Global gene expression analysis of H4T80A mutants vs WT H4 in Saccharomyces cerevisiae
Ontology highlight
ABSTRACT:
INSTRUMENT(S): NextSeq 500
ORGANISM(S): Saccharomyces cerevisiae
SUBMITTER: Sam Robson
PROVIDER: E-MTAB-7090 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:This SuperSeries is composed of the SubSeries listed below. Refer to individual Series
2012-11-30 | E-GEOD-38384 | biostudies-arrayexpress
Project description:Anti-sense non-coding transcripts, genes-within-genes, and convergent gene pairs are prevalent among eukaryotes. The existence of such transcription units raises the question of what happens when RNA polymerase II (RNAPII) molecules collide head-to-head. Here we use a combination of biochemical and genetic approaches in yeast to show that polymerases transcribing opposite DNA strands cannot bypass each other. RNAPII stops, but does not dissociate upon head-to-head collision in vitro, suggesting that opposing polymerases represent insurmountable obstacles for each other. Head-to-head collision in vivo results in RNAPII stopping as well, and removal of collided RNAPII from the DNA template can be achieved via ubiquitylation-directed proteolysis. Indeed, in cells lacking efficient RNAPII poly-ubiquitylation, the half-life of collided polymerases increases, so that these can be detected between convergent genes by ChIP-Seq. These results provide new insight into fundamental mechanisms of gene traffic control, and point to an unexplored effect of anti-sense transcription on gene regulation via polymerase collision. ChIP-Seq of RNA polymerase II was performed with WT and Elongain C deletion mutant (elc1M-bM-^HM-^F) cells. 4H8 antibody against the Rpb1 C-terminal domain was used for RNA polymerase II immunoprecipitation, whilst mouse IgG antibody was used for control immunoprecipitations. Two biological replicates were performed for both WT and elc1M-bM-^HM-^F.
2012-11-30 | E-GEOD-38382 | biostudies-arrayexpress
Project description:Anti-sense non-coding transcripts, genes-within-genes, and convergent gene pairs are prevalent among eukaryotes. The existence of such transcription units raises the question of what happens when RNA polymerase II (RNAPII) molecules collide head-to-head. Here we use a combination of biochemical and genetic approaches in yeast to show that polymerases transcribing opposite DNA strands cannot bypass each other. RNAPII stops, but does not dissociate upon head-to-head collision in vitro, suggesting that opposing polymerases represent insurmountable obstacles for each other. Head-to-head collision in vivo results in RNAPII stopping as well, and removal of collided RNAPII from the DNA template can be achieved via ubiquitylation-directed proteolysis. Indeed, in cells lacking efficient RNAPII poly-ubiquitylation, the half-life of collided polymerases increases, so that these can be detected between convergent genes by ChIP-Seq. These results provide new insight into fundamental mechanisms of gene traffic control, and point to an unexplored effect of anti-sense transcription on gene regulation via polymerase collision. Total RNA was extracted from WT or Elongin C deletion mutant (elc1M-bM-^HM-^F) cells and strand-specific RNA-Seq was performed. Three biological replicates were performed for WT and elc1M-bM-^HM-^F.
