Beckerus appressus
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA1305414 | ENA |
REPOSITORIES: ENA
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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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Project description:Genotoxic estrogen metabolites generate various endogenous DNA lesions but their carcinogenesis mechanisms have been overlooked by well-known cell proliferation pathways through estrogen receptor (ER). Genome-wide sequencing using click probe enrichment coupled with liquid chromatography-mass spectrometry (click probe-Seq/LCMS2) was developed to identify damaged genes and characterize global generation profiles of depurinating and stable adducts induced by 4-hydrolyxyl estradiol (4OHE2) in MCF-7 chromatin. Both data were combined to show guanine nucleobase in GC-rich transcription-relevant domain are main target sites. The damage abundance exhibited positive correlation with DNase hypersensitive sites, indicating 4OHE2 attacks chromatin exposure region beyond ER binding. Cell-based comparability studies indicated accumulated 4OHE2 caused suppressed transcription of target genes, in-effective damage repair, and decreased cell viability, differing from uncontrolled cell growth by extensive ER-signaling. Click probe-Seq/LCMS2 approach revealed the first chromatin damage map by endogenous metabolites, exposing a previously unexplored landscape in cancer research and being applicable to other genotoxic species.
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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.
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