other than the rDNA, silencing also requires additional Sir proteins, Sir1, Sir3, and Sir4 that together form a repressive heterochromatin-like structure termed silent chromatin. The mechanisms of silent chromatin establishment, maintenance, and inheritance have been investigated extensively over the last 25 years, and these studies have revealed numerous paradigms for transcriptional repression, chromatin organization, and epigenetic gene regulation. Studies of Sir2-dependent silencing at the rDNA have also contributed to understanding the mechanisms for maintaining the stability of repetitive DNA and regulating replicative cell aging. The goal of this comprehensive review is to distill a wide array of biochemical, molecular genetic, cell biological, and genomics studies down to the "nuts and bolts" of silent chromatin and the processes that yield transcriptional silencing.
| S-EPMC4981263 | biostudies-literature
Project description:Silent chromatin in Saccharomyces cerevisiae is established in a stepwise process involving the SIR complex, comprised of the histone deacetylase Sir2 and the structural components Sir3 and Sir4. The Sir3 protein, which is the primary histone-binding component of the SIR complex, forms oligomers in vitro and has been proposed to mediate the spreading of the SIR complex along the chromatin fiber. In order to analyze the role of Sir3 in the spreading of the SIR complex, we performed a targeted genetic screen for alleles of SIR3 that dominantly disrupt silencing. Most mutations mapped to a single surface in the conserved N-terminal BAH domain, while one, L738P, localized to the AAA ATPase-like domain within the C-terminal half of Sir3. The BAH point mutants, but not the L738P mutant, disrupted the interaction between Sir3 and nucleosomes. In contrast, Sir3-L738P bound the N-terminal tail of histone H4 more strongly than wild-type Sir3, indicating that misregulation of the Sir3 C-terminal histone-binding activity also disrupted spreading. Our results underscore the importance of proper interactions between Sir3 and the nucleosome in silent chromatin assembly. We propose a model for the spreading of the SIR complex along the chromatin fiber through the two distinct histone-binding domains in Sir3.
| S-EPMC2573294 | biostudies-literature
Project description:Project abstract : The trimethylation of histone H3 lysine 4 (H3K4me3) is a crucial factor in defining the promoter regions of active genes in all eukaryotes ranging from Saccharomyces cerevisiae (yeast) to humans. In budding yeast, this trimethylation process facilitated by the Set1 complex results in H3K4me3 requiring a prior mono-ubiquitination at the histone H2BK123 residue (H2Bub) by E2 enzyme Rad6 and E3 enzyme Bre1. A previous in vitro study suggested that ubiquitinated H2B directly facilitates H3K4me3. However, even low levels of global H2Bub is sufficient for the required H3K4me3 in yeast cells, thereby indicating that other factors resulting in the H2Bub-dependent H3K4me3 remain unknown. This study revealed the high level of correlation of H3K4me3 with chromatin recruitment of Rad6 at the genome-wide level. Rad6 is confirmd to interact and co-localize with Swd2/Cps35, a key factor for the H2Bub-dependent H3K4me3 in genes with high levels of H3K4me3 and intronic genes rather than non-intronic genes. This study therefore provides a mechanistic insight of the H2Bub-Rad6- Swd2/Cps35-H3K4me3 axis and its potential role in RNA biogenesis.
2022-06-07 | GSE146296 | GEO