Click-code-seq mapping of oxidized guanines in cells exposed to DNMT1 inhibitor GSK-3484862. Click-code-seq mapping of oxidized guanines and abasic sites in plasmids with site-specific DNA modifications.
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PROVIDER: PRJNA1253719 | ENA |
REPOSITORIES: ENA
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Project description:By relating the numbers of oxidized guanines in different sequence contexts to the abundance of these contexts in the human genome, we observed that the probability of guanine to be or remain oxidized is low if this guanine is preceded by cytosine (CpG sites) compared to other nucleotides. As CpG sites are substrates of epigenetic cytosine methylation, we investigated the influence of cytosine methylation status on guanine oxidation level in CpG sites. For this, we exposed HAP1 and U2OS cells for three days to GSK-3484862, a novel DNA methyltransferase 1 (DNMT1) inhibitor causing the degradation of this enzyme within 24 hours and having low cellular toxicity. Using Infinium MethylationEPIC v2.0 array, we observed a drastic hypomethylation after the exposure in both cell lines such that the peak of highly methylated CpG sites (beta values 0.8–1) found in vehicle (DMSO) exposed cells is absent in GSK-3484862-exposed cells. In contrast, the oxidation level of guanines in the CpG sites profiled by the array did not change in response to the inhibitor and was virtually constant across CpG sites with different methylation levels. In addition, on the level of all CpG sites in the genome, guanine-oxidation did not differ between DMSO and GSK-3484862 exposures. Thus, in CpG sites, guanine oxidation level likely does not depend on cytosine methylation status. As a control for the click-code-seq protocol, we successfully called 8-oxoG incorporated at specific sites in a plasmid (~25 fmol) that was mixed with ~28-times bigger amount of the unmodified vector. As a control for click-code-seq detection of abasic sites, we inserted uracils in a plasmid, converted them to abasic sites by uracil-DNA glycosylase (UDG) and successfully called them in the expected locations.
2025-08-22 | GSE295218 | GEO
Project description:DNA alterations are drivers of aging, neurodegeneration, carcinogenesis, and chemotherapy drug action. To understand the functional genomic roles of DNA modifications, it is critical to accurately map their diverse chemical forms with single-nucleotide precision in complex genomes, which however remains challenging. Click-code-seq is the click-chemistry-aided single-nucleotide-resolution method introduced to map oxidation in yeast DNA. Here, we upgraded click-code-seq to enable its first application for sequencing DNA oxidation and depurination in the human genome. For this, we developed a companion fluorescence tagging assay, click-fluoro-quant, to rapidly quantify common modifications. Moreover, we devised novel adapters to minimize false modification detection and assess modification frequency in cell populations. We uncovered that endogenous DNA oxidation in a human cell line has a highly similar pattern to the cancer mutational signatures associated with the effects of reactive oxygen species. We established that the chemotherapy drug irofulven preferentially depurinates ApA dimers and promoter regions. Intriguingly, we revealed that oxidized guanines and apurinic sites, both irofulven-induced and endogenous, are depleted in the transcribed strand of genes, and the strand bias widens with increasing gene expressions. This work advances click-code-seq for deciphering the impacts of key DNA modifications in the human genome on cellular physiology and toxicological responses.
2024-07-21 | GSE272366 | GEO
Project description:Abasic (AP) sites are one of the most common forms of DNA damage which can lead to polymerase stalling, strand breaks and mutations. We developed snA-seq, a mapping method that reveals the location of abasic sites at base-resolution. Using synthetic DNA, we show that high selectivity for AP DNA is achieved. We use this method to explore the distribution of thymine modifications in the Leishmania major genome, by converting these into abasic sites using a glycosylase enzyme. We also apply snAP-seq to the human genome to study the distribution of endogenous AP sites, in both APE1 knockdown and control cells.
2019-06-12 | E-MTAB-7152 | biostudies-arrayexpress
Project description:Mapping effective microRNA pairing beyond the seed using abasic mutations
| PRJNA1131823 | ENA
Project description:Single nucleotide resolution sequencing of DNA damage is required to decipher the complex causal link between the identity and location of DNA adduct and mutation patterns in the genome. We coupled the specificity of the repair enzyme with the efficiency of a one-pot click-DNA ligation reaction to insert a readable oligonucleotide code sequence. The biocompatible code enabled high-throughput, base resolution sequencing of the 8-oxoguanine site and thus have named the method click-code-seq. By applying click-code-seq to a eukaryotic (yeast) genome, we uncovered thousands of 8-oxoguanine sites with features and patterns suggesting a potential relationship to chromatin formation and transcription.
