DamID: mapping of in vivo protein-genome interactions using tethered DNA adenine methyltransferase.
A large variety of proteins bind to specific parts of the genome to regulate gene expression, DNA replication, and chromatin structure. DamID is a powerful method used to map the genomic interaction sites of these proteins in vivo. It is based on fusing a protein of interest to Escherichia coli DNA adenine methyltransferase (dam). Expression of this fusion protein in vivo leads to preferential methylation of adenines in DNA surrounding the native binding sites of the dam fusion partner. Because adenine methylation does not occur endogenously in most eukaryotes, it provides a unique tag to mark protein interaction sites. The adenine-methylated DNA fragments are isolated by selective polymerase chain reaction amplification and can be identified by microarray hybridization. We and others have successfully applied DamID to the genome-wide identification of interaction sites of several transcription factors and other chromatin-associated proteins. This chapter discusses DamID technology in detail, and a step-by-step experimental protocol is provided for use in Drosophila cell lines.
Greil
Frauke
F
Division of Molecular Biology, Netherlands Cancer Institute, Amsterdam, The Netherlands.
Moorman
Celine
C
van Steensel
Bas
B
D000818:Animals; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D023281:Genomics / Q000379:methods*; D006801:Humans; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific)
D004268:DNA-Binding Proteins; D004247:DNA; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific)