Similar Datasets
Project description:Sequence-specific DNA-binding proteins including transcription factors (TFs) are key determinants of gene regulation and chromatin architecture. Formaldehyde cross-linking and sonication followed by Chromatin ImmunoPrecipitation (X-ChIP) and sequencing is widely used for genome-wide profiling of protein binding, but is limited by low resolution and poor specificity and sensitivity. We have implemented a simple genome-wide ChIP protocol that starts with micrococcal nuclease-digested uncross-linked chromatin followed by affinity purification and paired-end sequencing without size-selection. The resulting ORGANIC (Occupied Regions of Genomes from Affinity-purified Naturally Isolated Chromatin) profiles of the budding yeast TFs Abf1 and Reb1 achieved near-perfect accuracy, in contrast to other profiling methods, which were much less sensitive and specific. Unlike profiles produced using X-ChIP methods such as ChIP-exo, ORGANIC profiles are not biased toward identifying sites in accessible chromatin and do not require input normalization. We also demonstrate the high specificity of our method when applied to larger genomes by profiling Drosophila GAGA Factor and Pipsqueak. Taken together, these results suggest that ORGANIC profiling outperforms current X-ChIP methodologies for genome-wide profiling of TF binding sites. Chromatin immunoprecipitation of micrococcal nuclease-digested native chromatin followed by paired-end sequencing (Occupied Regions of Genomes from Affinity-purified Naturally Isolated Chromatin 'ORGANIC' profiling) of DNA-binding proteins Abf1 and Reb1 from S. cerevisiae and GAGA-binding factor (GAF) and Pipsqueak (Psq) from D. melanogaster S2 cells; and, Sono-seq (paired-end sequencing of formaldehyde cross-linked and sonicated chromatin) of yeast nuclei. Reb1 ORGANIC profiling was performed at three different salt (NaCl) concentrations (80, 150, and 600 mM) and Abf1 ORGANIC profiling was done at two different salt concentrations (80 and 600 mM) to achieve varying levels of stringency. GAF and Psq ORGANIC profiles were determined at 80 mM salt. Two replicates each of Reb1 and Abf1 600 mM ORGANIC experiments, mixed Drosophila S2 cell and S. cerevisiae nuclei Reb1 ORGANIC experiments, yeast Sono-seq, and GAF and Psq ORGANIC experiments were performed. Each S. cerevisiae and mixed S2 cell/yeast ORGANIC profiling experiment included separately sequenced input chromatin and ChIP samples. Total of 24 samples.
2013-12-14 | E-GEOD-45672 | biostudies-arrayexpress
Project description:The occupancy of nucleosomes governs access to the eukaryotic genomes and results from a combination of biophysical features and the effect of ATP-dependent remodeling complexes. Most promoter regions show a conserved pattern characterized by a nucleosome-depleted region (NDR) flanked by nucleosomal arrays. The conserved RSC remodeler was reported to be critical to establish NDR in vivo in budding yeast but other evidences suggested that this activity may not be conserved in fission yeast. By reanalysing and expanding previously published data, we propose that NDR formation is dependent on RSC in both yeast species. We also discuss the most prominent biological role of RSC and the possibility that non-essential subunits define alternate versions of the complex. Samples from mononucleosomal DNA from S. pombe strains h- kanR-tetO-snf21-Tap-natR ura4::rTetR-tup11 were sequenced (Illumina NextSeq 500 platform) using the pair-end read protocol
2016-08-24 | E-GEOD-84912 | biostudies-arrayexpress
Project description:Purpose: Saccharomyceatacea yeast are intron-poor species and they contain on average 300 introns in their genomes. We designed RNAseq experiment to investigate if splicing patterns in related yeast species are similar. Methods: Total RNA was extracted from wild type cells and processed by the RiboMinus Transcriptome Isolation Kit for Yeast and Bacteria (Invitrogen) to deplete the rRNA. cDNA libraries were prepared according to manufacturer's protocol and sequenced by SOLiD. Sequence reads were filtered and processed by TopHat. Results: We found 216, 163, 200 and 155 predicted introns with canonical splice signals in S. cerevisiae, S. kudriavzevii, S. bayanus and N. castellii respectively. Three introns in S. cerevisiae, four in S. bayanus and ten in S. castellii are novel compared to Saccharomyces Genome Database (SGD) annotations. The expression of introns and splicing shows very high correlation between species. Conclusion: Transcripts with introns in yeast species tested show similar levels of expression and splicing. We found few novel introns, which are conserved in yeast genomes.
