DamID: a methylation-based chromatin profiling approach.
Gene expression is a dynamic process and is tightly connected to changes in chromatin structure and nuclear organization (Schneider, R. and Grosschedl, R., 2007, Genes Dev. 21, 3027-3043; Kosak, S. T. and Groudine, M., 2004, Genes Dev. 18, 1371-1384). Our ability to understand the intimate interactions between proteins and the rapidly changing chromatin environment requires methods that will be able to provide accurate, sensitive, and unbiased mapping of these interactions in vivo (van Steensel, B., 2005, Nat. Genet. 37 Suppl, S18-24). One such tool is DamID chromatin profiling, a methylation-based tagging method used to identify the direct genomic loci bound by sequence-specific transcription factors, co-factors as well as chromatin- and nuclear-associated proteins genome wide (van Steensel, B. and Henikoff, S., 2000, Nat. Biotechnol. 18, 424-428; van Steensel, Delrow, and Henikoff, 2001, Nat. Genet. 27, 304-308). Combined with other functional genomic methods and bioinformatics analysis (such as expression profiles and 5C analysis), DamID emerges as a powerful tool for analysis of chromatin structure and function in eukaryotes. DamID allows the detection of the direct genomic targets of any given factor independent of antibodies and without the need for DNA cross-linking. It is highly valuable for mapping proteins that associate with the genome indirectly or loosely (e.g., co-factors). DamID is based on the ability to fuse a bacterial Dam-methylase to a protein of interest and subsequently mark the factor's genomic binding site by adenine methylation. This marking is simple, highly specific, sensitive, inert, and can be done in both cell culture and living organisms. Below is a short description of the method, followed by a step-by-step protocol for performing DamID in Drosophila cells and embryos. Due to space limitations, the reader is referred to recent reviews that compare the method with other profiling techniques such as ChIP-chip as well as protocols for performing DamID in mammalian cells (NSouthall, T. D. and Brand, A. H., 2007, Nat. Struct. Mol. Biol. 14, 869-871; Orian, A., 2006, Curr. Opin. Genet. Dev. 16, 157-164; Vogel, M. J., Peric-Hupkes, D. and van Steensel, B. 2007, Nat. Protoc. 2, 1467-1478).
Orian
Amir
A
Center for Vascular and Cancer Biology, The Rappaport Faculty of Medicine and Research Institute, Technion-Israel Institute of Technology, Haifa, Israel.
Abed
Mona
M
Kenyagin-Karsenti
Dorit
D
Boico
Olga
O
D000818:Animals; D002843:Chromatin / Q000737:chemistry / Q000378:metabolism; D047369:Chromatin Immunoprecipitation / Q000379:methods; D002874:Chromosome Mapping / Q000379:methods*; D019175:DNA Methylation / Q000235:genetics*; D004330:Drosophila / Q000196:embryology / Q000235:genetics / Q000378:metabolism; D004625:Embryo, Nonmammalian; D020869:Gene Expression Profiling / Q000379:methods*; D006801:Humans; D008954:Models, Biological; D017422:Sequence Analysis, DNA / Q000379:methods; D012984:Software
D002843:Chromatin