2012-11-30 | E-GEOD-38383 | biostudies-arrayexpress
Project description:Sequencing DNA fragments associated with proteins following in vivo cross-linking with formaldehyde (known as ChIP-seq) has been used extensively to describe the distribution of proteins across genomes. It is not widely appreciated that this method merely estimates a protein’s distribution and cannot reveal changes in occupancy between samples. To do this, we tagged with the same epitope orthologous proteins in Saccharomyces cerevisiae and Candida glabrata, whose sequences have diverged to a degree that most DNA fragments longer than 50 bp are unique to just one species. By mixing defined numbers of C.glabrata cells (the calibration genome) with S.cerevisiae samples (the experimental genomes) prior to chromatin fragmentation and immunoprecipitation, it is possible to derive a quantitative measure of occupancy (the occupancy ratio – OR) that enables a comparison of occupancies not only within but also between genomes. We demonstrate for the first time that this “internal standard” calibration method satisfies the sine qua non for quantifying ChIP-seq profiles, namely linearity over a wide range. Crucially, by employing functional tagged proteins, our calibration process describes a method that distinguishes genuine association within ChIP-seq profiles from background noise. Our method is applicable to any protein, not merely highly conserved ones, and obviates the need for the time consuming, expensive, and technically demanding quantification of ChIP using PCR, which can only be performed on individual loci. As we demonstrate for the first time in this paper, calibrated ChIP-seq represents a major step towards documenting the quantitative distributions of proteins along chromosomes in different cell states, which we term biological chromodynamics. Develop a method for quantitative ChIP-seq
2015-07-10 | E-GEOD-69907 | biostudies-arrayexpress
Project description:Sister chromatid cohesion conferred by entrapment of sister DNAs within a tripartite ring formed between cohesinâs Scc1, Smc1, and Smc3 subunits is generated during S and eventually destroyed at anaphase through cleavage of Scc1 by separase. Throughout the cell cycle, cohesinâs association with chromosomes is controlled by opposing activities: loading by the Scc2/4 complex and release by a separase independent releasing activity. Co-entrapment of sister DNAs during replication is accompanied by acetylation of Smc3 by Eco1, which blocks releasing activity and ensures that sisters remain stably connected. Because fusion of Smc3 to Scc1 prevents release and bypasses the requirement for Eco1, we suggested that release is mediated by disengagement of the Smc3/Scc1 interface. We now show that all mutations capable of bypassing Eco1, be they in cohesinâs Smc1, Smc3, Scc1,Wapl, Pds5, or Scc3 subunits, greatly reduce dissociation of N-terminal cleavage fragments of Scc1 (NScc1) from Smc3. We show that this process involves interaction between Smc ATPase heads and is inhibited by Smc3 acetylation Effect of mutations QQ and EQ in Smc3 on cohesin loading onto chromosomes
2016-02-18 | E-GEOD-76890 | biostudies-arrayexpress
Project description:Cohesin stably holds together the sister chromatids from S phase until mitosis. To do so, cohesin must be protected against its cellular antagonist Wapl. Eco1 acetylates cohesinâs Smc3 subunit, which locks together the sister DNAs. We used yeast genetics to dissect how Wapl drives cohesin from chromatin and identified mutants of cohesin that are impaired in ATPase activity but remarkably confer robust cohesion that bypasses the need for the cohesin protectors Eco1 in yeast and Sororin in human cells. We uncover an unexpected functional asymmetry within the heart of cohesinâs highly conserved ABC-like ATPase machinery and show that an activity associated with one of cohesinâs two ATPase sites drives DNA release from cohesin rings. This key mechanism is conserved from yeast to humans. We propose that Eco1 locks cohesin rings around the sister chromatids by counteracting an asymmetric cohesin-associated ATPase activity. Effect of mutations in Smc1 and Smc3 on cohesin loading onto chromosomes
2016-02-18 | E-GEOD-76891 | biostudies-arrayexpress
Project description:Nuclear pore complexes (NPCs) influence gene expression besides their established function in nuclear transport. The TREX-2 complex localizes to the NPC basket and affects gene-NPC interactions, transcription and mRNA export. How TREX-2 regulates the gene expression machinery is unknown. Here, we show that TREX-2 interacts with the Mediator complex, an essential regulator of RNA Polymerase (Pol) II. Structural and biochemical studies identify a conserved region on TREX-2, which directly binds the Mediator Med31/Med7N submodule. TREX-2 regulates assembly of Mediator with its Cdk8 kinase and is required for recruitment and site-specific phosphorylation of Pol II. Transcriptome and phenotypic profiling confirm that TREX-2 and Med31 are functionally interdependent. TREX-2 additionally uses its Mediator-interacting surface to regulate mRNA export suggesting a mechanism for coupling transcription initiation and early steps of mRNA processing at the Mediator level. In sum, we provide insight into how NPC-associated adaptor complexes can access the core transcription machinery. RNAseq was performed from WT, sac3∆, cdk8∆ and Sac3 R288D mutant cells. For each strain triplicates were analyzed. WT strain was sac3∆ transformed with pRS315 SAC3 WT
2015-08-27 | E-GEOD-67431 | biostudies-arrayexpress
Project description:The production of Tobacco Acid Pyrophosphatase (TAP), an enzyme commonly used for the removal of the 5â??cap of eukaryotic mRNAs, has been recently discontinued. Here we performed a comparison of current alternatives for the mapping of 5â??cap mRNAs and the associated transcription start sites in Sacharomyces cerevisiae. Specifically we compared TAP with Cap-clip and a Decapping Pyrophosphohydrolase. Our results suggest that Cap-clip is a good alternative for TAP. We used two biological replicates of S. cerevisiae that was grown to exponential phase (OD600 ~1) in rich media (YPAD). Samples where processed until the dephosphorylation step (CIP treatment). After that each sample was split in 4 aliquots: TAP treatment, Cap-Clip treatment, Decapping Pyrophosphohydrolase treatment or no treatment (negative control). From that step all samples are processed in parallel.
