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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Oberley, M.J., Tsao, J., Yau, P. & Farnham, P.J. High-throughput screening of chromatin immunoprecipitates using CpG-island microarrays. Methods Enzymol. 376, 315–334 (2004).
Lee, T.I., Johnstone, S.E. & Young, R.A. Chromatin immunoprecipitation and microarray-based analysis of protein location. Nat. Protocols 1, 729–748 (2006).
Ren, B. & Dynlacht, B.D. Use of chromatin immunoprecipitation assays in genome-wide location analysis of mammalian transcription factors. Methods Enzymol. 376, 304–315 (2004).
Nelson, J.D., Denisenko, O., Sova, P. & Bomsztyk, K. Fast chromatin immunoprecipitation assay. Nucleic Acids Res. 34, e2 (2006).
Wei, C.L. et al. A global map of p53 transcription-factor binding sites in the human genome. Cell 124, 207–219 (2006).
van Steensel, B. & Henikoff, S. Identification of in vivo DNA targets of chromatin proteins using tethered dam methyltransferase. Nat. Biotechnol. 18, 424–428 (2000).
van Steensel, B., Delrow, J. & Henikoff, S. Chromatin profiling using targeted DNA adenine methyltransferase. Nat. Genet. 27, 304–308 (2001).
Vogel, M.J. et al. Human heterochromatin proteins form large domains containing KRAB-ZNF genes. Genome Res. 16, 1493–1504 (2006).
Greil, F., Moorman, C. & van Steensel, B. DamID: mapping of in vivo protein–genome interactions using tethered DNA adenine methyltransferase. Methods Enzymol. 410, 342–359 (2006).
Orian, A. Chromatin profiling, DamID and the emerging landscape of gene expression. Curr. Opin. Genet. Dev. 16, 157–164 (2006).
Moorman, C. et al. Hotspots of transcription factor colocalization in the genome of Drosophila melanogaster . Proc. Natl. Acad. Sci. USA 103, 12027–12032 (2006).
Pickersgill, H. et al. Characterization of the Drosophila melanogaster genome at the nuclear lamina. Nat. Genet. 38, 1005–1014 (2006).
Tolhuis, B. et al. Genome-wide profiling of PRC1 and PRC2 Polycomb chromatin binding in Drosophila melanogaster . Nat. Genet. 38, 694–699 (2006).
Germann, S., Juul-Jensen, T., Letarnec, B. & Gaudin, V. DamID, a new tool for studying plant chromatin profiling in vivo, and its use to identify putative LHP1 target loci. Plant J. 48, 153–163 (2006).
Sun, L.V. et al. Protein–DNA interaction mapping using genomic tiling path microarrays in Drosophila . Proc. Natl. Acad. Sci. USA 100, 9428–9433 (2003).
Buck, M.J. & Lieb, J.D. ChIP-chip: considerations for the design, analysis, and application of genome-wide chromatin immunoprecipitation experiments. Genomics 83, 349–360 (2004).
Boyer, L.A. et al. Core transcriptional regulatory circuitry in human embryonic stem cells. Cell 122, 947–956 (2005).
Negre, N. et al. Chromosomal distribution of PcG proteins during Drosophila development. PLoS Biol. 4, e170 (2006).
Tiscornia, G., Singer, O. & Verma, I.M. Production and purification of lentiviral vectors. Nat. Protocols 1, 241–245 (2006).
Smyth, G.K. Linear models and empirical bayes methods for assessing differential expression in microarray experiments. Stat. Appl. Genet. Mol. Biol. 3, Article3 (2004).
Huber, W., von Heydebreck, A., Sultmann, H., Poustka, A. & Vingron, M. Variance stabilization applied to microarray data calibration and to the quantification of differential expression. Bioinformatics 18 (suppl. 1): S96–S104 (2002).
Kreil, D.P. & Russell, R.R. There is no silver bullet—a guide to low-level data transforms and normalisation methods for microarray data. Brief Bioinform. 6, 86–97 (2005).
Wit, E. & McClure, J. Statistics for Microarrays: Design, Analysis, and Inference (John Wiley & Sons Ltd, Chichester, England, 2004).
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.
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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