Genome-wide maps of apurinic sites (AP-sites) and 8-oxoG derived secondary AP-sites
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PROVIDER: PRJNA497112 | ENA |
REPOSITORIES: ENA
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Project description:We developed a novel method, AP-seq, capable of mapping apurinic sites and 8-oxo-7,8-dihydroguanine bases at approximately 250bp resolution on a genome-wide scale. We directly demonstrate that the accumulation rate of apurinic sites varies widely across the genome, with hot spots acquiring many times more damage than cold spots. The experiment was performed in triplicates on HepG2 cells, 30 min after X-ray treatment (6Gy). AP-Seq is an approach that specifically enriches AP-sites via a biotin-labelled aldehyde-reactive probe under pH neutral conditions, which has been well established for the specific detection of AP-sites since its development by Kubo et al. in 1992. Under neutral conditions (pH7), 1h at 37ºC, the probe is highly specific for the aldehydes occurring at AP-sites. After biotin labelling of genomic DNA, DNA is sonicated into ~250 bp fragments, enriched, in vitro repaired (PreCR, NEB), and used for Illumina short read sequencing (125 bp, paired end, HiSeq2000). To assess oxidative damage as the sum of AP-sites and 8-oxoG, we applied recombinant OGG1 in vitro to the extracted DNA (OGG1-AP-seq). Under the conditions chosen, any remaining 8-oxoG is excised in a largely sequence-independent fashion after DNA extraction to result in a set of secondary AP-sites and to a lesser extent the associated beta-elimination product.
2018-10-22 | GSE121005 | GEO
Project description:We observed that transcriptional activity and the number of active genes were significantly correlated with the distribution of 8-oxoG in gene promoter regions, as determined by liquid chromatography/mass spectrometry (LC/MS), and 8-oxoG and RNA sequencing
2019-06-30 | GSE124712 | GEO
Project description:We observed that transcriptional activity and the number of active genes were significantly correlated with the distribution of 8-oxoG in gene promoter regions, as determined by liquid chromatography/mass spectrometry (LC/MS), and 8-oxoG and RNA sequencing
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Project description:Various environmental factors contribute to dysregulated spermatogenic gene network, abnormal spermatogenesis, and eventually impaired sperm fertility. 8-hydroxyguanine (8-oxoG) is the most common and well-studied oxidative DNA lesion of the four DNA bases and unrepaired 8-oxoG modifications are associated with DNA fragmentation in sperms. However, the molecular effects of 8-oxoG generated in sperm DNA by oxidative stress on spermatogenesis are not entirely understood. Here, we identified one infertile Korean native striped cattle at 6-year old (C14) due to asthenozoospermia and teratozoospermia. We compared global concentration of 8-oxoG by reverse-phase liquid chromatography/mass spectrometry (RP-LC/MS), the genomic distribution of 8-oxoG by next-generation sequencing (OG-seq), and expression of sperm proteins by 2-dimensional electrophoresis followed by mass spectrometry (2D/MS) in sperms of C14 with those of normal counterpart (C13). We found that averaged levels of 8-oxoG in C13 and C14 sperms were 0.027% and 0.044% of total dG and 8-oxoG was significantly greater in infertile sperm DNA (p = 0.0028). More than 81% of the 8-oxoG loci were distributed around the transcription start site (TSS) and 8-oxoG harboring 165 genes, including phospholipases, were annotated exclusively into infertile sperm DNA. Gene set enrichment analysis (GSEA) and the protein-protein interaction networking revealed that the Golgi apparatus was significantly enriched with the 8-oxoG genes of infertile sperm (q = 2.20E-07). Proteomic analysis verified that acrosome-related proteins including Acrosin-binding protein (ACRBP) and Sperm equatorial segment protein 1 (SPESP1) were 1.6-fold and 2.6-fold down-regulated in infertile sperm, respectively. These results suggest that 8-oxoG formation during spermatogenesis would dysregulate the acrosome-related gene network, causing structural and functional defects of sperm, leading to infertility.
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Project description:8-oxo-7,8-dihydroguanine (8-oxoG) is a common RNA oxidation that has been implicated in carcinogenesis, neurodegeneration, and aging. Some? Many? RNA-binding proteins have shown binding preference for 8-oxoG modified RNA; such binding? can protect cells from oxidative stress. To date, the presence of specific 8-oxoG-recognizing proteins is only known in model organisms, but, how these proteins cooperate is still unclear. Here, we use RNA affinity chromatography and mass spectrometry to search for proteins that specifically bind 8-oxoG-modified RNA in Deinococcus radiodurans, an extremophilic bacterium with extraordinary resistance to oxidative stresses. We identified four proteins that selectively bind to oxidized RNA: polynucleotide phosphorylase (DR_2063/PNPase), ATP-dependent DEAD-box RNA helicase (DR_0335/RhlB), ribosomal protein S1 (DR_1983/RpsA), and transcriptional termination factor (DR_1338/Rho). The knockout of PNPase and RhlB in D. radiodurans caused increased sensitivity to hydrogen peroxide and a significant accumulation of 8-oxoG modified RNA. Importantly, PNPase directly interacts with RhlB in D. radiodurans; however, no additional phenotypic effect was observed for the double deletion of pnp and rhlB compared to the individual single deletions. Overall, our findings suggest a role of the PNPase-RhlB complex in targeting 8-oxoG-modified RNAs for rapid degradation and thereby constitutes an important component of D. radiodurans resistance to oxidative stress.
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Project description:The polarization and activation of macrophages are controlled synergistically by transcription factors such as NF-κB and AP-1 transcription factor members. Surprisingly, little is known about the role of the AP-1 member, Fra-1, during macrophage responses. To determine the full spectrum of Fra-1 binding sites in macrophages, chromatin immunoprecipitation sequencing analysis was performed in macrophages pooling down for the transcription factor Fra-1.
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Project description:Identification of Fra-1/AP-1 binding sites in macrophages
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Project description:We applied deep-sequencing based technique, 3'-Seq, to obtain comprehansive maps of poly-A sites in human cells. 3'-Seq was applied to two cell lines (U2OS and RPE-1), in control and PABPN1 knockdown cells Examination of poly-A sites in control and PABPN1kd cells (in two different cell lines)
2012-04-15 | E-GEOD-27452 | biostudies-arrayexpress
Project description:We applied deep-sequencing based technique, 3'-Seq, to obtain comprehansive maps of poly-A sites in OPMD-model mice expressing trePABPN1(A17) mutant and control mice Examination of poly-A sites in control and OPMD-model mice (in two ages: 20 weeks and 12 months)
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Project description:DNA damage plays critical role in biology and disease, however, precise understanding of how different types of DNA damage affect cellular functions is far from clear. A major underlying reason for this is paucity of high-resolution methods that can map locations of different types of DNA damage in complex genomes such as those of mammals. Here, we present development and validation of SSiNGLe-AP method that can map a common type of DNA damage, abasic (AP) sites, genome-wide and with high-resolution. We applied this method to six different tissues of mice of different ages and human cancer cell lines. We found non-random distribution of AP sites in mammalian genome that exhibits dynamic enrichment at specific genomic locations, and is significantly influenced by gene expression, age and especially by tissue-type. Overall, these results suggest that we are at the very beginning of understanding the true complexities of genomic patterns of DNA damage.
2022-09-15 | GSE190955 | GEO