DNA-protein Crosslinking Sequencing for Genome-wide Mapping of Thymidine Glycol
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ABSTRACT:
PROVIDER: PRJNA763568 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR15905054.fastq.gz | Fastqsanger.gz | |||
| SRR15905055.fastq.gz | Fastqsanger.gz | |||
| SRR15905056.fastq.gz | Fastqsanger.gz | |||
| SRR15905057.fastq.gz | Fastqsanger.gz | |||
| SRR15905058.fastq.gz | Fastqsanger.gz |
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Project description:Thymidine glycol (Tg) is the most prevalent form of oxidatively induced pyrimidine lesion in DNA. Tg can arise from direct oxidation of thymidine in DNA. In addition, 5-methyl-2¢-deoxycytidine (5-mdC) can be oxidized to 5-mdC glycol and its subsequent deamination also yields Tg. However, its distribution in the human genome remains unknown. Here, we presented a DNA-protein cross-linking sequencing (DPC-Seq) method for genome-wide mapping of Tg in human cells. Our approach is capitalized on the specificity of a DNA glycosylase, i.e., NTHL1, for the covalent labeling, as well as DPC pulldown, SDS-PAGE fractionation, and membrane transfer for highly efficient and selective enrichment of Tg-bearing DNA. By employing DPC-Seq, we detected thousands of Tg sites in HEK293T cells and the isogenic NTHL1 and NEIL1 double knock out cell lines. We found that Tg is depleted in genomic regions associated with active transcription, but enriched at the nucleosome-binding sites, especially at heterochromatin sites. Collectively, our approach allows for comprehensive analysis of Tg in the human genome and provides a robust tool for future functional studies of Tg in DNA. It can be envisaged that the method can be adapted for mapping other modified nucleosides in genomic DNA in the future.
2021-09-18 | GSE184204 | GEO
Project description:Thymidine glycol (Tg) is the most prevalent form of oxidatively induced pyrimidine lesions in DNA. Tg can arise from direct oxidation of thymidine in DNA. In addition, 5-methyl-2'-deoxycytidine (5-mdC) can be oxidized to 5-mdC glycol, and its subsequent deamination also yields Tg. However, Tg's distribution in the human genome remains unknown. Here, we presented a DNA-protein cross-linking sequencing (DPC-Seq) method for genome-wide mapping of Tg in human cells. Our approach capitalizes on the specificity of a bifunctional DNA glycosylase, i.e., NTHL1, for the covalent labeling, as well as DPC pulldown, SDS-PAGE fractionation, and membrane transfer for highly efficient and selective enrichment of Tg-bearing DNA. By employing DPC-Seq, we detected thousands of Tg sites in the human genome, where dual ablation of NTHL1 and NEIL1, the major DNA glycosylases responsible for Tg repair, led to pronounced increases in the number of Tg peaks. In addition, Tg is depleted in genomic regions associated with active transcription but enriched at nucleosome-binding sites, especially at heterochromatin sites marked with H3K9me2. Collectively, we developed a DPC-Seq method for highly efficient enrichment of Tg-containing DNA and for genome-wide mapping of Tg in human cells. Our work offers a robust tool for future functional studies of Tg in DNA, and we envision that the method can also be adapted for mapping other modified nucleosides in genomic DNA in the future.
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Project description:Integrating crosslinking mass spectrometry (XL-MS) into structural biology workflows provides valuable information about the spatial arrangement of amino acid stretches, which can guide elucidation of protein assembly architecture. Additionally, combination of XL-MS with peptide quantitation techniques is a powerful approach to delineate protein interface dynamics across diverse conditions. While XL-MS is increasingly effective with isolated proteins or small complexes, its application to whole-cell samples poses technical challenges related to analysis depth and throughput. The use of enrichable crosslinkers has greatly improved the detectability of protein interfaces in a proteome-wide scale, facilitating global protein-protein interaction mapping. Therefore, bringing together enrichable crosslinking and multiplexed peptide quantification is an appealing approach to enable comparative characterization of structural attributes of proteins and protein interactions. Here, we combined phospho-enrichable crosslinking with TMT labelling to develop a streamline workflow (PhoXplex) for the detection of differential structural features across a panel of cell lines in a global scale. We achieved deep coverage with quantification of over 9000 crosslinking events including potentially novel interactions. Overlaying AlphaFold predictions and disorder protein annotations enables exploration of the quantitative crosslinking dataset, to reveal possible associations between mutations and protein structures. Lastly, we discuss current shortcomings and perspectives for deep whole-cell profiling of protein interfaces at large-scale.
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Project description:Transcription factors (TFs) regulate target genes by specific interaction with DNA sequences. Detecting and understanding these interactions on molecular level is of fundamental interest in both biological and clinical context. Crosslinking mass spectrometry is a powerful tool to assist structure prediction of protein complexes by providing direct evidence for physical contacts on sub-molecular level. Yet, until now it has been limited to the study of protein-protein and protein-RNA interactions. Here, we developed a femtosecond laser induced crosslinking mass spectrometry (fLiX-MS) workflow, which allows mapping of protein-DNA contacts at single nucleotide and up to single amino acid resolution. Applying the method to in-vitro assembled nucleosomes and recombinant TF-DNA complexes, our method proved both versatile and highly specific for mapping DNA binding domains. While for Nuclear factor 1 (NF1) the detected crosslinks were in perfect agreement with previous biochemical data on DNA binding, crosslinks for the TATA box binding protein (TBP) fitted to the known crystal structure. We further identified several protein-DNA interactions for the nucleosome and TBP, which were rather distant in the crystal-structure, indicating, that our method is capable to detect structural flexibility reflected in distinct conformational states. Moreover, applying the fLiX-MS workflow to cells, we detected several bona-fide crosslinks on DNA-binding domains, proving the applicability of the method for future large scale in-vivo experiments.
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