title: ChIP bias as a function of cross-linking time.
abstract: The chromatin immunoprecipitation (ChIP) assay is widely used to capture interactions between chromatin and regulatory proteins in vivo. Formaldehyde cross-linking of DNA and proteins is a critical step required to trap their interactions inside the cells before immunoprecipitation and analysis. Yet insufficient attention has been given to variables that might give rise to artifacts in this procedure, such as the duration of cross-linking. We analyzed the dependence of the ChIP signal on the duration of formaldehyde cross-linking time for two proteins: DNA topoisomerase 1 (Top1) that is functionally associated with the double helix in vivo, especially with active chromatin, and green fluorescent protein (GFP) that has no known bona fide interactions with DNA. With short time of formaldehyde fixation, only Top1 immunoprecipation efficiently recovered DNA from active promoters, whereas prolonged fixation augmented non-specific recovery of GFP dramatizing the need to optimize ChIP protocols to minimize the time of cross-linking, especially for abundant nuclear proteins. Thus, ChIP is a powerful approach to study the localization of protein on the genome when care is taken to manage potential artifacts.
authors.affiliation: Laboratory of Pathology, NCI/NIH, Bethesda, MD, 20892, USA. baranellolf@mail.nih.gov.
authors.forename: Laura
authors.initials: L
authors.lastname: Baranello
authors.affiliation: Laboratory of Pathology, NCI/NIH, Bethesda, MD, 20892, USA.
authors.forename: Fedor
authors.initials: F
authors.lastname: Kouzine
authors.affiliation: Laboratory of Pathology, NCI/NIH, Bethesda, MD, 20892, USA.
authors.forename: Suzanne
authors.initials: S
authors.lastname: Sanford
authors.affiliation: Laboratory of Pathology, NCI/NIH, Bethesda, MD, 20892, USA.
authors.forename: David
authors.initials: D
authors.lastname: Levens
mesh_terms: D045744:Cell Line, Tumor; D002843:Chromatin / Q000737:chemistry*; D047369:Chromatin Immunoprecipitation / Q000379:methods*; D003432:Cross-Linking Reagents / Q000737:chemistry*; D004247:DNA / Q000737:chemistry*; D004264:DNA Topoisomerases, Type I / Q000737:chemistry*; D004268:DNA-Binding Proteins / Q000737:chemistry; D005557:Formaldehyde / Q000737:chemistry*; D049452:Green Fluorescent Proteins / Q000737:chemistry*; D045325:HCT116 Cells; D006801:Humans; D013997:Time Factors
chemical_list: D002843:Chromatin; D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins; D049452:Green Fluorescent Proteins; D005557:Formaldehyde; D004247:DNA; D004264:DNA Topoisomerases, Type I; C528909:TOP1 protein, human
keywords: Chromatin immunoprecipitation; DNA topoisomerase 1; Formaldehyde cross-linking; Green fluorescent protein