2014-09-15 | GSE58884 | GEO
Project description:The occupancy of nucleosomes governs access to the eukaryotic genomes and results from a combination of biophysical features and the effect of ATP-dependent remodeling complexes. Most promoter regions show a conserved pattern characterized by a nucleosome-depleted region (NDR) flanked by nucleosomal arrays. The conserved RSC remodeler was reported to be critical to establish NDR in vivo in budding yeast but other evidences suggested that this activity may not be conserved in fission yeast. By reanalysing and expanding previously published data, we propose that NDR formation is dependent on RSC in both yeast species. We also discuss the most prominent biological role of RSC and the possibility that non-essential subunits define alternate versions of the complex.
2016-08-24 | GSE84912 | GEO
Project description:Whether synthetic genomescan power life has attracted broad interest in the synthetic biology field, especially when the synthetic genomes are extensively modified with thousands of designer features. Here we reportde novosynthesis of the largest eukaryotic chromosome thus far, synIV, a 1,454,621-bpSaccharomyces cerevisiaechromosome resulting from extensive genome streamlining and modification. During the construction ofsynIV, we developed megachunk assembly combined with a hierarchical integration strategy, which significantly increased the accuracy and flexibility of synthetic chromosome construction and facilitated chromosome debugging. In addition to the drastic sequence changes made to synIV by rewriting it, we further manipulated the three-dimensional structure of synIV in the yeast nucleus to explore spatial gene regulation within the nuclear space. Surprisingly, we found few gene expression changes, suggesting that positioning inside the yeast nucleoplasm plays a minor role in gene regulation. Lastly, we tethered synIV to the inner nuclear membrane via its hundreds of loxPsym sites and observed transcriptional repression of the entire chromosome, demonstrating chromosome-wide transcription manipulation without changing the DNA sequences. Our manipulation of the spatial structure of the largest synthetic yeast chromosome shed light on higher-order architectural design of the synthetic genomes.
2023-08-04 | GSE239535 | GEO
Project description:Mitochondrial DNA (mtDNA) in budding yeast is biparentally inherited, but colonies rapidly lose one type of parental mtDNA, becoming homoplasmic. Therefore, hybrids between different yeast species possess two homologous nuclear genomes, but only one type of mitochondrial DNA. We hypothesise that the choice of mtDNA retention is influenced by its contribution to hybrid fitness in different environments, and that the allelic expression of the two nuclear sub-genomes is affected by the presence of different mtDNAs in hybrids. Here, we crossed Saccharomyces cerevisiae with S. uvarum under different environmental conditions and examined the plasticity of the retention of mtDNA in each hybrid.
2020-04-16 | E-MTAB-7922 | biostudies-arrayexpress
Project description:Mitochondrial DNA (mtDNA) in budding yeast is biparentally inherited, but colonies rapidly lose one type of parental mtDNA, becoming homoplasmic. Therefore, hybrids between different yeast species possess two homologous nuclear genomes, but only one type of mitochondrial DNA. We hypothesise that the choice of mtDNA retention is influenced by its contribution to hybrid fitness in different environments, and that the allelic expression of the two nuclear sub-genomes is affected by the presence of different mtDNAs in hybrids. Here, we crossed Saccharomyces cerevisiae with S. uvarum under different environmental conditions and examined the plasticity of the retention of mtDNA in each hybrid.
2020-10-26 | E-MTAB-8900 | biostudies-arrayexpress