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Detection of in vivo protein–DNA interactions using DamID in mammalian cells

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Abstract

Understanding gene regulatory networks in mammalian cells requires detailed knowledge of protein–DNA interactions. Commonly used methods for genome-wide mapping of these interactions are based on chromatin immunoprecipitation. However, these methods have some drawbacks, such as the use of crosslinking reagents, the need for highly specific antibodies and relatively large amounts of starting material. We present DamID, an alternative technique to map genome-wide occupancy of interaction sites in vivo, that bypasses these limitations. DamID is based on the expression of a fusion protein consisting of a protein of interest and DNA adenine methyltransferase (Dam). This leads to methylation of adenines near sites where the protein of interest interacts with the DNA. These methylated sequences are subsequently amplified by a methylation-specific PCR protocol and identified by hybridization to microarrays. Using DamID, genome-wide maps of the binding of DNA-interacting proteins in mammalian cells can be constructed efficiently. Depending on the strategy used for expression of the Dam-fusion proteins, genome-wide binding maps can be obtained in as little as 2 weeks.

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Figure 1: DamID.
Figure 2
Figure 3: Typical example of PCR-amplified methylated DNA fragments, run on a 1% agarose gel and stained with EtBr.

Change history

  • 06 September 2007

    The version of this article originally published indicated that the authors had no competing financial interests. The Competing Interests Statement should have read: “B.V.S. is listed as an inventor on a patent application for the DamID technology.” The error has been corrected in the HTML and PDF versions of the article.

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Acknowledgements

We thank Lars Guelen for development of DamID using lentiviruses and critical reading of the manuscript; Martin Lodén, Richard Heideman and Anja Duursma for useful suggestions and sharing results; and members of our laboratory for help with the development of DamID in mammalian cells. We thank the staff of the NKI Central Microarray Facility for extensive help with protocol development and technical support.

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Correspondence to Bas van Steensel.

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B.V.S. is listed as an inventor on a patent application for the DamID technology.

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Vogel, M., Peric-Hupkes, D. & van Steensel, B. Detection of in vivo protein–DNA interactions using DamID in mammalian cells. Nat Protoc 2, 1467–1478 (2007). https://doi.org/10.1038/nprot.2007.148

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