title: Chromatin composition is changed by poly(ADP-ribosyl)ation during chromatin immunoprecipitation.
abstract: Chromatin-immunoprecipitation (ChIP) employs generally a mild formaldehyde cross-linking step, which is followed by isolation of specific protein-DNA complexes and subsequent PCR testing, to analyze DNA-protein interactions. Poly(ADP-ribosyl)ation, a posttranslational modification involved in diverse cellular functions like repair, replication, transcription, and cell death regulation, is most prominent after DNA damage. Poly(ADP-ribose)polymerase-1 is activated upon binding to DNA strand-breaks and coordinates repair by recruitment or displacement of proteins. Several proteins involved in different nuclear pathways are directly modified or contain poly(ADP-ribose)-interaction motifs. Thus, poly(ADP-ribose) regulates chromatin composition. In immunofluorescence experiments, we noticed artificial polymer-formation after formaldehyde-fixation of undamaged cells. Therefore, we analyzed if the formaldehyde applied during ChIP also induces poly(ADP-ribosyl)ation and its impact on chromatin composition. We observed massive polymer-formation in three different ChIP-protocols tested independent on the cell line. This was due to induction of DNA damage signaling as monitored by γH2AX formation. To abrogate poly(ADP-ribose) synthesis, we inhibited this enzymatic reaction either pharmacologically or by increased formaldehyde concentration. Both approaches changed ChIP-efficiency. Additionally, we detected specific differences in promoter-occupancy of tested transcription factors as well as the in the presence of histone H1 at the respective sites. In summary, we show here that standard ChIP is flawed by artificial formation of poly(ADP-ribose) and suppression of this enzymatic activity improves ChIP-efficiency in general. Also, we detected specific changes in promoter-occupancy dependent on poly(ADP-ribose). By preventing polymer synthesis with the proposed modifications in standard ChIP protocols it is now possible to analyze the natural chromatin-composition.
authors.affiliation: Molecular Toxicology, University of Konstanz, Konstanz, Germany. sascha.beneke@vetpharm.uzh.ch
authors.forename: Sascha
authors.initials: S
authors.lastname: Beneke
authors.forename: Kirstin
authors.initials: K
authors.lastname: Meyer
authors.forename: Anja
authors.initials: A
authors.lastname: Holtz
authors.forename: Katharina
authors.initials: K
authors.lastname: Hüttner
authors.forename: Alexander
authors.initials: A
authors.lastname: Bürkle
mesh_terms: D016475:3T3 Cells; D000818:Animals; D002460:Cell Line; D002843:Chromatin / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D047369:Chromatin Immunoprecipitation / Q000379:methods*; D004249:DNA Damage; D005404:Fixatives / Q000494:pharmacology; D005557:Formaldehyde / Q000494:pharmacology; D006367:HeLa Cells; D006657:Histones / Q000378:metabolism; D006801:Humans; D051379:Mice; D008856:Microscopy, Fluorescence; D010616:Phenanthrenes / Q000494:pharmacology; D011064:Poly Adenosine Diphosphate Ribose / Q000378:metabolism*; D000067856:Poly(ADP-ribose) Polymerase Inhibitors; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011485:Protein Binding / Q000187:drug effects
chemical_list: D002843:Chromatin; D005404:Fixatives; C468783:H2AX protein, human; D006657:Histones; C434926:N-(oxo-5,6-dihydrophenanthridin-2-yl)-N,N-dimethylacetamide hydrochloride; D010616:Phenanthrenes; D000067856:Poly(ADP-ribose) Polymerase Inhibitors; D005557:Formaldehyde; D011064:Poly Adenosine Diphosphate Ribose; D011065:Poly(ADP-ribose) Polymerases