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Click-chemistry-aided quantitation and sequencing of oxidized guanines and apurinic sites uncovers their transcription-linked strand bias in human cells

ABSTRACT: Click-chemistry-aided quantitation and sequencing of oxidized guanines and apurinic sites uncovers their transcription-linked strand bias in human cells

PROVIDER: PRJNA1136520 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR29848888.fastq.gz	Fastqsanger.gz
	SRR29848889.fastq.gz	Fastqsanger.gz
	SRR29848890.fastq.gz	Fastqsanger.gz
	SRR29848891.fastq.gz	Fastqsanger.gz
	SRR29848892.fastq.gz	Fastqsanger.gz

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Click-chemistry-aided quantitation and sequencing of oxidized guanines and apurinic sites uncovers their transcription-linked strand bias in human cells

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.

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Click chemistry enables comprehensive preclinical evaluation of targeted epigenetic therapies [Click-Seq 2]

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

Click chemistry enables comprehensive preclinical evaluation of targeted epigenetic therapies [Click-Seq 1]

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Click chemistry enables comprehensive preclinical evaluation of targeted epigenetic therapies [RNA-seq]

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Click chemistry enables comprehensive preclinical evaluation of targeted epigenetic therapies [ChIP-seq 1]

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Click chemistry enables comprehensive preclinical evaluation of targeted epigenetic therapies [ChIP-seq 2]

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Click-linking: a cell-compatible protein crosslinking method based on click chemistry

Project description:We developed a novel approach to in situ crosslinking mass spectrometry (XL-MS) where the reaction is split into two sequential and orthogonal coupling events. The method involves pre-stabilization of the proteome using a fixation protocol, followed by a two-step process that begins with extensive labeling of surface-available lysines with click-chemistry compatible reagents modified with NHS Esters. The second step involves the copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction of the installed precursors, which generates crosslinks with high efficiency. We observed approximately a 20-fold increase in protein-protein interactions detected by this approach when compared to standard DSS crosslinkers, and our Click-linking strategy even outperformed enrichable crosslinkers such as PhoX.

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Click chemistry enables comprehensive preclinical evaluation of targeted epigenetic therapies

Project description:Click chemistry enables comprehensive preclinical evaluation of targeted epigenetic therapies

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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.

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

Impact of azide metabolic glycoengineering and bioorthogonal click chemistry cell-cell tethering in adipose tissue-derived mesenchymal stromal cells

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2024-04-15 | GSE231507 | GEO

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