IMUT-seq analysis of over 20 different DSB repair siRNA mediated knockdowns or small molecule inhibitors
Ontology highlight
ABSTRACT:
INSTRUMENT(S): NextSeq 500
ORGANISM(S): Homo sapiens
SUBMITTER: Aldo Bader
PROVIDER: E-MTAB-11259 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:DNA Double Strand Breaks (DSBs) repair is essential to safeguard genome integrity but little is known about the contribution of chromosome folding into these processes. Here we unveiled basic principles of chromosome dynamics occurring post-DSB both locally and at a genome wide scale in mammalian cells. We report that topologically associating domains (TAD) that experience a DSB undergo acute ATM-dependent but DNAPK- independent changes. Within these damaged TADs, DSB-induced loop extrusion ensures local transcriptional regulation in response to DSBs. Damaged TADs further coalesce in an ATM-dependent manner, forming a new “D” compartment, where upregulated genes of the DNA damage response (DDR) physically localize suggesting a function of DSB clustering in activating the DNA damage Response. However, these alterations of chromosome folding induced by DSB also come at the expense of an increased translocations rate. Our work highlights the critical impact of chromosome conformation in the maintenance of genome integrity.
2023-11-17 | E-MTAB-10865 | biostudies-arrayexpress
Project description:Differentiation into diverse cell lineages requires orchestration of gene regulatory networks guiding cell fate choices. Genetic factors acting through changes in transcriptional levels can contribute to cardiovascular disease risk by impacting early stages of development and have cell type-specific effects. We set out to characterize lineage trajectory progression of subpopulations and identify potential disease-related genes by examining their expression changes in single cells during early stages of cardiac lineage specification. Using 43,168 single-cell transcriptomes, we developed novel classification and trajectory analysis methods to dissect cellular composition and gene networks across five discrete time points underlying lineage derivation of mesoderm, definitive endoderm, vascular endothelium, cardiac precursors, and definitive cell types that comprise cardiomyocytes and a previously unrecognized cardiac outflow tract population.
2018-10-06 | E-MTAB-6268 | biostudies-arrayexpress
Project description:Sister chromatid cohesion, established during replication by the protein complex Cohesin, is essential for both chromosome segregation and double-strand break (DSB) repair. Normally cohesion formation is strictly limited to the S-phase of the cell cycle, but DSBs can trigger cohesion also after DNA replication has been completed. The function of this damage-induced cohesion remains unknown. In this investigation we show that it is essential for repair in post-replicative cells in yeast. Furthermore, it is established genome-wide after induction of a single DSB, and controlled by the DNA damage response and Cohesin regulating factors. We thus define a cohesion establishment pathway that is independent of DNA duplication and acts together with cohesion formed during replication in sister chromatid-based DSB repair. Keywords: ChIP-chip analysis Scc2, Scc1, and rH2AX distribution with or without DSB in wild type and mutant strains. DSB was induced by galactose promoter driven HO. All yeast strains were haploid and of W303 origin (ade2-1, trp1-1, can1-100, leu2-3, 112, his3-11, 15, ura3, RAD5). DNA was purified and amplified as previously described by Katou et al. (nature, 2003), and hybridized to SC3456a 52005F or S.cerevisiae Tiling 1.0F arrays, Part#520286 (Affymetrix). Scc2 was C- terminally tagged with 6HIS-3xFLAG, while Mcd1UNCL was marked with 3HA epitopes. Anti-FLAG antibody M2 (Sigma), anti-HA antibody (Babco), or anti γ-H2A-antobodies kindly provided by A. Verreault were used for immuno-precipitations.
2010-06-10 | E-GEOD-8270 | biostudies-arrayexpress
Project description:Spo11-mediated DNA double strand breaks (DSBs) that initiate meiotic recombination are temporally and spatially controlled. The meiotic cohesin Rec8 has been implicated in regulating DSB formation, but little is known about the features of their interplay. To shed light on this point, we investigated the genome-wide localization of Spo11 in budding yeast during early meiosis by chromatin immunoprecipitation using high-density tiling arrays. We found that Spo11 is dynamically localized to meiotic chromosomes. Spo11 initially accumulated around centromeres and thereafter localized to arm regions as premeiotic S-phase proceeded. During this stage, a substantial proportion of Spo11 bound to Rec8 binding sites. Eventually, some of Spo11 further bound to both DSB and Rec8 sites. We also showed that such a change in a distribution of Spo11 is affected by hydroxyurea (HU) treatment. Interestingly, deletion of REC8 influences the localization of Spo11 to centromeres and in some of the intervals of the chromosomal arms. Thereby we observed a lack of DSB formation in a region-specific manner. These observations suggest that Rec8 would prearrange the distribution of Spo11 along chromosomes and will provide clues to understanding temporal and spatial regulation of DSB formation. Keywords: ChIP-chip â?¢ The goal of the experiment Genome-wide localization of Spo11, Mre11, Rec8, and DSB sites on meiotic chromosomes in Saccharomyces cerevisiae â?¢ Keywords Meiosis, Meiotic homologous recombination, Premeiotic DNA replication, cohesin, Saccharomyces cerevisiae, Genome tilling array (chromosome III, IV, V, VI), Spo11, Mre11, Rec8, DSB (Double strand break) â?¢ Experimental factor Distribution of Spo11, Mre11, and Rec8 in wild type in early meiosis (1.5 hrs, 2 hrs, 3 hrs, 4 hrs, and 5 hrs in sporulation medium) Distribution of Spo11 in rec8delta cells in early meiosis (1.5 hrs, 2 hrs, 3 hrs, 4 hrs, and 5 hrs in sporulation medium) Distribution of Spo11 in wild type in the presence of HU (2hrs and 4 hrs in sporulation medium containing HU) Distribution of DSB sites in rad50S mutant cells at 7 hrs in sporulation medium Distribution of DSB sites in rec8delta rad50S mutant cells at 7 hrs in sporulation medium â?¢ Experimental design ChIP analyses: SK1 background cells expressing FLAG tagged protein were used for the ChIP using anti-FLAG M2 antibody. ChIP-chip analyses: In all cases, hybridization data for ChIP fraction was compared with WCE (whole cell extract) fraction. Saccharomyces cerevisiae affymetrix genome tiling array (SC3456a520015F for chromosome III, IV, V, VI and rikDACF for chromosome VI) were used. Mapping of DSB sites: DSB rich fraction was concentrated by ChIP of Spo11-FLAG in rad50S mutant without crosslinking. In the mutant, DSBs ramain unrepaired with covalently attached Spo11.Meiotic cells (at 7 hours in sporulation medium) were used for the analyses. â?¢ Quality control steps taken Confirmation of several loci by quantitative real time PCR. Southern blotting of several DSB sites.
2010-05-18 | E-GEOD-8422 | biostudies-arrayexpress
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2015-08-21 | E-GEOD-67910 | biostudies-arrayexpress
Project description:The nonrandom distribution of meiotic recombination shapes patterns of inheritance and genome evolution, but chromosomal features governing this distribution are poorly understood. Formation of the DNA double-strand breaks (DSBs) that initiate recombination results in accumulation of Spo11 protein covalently bound to small DNA fragments. We show here that sequencing these fragments provides a genome-wide DSB map of unprecedented resolution and sensitivity. We use this map to explore the influence of large-scale chromosome structures, chromatin, transcription factors, and local sequence composition on DSB distributions. Our analysis supports the view that the recombination terrain is molded by combinatorial and hierarchical interaction of factors that work on widely different size scales. Mechanistic aspects of DSB formation and early processing steps are also uncovered. This map illuminates the occurrence of DSBs in repetitive DNA elements, repair of which can lead to chromosomal rearrangements. We discuss implications for evolutionary dynamics of recombination hotspots. We deep sequenced 4 samples of Spo11 oligos on Roche 454 platform. Three samples are technical replicates of Spo11 oligo products prepared from one meiotic culture and the fourth sample was prepared from an independent culture.
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2014-03-31 | E-GEOD-52858 | biostudies-arrayexpress
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Project description:Meiotic chromosome architecture called M-bM-^@M-^\axis-loop structuresM-bM-^@M-^] and histone modifications have been demonstrated to regulate the Spo11-dependent formation of DNA double-strand breaks (DSBs) that trigger meiotic recombination. Using genome-wide chromatin immunoprecipitation (ChIP) analyses followed by deep sequencing, we compared the genome-wide distribution of the axis protein Rec8 (the kleisin subunit of meiotic cohesin) with that of oligomeric DNA covalently bound to Spo11, indicative of DSB sites. The frequency of DSB sites is overall constant between Rec8 binding sites. However, DSB cold spots are observed in regions spanning M-BM-10.8 kb around Rec8 binding sites. The axis-associated cold spots are not due to exclusion of Spo11 localization from the axis, since ChIP experiments revealed that substantial Spo11 persists at Rec8 binding sites during DSB formation. Spo11 fused with Gal4 DNA binding domain (Gal4BD-Spo11) tethered in close proximity (M-bM-^IM-$0.8 kb) to Rec8 binding sites hardly forms meiotic DSBs, in contrast with other regions. In addition, H3K4 tri-methylation (H3K4me3) remarkably decreases at Rec8 binding sites. These results suggest that reduced histone H3K4me3 in combination with inactivation of Spo11 activity on the axis discourages DSB hot spot formation. ChIP-seq analyses of Rec8, Spo11, and Gal4BD-Spo11 on budding yeast meiotic chromosomes M-bM-^@M-" Distribution of Rec8 in wt and Gal4BD-Spo11-expressing cells at 4h after meiotic induction M-bM-^@M-" Distribution of Spo11 at 3h, 4h, and 5h after meiotic induction M-bM-^@M-" Distribution of Gal4BD-Spo11 at 0h after meiotic induction
2014-03-31 | E-GEOD-52862 | biostudies-arrayexpress