Break-Seq and RNA-seq analysis of gene expression during and after exposure to hydroxyurea in WT and mec1 cells in Saccharomyces cerevisiae
Ontology highlight
ABSTRACT:
ORGANISM(S): Saccharomyces cerevisiae
SUBMITTER: Wenyi Feng
PROVIDER: E-GEOD-63517 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
Similar Datasets
Project description:Break-chip (microarray-based double strand break mapping) analysis of mec1 cells recovering from 200 mM hydroxyurea in the presence or absence of 0.8 micromolar bathophenanthroline sulfonate (BPS). We asked if the presence of an iron chelator, BPS, during cell recovery from transient exposure to 200 mM hydroxyurea changes the global patterns of DNA double strand breaks (DSBs). We used a yeast checkpoint mutant, mec1, which has been shown to produce DSBs at replication forks after hydroxyurea was removed from the cell culture. We synchronously released cells from the G1/S transition into S phase in the presence of 200 mM hydroxyurea. After 1h treatment, the drug was removed and cells were allowed to recover in fresh medium for 1h in the presence or absence of 0.8 micromolar BPS. Recover (R) samples, R-1h-BPS and R-1h+BPS, as well as the G1 control samples were collected. Break-chip analysis was performed as previously described (Feng et al., G3(Bethesda) 2011 Oct;1(5):327-35. doi: 10.1534/g3.111.000554).
2015-01-12 | E-GEOD-64446 | biostudies-arrayexpress
Project description:The DNA double-strand breaks that initiate homologous recombination during meiosis are subject to extensive 5ʹâ??3â?² exonucleolytic processing. This resection is a central and conserved feature of recombination, yet its mechanism is poorly understood. Using a purpose-made deep-sequencing method, namely S1Seq, we mapped meiotic resection endpoints genome-wide at high spatial resolution in Saccharomyces cerevisiae. Generating full-length resection tracts requires Exo1 exonuclease activity and the DNA-damage responsive kinase Tel1, but not the helicase Sgs1. Tel1 is also required for efficient and timely initiation of resection. We find that distributions of resection endpoints at individual genomic loci display pronounced heterogeneity that reflects a tendency for nucleosomes to block Exo1 in vivo, yet modeling experiments indicate that Exo1 digests chromatin with high apparent processivity and at rates approaching those for naked DNA in vitro. This paradox points to nucleosome destabilization or eviction as a determining feature of the meiotic resection landscape. 36 samples total: two biological replicate S1Seq maps of S.cerevisiae for each genetic background and time point in meiosis
2016-08-06 | E-GEOD-85253 | biostudies-arrayexpress
Project description:We previously demonstrated that inactivation of the replication checkpoint via a mec1 mutation led to chromosome breakage at replication forks initiated from virtually all origins of replication, after transient exposure to hydroxyurea (HU), an inhibitor of ribonucleotide reductase. Furthermore, we have shown that chromosomes break at replication forks that have suffered single-stranded DNA (ssDNA) formation. Here we sought to determine whether all replication forks containing ssDNA gaps have equal probability of producing double strand breaks (DSBs) when cells attempt to recover from HU exposure. We devised a new methodology, Break-Seq, that combines our previously described DSB labeling with NextGen sequencing to map chromosome breaks with improved sensitivity and resolution. We show that DSBs preferentially occur at genes transcriptionally induced by HU. Notably, different subsets of the HU-induced genes produced DSBs in MEC1 and mec1 cells as replication forks traversed greater distance in MEC1 cells than in mec1 cells during the recovery from HU. Specifically, while MEC1 cells exhibited chromosome breakage at stress-response transcription factors, mec1 cells predominantly suffered chromosome breakage at transporter genes, many of which are the substrates of the said transcription factors. We propose that HU-induced chromosome fragility arises at higher frequency near HU-induced genes as a result of destabilized replication forks encountering transcription factor binding and/or the act of transcription. Our model provides an explanation for a long-standing problem in chromosome biology: why different replication inhibitors produce different spectra of chromosome breakage? We propose that different inhibitors elicit different transcription responses as well as destabilize replication forks, and, when the two processes collide, ssDNA at the replication fork suffers further strand breakage, causing DSBs. We queried the yeast genome for DSBs after cells were treated with 200 mM hydroxyurea during S phase. Samples were collected from 1) cells synchronized in G1 phase by alpha factor; 2) cells released from G1 into medium containing 200 mM hydroxyurea for 1 h; 3) cells recovering in fresh medium without hydroxyurea for 1 h after the 1 h exposure to HU. These samples are referred to as G1, HU 1h, and R 1h, respectively. The strains from which the samples were collected are indicated following the time point, e.g. G1_MEC1 or R 1h_mec1. The experiment with mec1 was done twice (Experiments A and B) and that with MEC1 was done once (Experiment C). In addition, a control experiment of in vitro digestion with BamHI using the G1_mec1 sample (G1_BamHI) was performed.
