title: A multi-parametric flow cytometric assay to analyze DNA-protein interactions.
abstract: Interactions between DNA and transcription factors (TFs) guide cellular function and development, yet the complexities of gene regulation are still far from being understood. Such understanding is limited by a paucity of techniques with which to probe DNA-protein interactions. We have devised magnetic protein immobilization on enhancer DNA (MagPIE), a simple, rapid, multi-parametric assay using flow cytometric immunofluorescence to reveal interactions among TFs, chromatin structure and DNA. In MagPIE, synthesized DNA is bound to magnetic beads, which are then incubated with nuclear lysate, permitting sequence-specific binding by TFs, histones and methylation by native lysate factors that can be optionally inhibited with small molecules. Lysate protein-DNA binding is monitored by flow cytometric immunofluorescence, which allows for accurate comparative measurement of TF-DNA affinity. Combinatorial fluorescent staining allows simultaneous analysis of sequence-specific TF-DNA interaction and chromatin modification. MagPIE provides a simple and robust method to analyze complex epigenetic interactions in vitro.
authors.affiliation: Division of Genetics, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, 77 Avenue Louis Pasteur, Boston, MA 02115, USA.
authors.forename: Mandana
authors.initials: M
authors.lastname: Arbab
authors.forename: Shaun
authors.initials: S
authors.lastname: Mahony
authors.forename: Hyunjii
authors.initials: H
authors.lastname: Cho
authors.forename: Joel M
authors.initials: JM
authors.lastname: Chick
authors.forename: P Alexander
authors.initials: PA
authors.lastname: Rolfe
authors.forename: John Peter
authors.initials: JP
authors.lastname: van Hoff
authors.forename: Viveca W S
authors.initials: VW
authors.lastname: Morris
authors.forename: Steven P
authors.initials: SP
authors.lastname: Gygi
authors.forename: Richard L
authors.initials: RL
authors.lastname: Maas
authors.forename: David K
authors.initials: DK
authors.lastname: Gifford
authors.forename: Richard I
authors.initials: RI
authors.lastname: Sherwood
mesh_terms: D000818:Animals; D004247:DNA / Q000378:metabolism*; D019175:DNA Methylation; D004268:DNA-Binding Proteins / Q000378:metabolism; D044127:Epigenesis, Genetic; D005434:Flow Cytometry / Q000379:methods*; D006657:Histones / Q000378:metabolism; D051379:Mice; D014157:Transcription Factors / Q000378:metabolism*
chemical_list: D004268:DNA-Binding Proteins; D006657:Histones; D014157:Transcription Factors; D004247:DNA