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Quantifying propagation of DNA methylation and hydroxymethylation with iDEMS
DNA methylation is a critical epigenetic mark in mammalian cells. Many aspects of DNA methylation maintenance have been characterized; however, the exact kinetics of post-replicative methylation maintenance remain a subject of debate. Here we develop isolation of DNA by 5-ethynyl-deoxyuridine labell...
Autores principales: | , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Nature Publishing Group UK
2023
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9859752/ https://www.ncbi.nlm.nih.gov/pubmed/36635504 http://dx.doi.org/10.1038/s41556-022-01048-x |
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author | Stewart-Morgan, Kathleen R. Requena, Cristina E. Flury, Valentin Du, Qian Heckhausen, Zoe Hajkova, Petra Groth, Anja |
author_facet | Stewart-Morgan, Kathleen R. Requena, Cristina E. Flury, Valentin Du, Qian Heckhausen, Zoe Hajkova, Petra Groth, Anja |
author_sort | Stewart-Morgan, Kathleen R. |
collection | PubMed |
description | DNA methylation is a critical epigenetic mark in mammalian cells. Many aspects of DNA methylation maintenance have been characterized; however, the exact kinetics of post-replicative methylation maintenance remain a subject of debate. Here we develop isolation of DNA by 5-ethynyl-deoxyuridine labelling for mass spectrometry (iDEMS), a highly sensitive, quantitative mass spectrometry-based method for measuring DNA modifications on metabolically labelled DNA. iDEMS reveals an unexpectedly hemi-methylated landscape on nascent DNA. Combining iDEMS with metabolic labelling reveals that methylation maintenance is outpaced by cell division in mouse embryonic stem cells. Our approach shows that hydroxymethylation is perpetually asymmetric between sister strands in favour of the parental, template strand. iDEMS can be coupled with immunoprecipitation of chromatin proteins, revealing features of DNA methylation–histone modification crosstalk and suggesting a model for interplay between methylation and nucleosome assembly. iDEMS therefore elucidates long-standing questions about DNA modification propagation and provides an important orthogonal technology to understanding this process in dynamic cellular contexts. |
format | Online Article Text |
id | pubmed-9859752 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | Nature Publishing Group UK |
record_format | MEDLINE/PubMed |
spelling | pubmed-98597522023-01-22 Quantifying propagation of DNA methylation and hydroxymethylation with iDEMS Stewart-Morgan, Kathleen R. Requena, Cristina E. Flury, Valentin Du, Qian Heckhausen, Zoe Hajkova, Petra Groth, Anja Nat Cell Biol Technical Report DNA methylation is a critical epigenetic mark in mammalian cells. Many aspects of DNA methylation maintenance have been characterized; however, the exact kinetics of post-replicative methylation maintenance remain a subject of debate. Here we develop isolation of DNA by 5-ethynyl-deoxyuridine labelling for mass spectrometry (iDEMS), a highly sensitive, quantitative mass spectrometry-based method for measuring DNA modifications on metabolically labelled DNA. iDEMS reveals an unexpectedly hemi-methylated landscape on nascent DNA. Combining iDEMS with metabolic labelling reveals that methylation maintenance is outpaced by cell division in mouse embryonic stem cells. Our approach shows that hydroxymethylation is perpetually asymmetric between sister strands in favour of the parental, template strand. iDEMS can be coupled with immunoprecipitation of chromatin proteins, revealing features of DNA methylation–histone modification crosstalk and suggesting a model for interplay between methylation and nucleosome assembly. iDEMS therefore elucidates long-standing questions about DNA modification propagation and provides an important orthogonal technology to understanding this process in dynamic cellular contexts. Nature Publishing Group UK 2023-01-12 2023 /pmc/articles/PMC9859752/ /pubmed/36635504 http://dx.doi.org/10.1038/s41556-022-01048-x Text en © The Author(s) 2023 https://creativecommons.org/licenses/by/4.0/Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) . |
spellingShingle | Technical Report Stewart-Morgan, Kathleen R. Requena, Cristina E. Flury, Valentin Du, Qian Heckhausen, Zoe Hajkova, Petra Groth, Anja Quantifying propagation of DNA methylation and hydroxymethylation with iDEMS |
title | Quantifying propagation of DNA methylation and hydroxymethylation with iDEMS |
title_full | Quantifying propagation of DNA methylation and hydroxymethylation with iDEMS |
title_fullStr | Quantifying propagation of DNA methylation and hydroxymethylation with iDEMS |
title_full_unstemmed | Quantifying propagation of DNA methylation and hydroxymethylation with iDEMS |
title_short | Quantifying propagation of DNA methylation and hydroxymethylation with iDEMS |
title_sort | quantifying propagation of dna methylation and hydroxymethylation with idems |
topic | Technical Report |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9859752/ https://www.ncbi.nlm.nih.gov/pubmed/36635504 http://dx.doi.org/10.1038/s41556-022-01048-x |
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