2015-01-12 | E-GEOD-58808 | biostudies-arrayexpress
Project description:This SuperSeries is composed of the following subset Series: GSE21820: Genome-wide characterization of PhoB binding profile in Escherichia coli (gene expression data) GSE21856: Genome-wide characterization of PhoB binding profile in Escherichia coli (ChIP-chip data) Refer to individual Series
2012-10-21 | E-GEOD-21857 | biostudies-arrayexpress
Project description:Chromatin immunoprecipitation was combined with high-density tiling array (ChIP-chip) and gene expression microarray to reveal the adaptive responses of Escherichia coli to phosphate starvation. The first sketch of the genome-wide distribution of PhoB binding profile was unveiled and 43 regions were identified as the PhoB binding regions. The presence of a significant common motif in these binding regions allowed us to reconstruct the PhoB binding pattern. By comparing the ChIP-chip and microarray datasets, we were also able to identify genes directly or indirectly affected through PhoB regulation. Nineteen out of the 287 differentially expressed genes in the presence and absence of PhoB activity were considered as the genes directly regulated by PhoB. The adaptive responses affected through PhoB regulation are discussed and these responses involve in several important biological functions including transcriptional regulation, transportation, membrane component arrangement, sigma factor modulation and DNA replication inhibition. Comparison of the E. coli K12 MG1655 wild type and a PhoB-FLAG fusion expressing strain (MG1655_PhoB_FLAG). The anti-FLAG antibody was used to recognize PhoB-FLAG fusion protein. Therefore, ChIPed DNA from the wild type strain was used as the control group. Three biological replicates were performed.
2012-10-21 | E-GEOD-21856 | biostudies-arrayexpress
Project description:The meiotic cell division reduces the chromosome number from diploid to haploid to form gametes for sexual reproduction. Although much progress has been made in understanding meiotic recombination and the two meiotic divisions, the processes leading up to recombination, including the prolonged pre-meiotic S phase (meiS) and the assembly of meiotic chromosome axes, remain poorly defined. We have used genome-wide approaches in Saccharomyces cerevisiae to measure the kinetics of pre-meiotic DNA replication, and to investigate the interdependencies between replication and axis formation. We found that replication initiation was delayed for a large number of origins in meiS compared to mitosis, and that meiotic cells were far more sensitive to replication inhibition, most likely due to the starvation conditions required for meiotic induction. Moreover, replication initiation was delayed even in the absence of chromosome axes, indicating replication timing is independent of the process of axis assembly. Finally, we found that cells were able to install axis components and initiate recombination on unreplicated DNA. Thus, although pre-meiotic DNA replication and meiotic chromosome axis formation occur concurrently, they are not directly coupled. The functional separation of these processes reveals a modular method of building meiotic chromosomes, and predicts that any crosstalk between these modules must occur through superimposed regulatory mechanisms. Multiple studies of meiotic chromosomes were undertaken. To study DNA replication, the locations of replicative helicase (Mcm2-7) were mapped in pre-meiotic and pre-mitotic cells, and DNA replication profiles were created for pre-meiotic S (meiS) and pre-mitotic S (mitS) phases. Early origins were mapped in hydroxyurea for wild-type cells in mitS + 200mM HU, and meiS +20mM HU for wild-type, sml1, rec8 and spo11 deletion cells. Rec8, Hop1 and Red1 binding to meiotic chromosomes was evaluated using ChIP-chip in wild-type cells with and without 20 mM HU, and in cdc6-mn and clb5 clb6 delete cells. Finally, meiotic DNA double-strand breaks (DSBs) were mapped in cdc6-mn dmc1 delete cells by measuring the ssDNA that accumulates at DSB hotspots. This SuperSeries is composed of the following subset Series: GSE35658: Chromatin IP for Mcm2-7, Rec8, Hop1 and Red1 GSE35662: S phase and HU profiles in wild-type and mutant cells GSE35666: DSB formation in replication compromised cells
2012-06-14 | E-GEOD-35667 | biostudies-arrayexpress
Project description:We previously demonstrated that inactivation of the replication checkpoint via a mec1 mutation led to chromosome breakage at replication forks initiated from virtually all origins of replication, after transient exposure to hydroxyurea (HU), an inhibitor of ribonucleotide reductase. Furthermore, we have shown that chromosomes break at replication forks that have suffered single-stranded DNA (ssDNA) formation. Here we sought to determine whether all replication forks containing ssDNA gaps have equal probability of producing double strand breaks (DSBs) when cells attempt to recover from HU exposure. We devised a new methodology, Break-Seq, that combines our previously described DSB labeling with NextGen sequencing to map chromosome breaks with improved sensitivity and resolution. We show that DSBs preferentially occur at genes transcriptionally induced by HU. Notably, different subsets of the HU-induced genes produced DSBs in MEC1 and mec1 cells as replication forks traversed greater distance in MEC1 cells than in mec1 cells during the recovery from HU. Specifically, while MEC1 cells exhibited chromosome breakage at stress-response transcription factors, mec1 cells predominantly suffered chromosome breakage at transporter genes, many of which are the substrates of the said transcription factors. We propose that HU-induced chromosome fragility arises at higher frequency near HU-induced genes as a result of destabilized replication forks encountering transcription factor binding and/or the act of transcription. Our model provides an explanation for a long-standing problem in chromosome biology: why different replication inhibitors produce different spectra of chromosome breakage? We propose that different inhibitors elicit different transcription responses as well as destabilize replication forks, and, when the two processes collide, ssDNA at the replication fork suffers further strand breakage, causing DSBs. We queried the yeast genome for gene expression after cells were treated with 200 mM hydroxyurea during S phase. Samples were collected from 1) cells synchronized in G1 phase by alpha factor; 2) cells released from G1 into medium containing 200 mM hydroxyurea for 1 h; 3) cells recovering in fresh medium without hydroxyurea for 30 and 60 min after the 1 h exposure to HU. These samples are referred to as G1, HU 1h, R30, and R60, respectively. The strains from which the samples were collected are indicated before the time point, e.g. mec1_G1 or MEC1_R30. Stranded mRNA libraries were prepared according to manufacturer's suggestion and sequenced on Illumina MiSeq with paired-end reads of 75 bp.
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2017-01-10 | E-MTAB-4722 | biostudies-arrayexpress
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2014-02-20 | E-GEOD-53921 | biostudies-arrayexpress