Abstract
Mapping the binding sites of DNA- or chromatin-interacting proteins is essential to understand many essential biological processes. Methyl Adenine Identification (MadID) is a proximity methylation-based assay that allows the visualization, quantification, and identification of binding sites from DNA-interacting proteins in eukaryotic cells. Chromatin-binding proteins of interest are fused to the newly described bacterial methyltransferase M.EcoGII. This enzyme catalyzes the methylation of adenine residues with no sequence specificity. Consequently, adenines within and in the vicinity of the protein binding sites will be decorated with a methyl group (m6A), a modification that can be further detected using different methods. M.EcoGII-dependent DNA methylation can be monitored in situ using immunostaining, at the genome-wide level using a combination of m6A-specific immunoprecipitation and whole-genome sequencing, or locally at DNA regions of interest purified by chromatin immunoprecipitation or probe-based capture techniques. MadID is conceptually similar to DNA adenine methyltransferase identification (DamID) that relies on the methylation of GATC motifs. However, MadID provides a higher resolution, deeper coverage, and opens ways for identification of binding sites in genomic regions that were largely inaccessible such as telomeres, centromeres, and repeated elements.
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Acknowledgments
We would like to thank all the members (past and present) of the Crabbé lab for helpful discussions, especially Sonia Stinus Ruiz de Gauna for critical reading of the manuscript. This work was supported by an ATIP starting grant from CNRS in the framework of Plan Cancer 2014–2019 (to L.C.), the ANR Tremplin ERC teloHOOK (ANR-16-TERC-0028-01 to L.C.), and a European Research Council (ERC) grant (TeloHOOK, 714653 to L.C.).
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Umlauf, D., Sobecki, M., Crabbe, L. (2020). Methyl Adenine Identification (MadID): High-Resolution Detection of Protein-DNA Interactions. In: Hancock, R. (eds) The Nucleus . Methods in Molecular Biology, vol 2175. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0763-3_10
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DOI: https://doi.org/10.1007/978-1-0716-0763-3_10
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