title: DamID-seq: Genome-wide Mapping of Protein-DNA Interactions by High Throughput Sequencing of Adenine-methylated DNA Fragments.
abstract: The DNA adenine methyltransferase identification (DamID) assay is a powerful method to detect protein-DNA interactions both locally and genome-wide. It is an alternative approach to chromatin immunoprecipitation (ChIP). An expressed fusion protein consisting of the protein of interest and the E. coli DNA adenine methyltransferase can methylate the adenine base in GATC motifs near the sites of protein-DNA interactions. Adenine-methylated DNA fragments can then be specifically amplified and detected. The original DamID assay detects the genomic locations of methylated DNA fragments by hybridization to DNA microarrays, which is limited by the availability of microarrays and the density of predetermined probes. In this paper, we report the detailed protocol of integrating high throughput DNA sequencing into DamID (DamID-seq). The large number of short reads generated from DamID-seq enables detecting and localizing protein-DNA interactions genome-wide with high precision and sensitivity. We have used the DamID-seq assay to study genome-nuclear lamina (NL) interactions in mammalian cells, and have noticed that DamID-seq provides a high resolution and a wide dynamic range in detecting genome-NL interactions. The DamID-seq approach enables probing NL associations within gene structures and allows comparing genome-NL interaction maps with other functional genomic data, such as ChIP-seq and RNA-seq.
authors: Department of Cell Biology, Yale University School of Medicine.
authors: Feinan
authors: F
authors: Wu
authors: Department of Cell Biology, Yale University School of Medicine.
authors: Brennan G
authors: BG
authors: Olson
authors: Department of Cell Biology, Yale University School of Medicine; yao.j@yale.edu.
authors: Jie
authors: J
authors: Yao
mesh_terms: D016475:3T3 Cells; D000225:Adenine / Q000378:metabolism; D000818:Animals; D002874:Chromosome Mapping / Q000379:methods*; D004247:DNA / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019175:DNA Methylation; D023281:Genomics / Q000379:methods; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D051379:Mice; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods; D017422:Sequence Analysis, DNA / Q000379:methods; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific) / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D004247:DNA; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific); D000225:Adenine