Shuffling the yeast genome using CRISPR/Cas9-generated DSBs that target the transposable Ty1 elements
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ABSTRACT:
PROVIDER: PRJNA844083 | ENA |
REPOSITORIES: ENA
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Project description:We explored how Cas9-induced double-strand breaks (DSBs) on Ty1 produce genomic alterations in the diploid yeast Saccharomyces cerevisiae. Following Cas9 induction, we observed a significant elevation of chromosome rearrangements (large deletions and duplications), loss of heterozygosity (gene conversions, crossovers, and break-induced replication), and aneuploidy. Almost all of the chromosomal rearrangements reflect the repairing of DSBs at Ty1 elements by homologous recombination.
2022-06-01 | GSE205199 | GEO
Project description:To identify yeast proteins associated with Ty1 integrase (IN) that could regulate Ty1 replication, we co-purified IN partners using the tandem chromatin affinity purification procedure after in vivo cross-link (TChAP), which we developed previously (Nguyen et al. 2014). We first identified RNA Pol I and Pol III complexes and also a small subset of additional evolutionary conserved complexes, including PAF1 (Polymerase-Associated Factor 1),FACT (FAcilitates Chromatin Transcription), the proteasome and the CK2 kinase. We next confirmed that CK2 interacts with Ty1 integrase in vivo and repress Ty1 retromobility. We showed that Ty1 IN is a substrate of CK2 in vitro and identified 12 phosphorylated residues. In vivo approaches showed that only part of the protein was phosphorylated in the cells and did not demonstrate any direct evidence between Ty1 IN phosphorylation and retromobility inhibition.
2022-11-17 | PXD027420 | Pride
Project description:Analysis of the genome-wide distribution of the histone H2A, using TY1-H2A as a Proxy
2020-02-21 | GSE145625 | GEO
Project description:We identified robust cleavage by non-canonically-targeted SpCas9 that target protospacer with a few bases from the canonical NGG PAM in biochemistry assay. To determine whether this spaced SpCas9 cleavage exists in human cells, we employed high-throughput genome-wide translocation sequencing (LAM-HTGTS) using SpCas9 with three independent canonical sgRNAs served as bait (bait-A, bait-L and bait-D) to compared the cleavage activities of canonically-targeted and non-canonically-targeted SpCas9 in eight locus (RAG1B-D, RAG1K-O) in chromosome 11. We found that almost all 1bp-target shifted SpCas9 generated substantial DSBs as the canonical controls. While detected less frequently, 2bp-targeted shifted SpCas9 generated significant amounts of DSBs in RAG1D and RAG1L locus. These findings underscore the spaced PAM-mediated DSBs occurred in living cells.
2022-02-28 | GSE192459 | GEO
Project description:P. aeruginosa TY1-PA3898 ChIP-seq
| PRJNA491517 | ENA
Project description:Using a TIP-seq protocol (specifically isolating transposon insertion junctions) we determined that the Ty1 retrotransposon targets tRNA genes and, in particular, we determined that the transposon inserts into nucleosomal DNA in an asymmetric pattern.
2012-01-01 | GSE33986 | GEO
Project description:Chromatin undergoes major remodeling around DNA double strand breaks (DSB) to promote repair and DNA damage response (DDR) activation. We recently reported a high resolution map of gammaH2AX around multiple breaks on the human genome, using a new cell-based DSB inducible system. In an attempt to further characterize the chromatin landscape induced around DSBs, we now report the profile of SMC3, a subunit from the cohesin complex, previously characterized as required for repair by homologous recombination. We found that the recruitment of cohesin is moderate and restricted to the immediate vicinity of DSBs. In addition, we show that the cohesin complex, which was also recently proposed to be a key player in chromosome organisation and chromatin looping, controls gammaH2AX distribution within domains. Indeed, as we reported for transcription, cohesin binding antagonizes gammaH2AX spreading. Remarkably, depletion of cohesin leads to an increase of gammaH2AX at cohesin-bound genes (revelead by gammaH2AX mapping in upon SCC1 siRNA), associated with a decrease in their expression level after DSB induction. Thus our study identifies a novel role for the cohesin complex in protecting the genes located in gammaH2AX domains from both gammaH2AX spreading and transcriptional shut-down after DSB induction.
2012-01-01 | E-TABM-1164 | biostudies-arrayexpress
Project description:Enterococcus faecalis strain:TY1 | isolate:flatfish Genome sequencing
| PRJNA480550 | ENA
Project description:For genome-wide identification and analysis of endogenous DSBs in HEK293T cells chromosomes the ligation-mediated PCR and deep sequencing of the DSBs library were used. The cells were included in low-melt agarose, and the plugs were used for isolation of DNA in a solid phase using incubation in the solution containing 0.5 M EDTA (pH 9.5), 1% sodium laurylsarcosine, and 1-2 mg of proteinase K per ml for 40-48 hr at 50°C. Then DNA was isolated by electro-elution and used for ligation with biotinylated ds-adapter. To shorten large DNA molecules the digestion with Sau3A endonuclease was performed. Then the short regions of DNA attached to DSBs sites were selected using the streptavidin magnespere paramagnetic particles. To prepare samples for amplification of DSBs library the Sau3A adapter was ligated at the opposite to DSBs sites. PCR amplification was performed using the primers corresponding to biotinylated and Sau3A adapters. The library was used for paired end sequencing. The corresponding reads were processed and used for analysis of their distribution in human genome.
2022-07-15 | GSE201829 | GEO
Project description:Background: The small RNAs that Transposable Elements generate are vastly different when they are transcriptionally silenced compared to when they are transcriptionally activated. We performed the deep sequencing of small RNAs in a number of small RNA biogenesis mutants in both Transposable Element-silenced and Transposable Element-active epigenome backgrounds. Results: We found that Transposable Elements generate large amounts of 21-22nt siRNAs only when they are transcriptionally active. These 21-22nt siRNAs are incorporated into the AGO6 protein. Conclusion: Ago6 is the key protein that bridges the post-transcriptional degradation of Transposable Element mRNAs and the establishment of DNA methylation. Examination of flower bud small RNAs from wild type and 5 single or double mutant combinations, many of which have biological replicates. In addition, IP purification of the AGO6 protein (and mock no-antigen controls) followed by sequencing of the incorporated small RNAs. Replicate A for Col and ddm1 are submitted in GSE41755
2014-11-14 | E-GEOD-57191 | biostudies-arrayexpress