2018-06-28 | GSE112212 | GEO
Project description:Single-nucleotide resolution mapping of DNA abasic sites by snAP-seq
| PRJEB28471 | ENA
Project description:Understanding the genomic locations of DNA modifications is pivotal for deciphering their roles in gene regulation, mutagenesis, and pathology. The affinity probe-based enrichment of lesion-containing DNA represents a key strategy for sequencing DNA modifications. Existing methods are limited in the specificity of labeling and enrichment of abasic (AP) sites, a prevalent DNA modification and repair intermediate. Herein, we devise a novel approach, termed dual chemical labeling-assisted sequencing (DCL-seq), for mapping AP sites. DCL-seq features two designer compounds for enriching and mapping AP sites specifically at single-nucleotide resolution. For proof of principle, we mapped AP sites in mitochondrial DNA (mtDNA) from cultured human cells under different biological conditions. Also, we demonstrated the broader applicability of the method in sequencing additional DNA modifications in mtDNA, such as N7-methyl-2'-deoxyguanosine and N3-methyl-2'-deoxyadenosine, when coupled with a lesion-specific repair enzyme. Together, DCL-seq holds the promise to sequence multiple DNA modifications in various biological samples.
2022-09-24 | GSE213585 | GEO
Project description:The AP-seq provides a genome-wide method for detecting abasic sites among the human and mouse genomes Purpose: Abasic sites are one of the most frequent DNA damage among the genome , which often carries harmful consequences for the genome stability and the normal cell functions. So it is necessary to develop a whole genome abasic sites (AP sites) profiling method to detect all the AP sites among the genome, which we called the AP-seq. Methods: The human and mouse genome are processed with enzymatic fragmentation, damage repair, biotin-dU labeling, and pull-down steps. After the sequencing library preparation, the FASTQ files are produced by the Illumina HiSeq XTen platform. To guarantee the reproducibility, each case of the experiment is present with two biological replicates. Results: The reproducibility of our two biological replicates shows a high correlation which larger than 0.95. And the overlap AP site peaks between the HEK293T and MCF-7 cell line are over 50%, which show a great similarity between the different cell lines. Totally, we identified more than 2000 and 1000 abasic site peaks in human, and mouse cell lines separately. Moreover, the distribution of these AP sites prefer to locate in the intergenic regions and introns.
2020-12-01 | GSE125254 | GEO
Project description:The success of targeted therapies hinges on our ability to understand the molecular and cellular mechanism of action of these agents. Here we modify various BET bromodomain inhibitors, an exemplar novel targeted therapy, to create functionally conserved compounds that are amenable to click-chemistry and can be used as molecular probes in vitro and in vivo. Using click-proteomics and click-sequencing we provide new mechanistic insights to explain the gene regulatory function of BRD4 and the transcriptional changes invoked by BET inhibitors. In mouse models of acute leukaemia, we use high resolution microscopy and flow cytometry to highlight the underappreciated heterogeneity of drug activity within tumour cells located in different tissue compartments. We also demonstrate the differential distribution and effects of the drug in normal and malignant cells in vivo. These data provide critical insights that reveal the cellular and molecular details for the efficacy and limitations of these agents. This study provides a framework for the pre-clinical assessment of other conventional and targeted therapies.
2017-07-13 | GSE88749 | GEO
Project description:The success of targeted therapies hinges on our ability to understand the molecular and cellular mechanism of action of these agents. Here we modify various BET bromodomain inhibitors, an exemplar novel targeted therapy, to create functionally conserved compounds that are amenable to click-chemistry and can be used as molecular probes in vitro and in vivo. Using click-proteomics and click-sequencing we provide new mechanistic insights to explain the gene regulatory function of BRD4 and the transcriptional changes invoked by BET inhibitors. In mouse models of acute leukaemia, we use high resolution microscopy and flow cytometry to highlight the underappreciated heterogeneity of drug activity within tumour cells located in different tissue compartments. We also demonstrate the differential distribution and effects of the drug in normal and malignant cells in vivo. These data provide critical insights that reveal the cellular and molecular details for the efficacy and limitations of these agents. This study provides a framework for the pre-clinical assessment of other conventional and targeted therapies.
2017-07-13 | GSE88748 | GEO