title: Identification of holocarboxylase synthetase chromatin binding sites in human mammary cell lines using the DNA adenine methyltransferase identification technology.
abstract: Holocarboxylase synthetase (HCS) is a chromatin protein that is essential for mediating the covalent binding of biotin to histones. Biotinylation of histones plays crucial roles in the repression of genes and repeats in the human genome. We tested the feasibility of DNA adenine methyltransferase identification (DamID) technology to map HCS binding sites in human mammary cell lines. Full-length HCS was fused to DNA adenine methyltransferase (Dam) for subsequent transfection into breast cancer (MCF-7) and normal breast (MCF-10A) cells. HCS docking sites in chromatin were identified by using the unique adenine methylation sites established by Dam in the fusion construct; docking sites were unambiguously identified using methylation-sensitive digestion, cloning, and sequencing. In total, 15 novel HCS binding sites were identified in the two cell lines, and the following 4 of the 15 overlapped between MCF-7 and MCF-10A cells: inositol polyphosphate-5-phosphatase A, corticotropin hormone precursor, ribosome biogenesis regulatory protein, and leptin precursor. We conclude that DamID is a useful technology to map HCS binding sites in human chromatin and propose that the entire set of HCS binding sites could be mapped by combining DamID with microarray technology.
authors.affiliation: Department of Biological Systems Engineering, University of Nebraska-Lincoln, Lincoln, NE 68583, USA.
authors.forename: Dipika
authors.initials: D
authors.lastname: Singh
authors.forename: Angela K
authors.initials: AK
authors.lastname: Pannier
authors.forename: Janos
authors.initials: J
authors.lastname: Zempleni
mesh_terms: D001665:Binding Sites; D019731:Carbon-Nitrogen Ligases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D002843:Chromatin / Q000737:chemistry*; D006801:Humans; D042361:Mammary Glands, Human / Q000166:cytology; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific) / Q000235:genetics / Q000378:metabolism*
chemical_list: D002843:Chromatin; D011993:Recombinant Fusion Proteins; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific); D019731:Carbon-Nitrogen Ligases; C023312:holocarboxylase synthetases