2016-01-06 | E-GEOD-73819 | biostudies-arrayexpress
Project description:It is generally assumed that mRNAs undergoing translation are protected from decay. Here, we show that mRNAs are, in fact, co-translationally degraded. This is a widespread and conserved process affecting most genes, where 5′–3′ transcript degradation follows the last translating ribosome, producing an in vivo ribosomal footprint. By sequencing the ends of 5′ phosphorylated mRNA degradation intermediates, we obtain a genome-wide drug-free measurement of ribosome dynamics. We identify general translation termination pauses in both normal and stress conditions. In addition, we describe novel codon-specific ribosomal pausing sites in response to oxidative stress that are dependent on the RNase Rny1. Our approach is simple and straightforward and does not require the use of translational inhibitors or in vitro RNA footprinting that can alter ribosome protection patterns. RNA samples were quantified according to their 5’modification. Samples were quantified using 2 alternative methods. 5PSeq (measures 5’P RNAs) and 5PSeq-fragmented (measures the sequencing bias present in any sample by randomly fragmenting RNA, re-phosphorilating the 5’ends and subjecting the sample to 5PSeq; it is used as a negative control). Different strains of S. cerevisiae and S. pombe samples were analyzed. Cells were grown both in rich media (YPD or YES) and synthetic defined media (SD). Cells were subjected to different treatments such as inhibition of translation elongation with cyclohexamide (CHX), oxidative stress (0.2 mM H2O2) or inhibition of histidine biosyntesys (3-AT; 100mM 3-amino-1,2,4-triazole). S. cerevisiae samples grown in rich media were also analyzed after CHX treatment and sucrose fractionation into monosome and polyribosome fractionation. All samples were analyzed in biological duplicates.
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Project description:LncRNAs represent a major transcriptional output of the human genome, but the function of many of these elements is unknown. In this experiment, we have used the ‘CUT&RUN’ technique to quantify the changes in histone mark enrichment across the genome upon depletion of the lncRNA LINC00899 in HeLa cells. LINC00899 is of interest as its depletion results in mitotic delay, suggesting a role in mitotic progression. The CUT&RUN procedure uses antibodies to target a nuclease to the relevant protein binding site, cleaving the surrounding DNA for sequencing and yielding output equivalent to that of ChIP-seq. This experiment contains 2 replicate batches where each batch contains a sample with LINC00899 knocked down by RNA interference (RNAi); a RNAi negative control; a sample with LINC00899 knocked down with locked nucleic acid antisense oligonucleotides (LNA); and a LNA negative control. This was performed using antibodies against H3K4me3, H3K27ac, H3K36me3 and H3K27me3, as well as an IgG (goat-derived anti-rabbit) control. All samples in each batch were generated at the same time. Within each batch, all CUT&RUN experiments with the same knockdown were performed on nuclei obtained from the same cell culture. Independent cell cultures were used for experiments with different knockdown or in different batches.
2019-03-29 | E-MTAB-7419 | biostudies-arrayexpress