title: Active promoters give rise to false positive 'Phantom Peaks' in ChIP-seq experiments.
abstract: Chromatin immunoprecipitation (ChIP) is widely used to identify chromosomal binding sites. Chromatin proteins are cross-linked to their target sequences in living cells. The purified chromatin is sheared and the relevant protein is enriched by immunoprecipitation with specific antibodies. The co-purifying genomic DNA is then determined by massive parallel sequencing (ChIP-seq).We applied ChIP-seq to map the chromosomal binding sites for two ISWI-containing nucleosome remodeling factors, ACF and RSF, in Drosophila embryos. Employing several polyclonal and monoclonal antibodies directed against their signature subunits, ACF1 and RSF-1, robust profiles were obtained indicating that both remodelers co-occupied a large set of active promoters.Further validation included controls using chromatin of mutant embryos that do not express ACF1 or RSF-1. Surprisingly, the ChIP-seq profiles were unchanged, suggesting that they were not due to specific immunoprecipitation. Conservative analysis lists about 3000 chromosomal loci, mostly active promoters that are prone to non-specific enrichment in ChIP and appear as 'Phantom Peaks'. These peaks are not obtained with pre-immune serum and are not prominent in input chromatin.Mining the modENCODE ChIP-seq profiles identifies potential Phantom Peaks in many profiles of epigenetic regulators. These profiles and other ChIP-seq data featuring prominent Phantom Peaks must be validated with chromatin from cells in which the protein of interest has been depleted.
authors.affiliation: Biomedical Center and Center for Integrated Protein Science Munich, Ludwig-Maximilians-University, Munich, Germany.
authors.forename: Dhawal
authors.initials: D
authors.lastname: Jain
authors.affiliation: Biomedical Center and Center for Integrated Protein Science Munich, Ludwig-Maximilians-University, Munich, Germany.
authors.forename: Sandro
authors.initials: S
authors.lastname: Baldi
authors.affiliation: Biomedical Center and Center for Integrated Protein Science Munich, Ludwig-Maximilians-University, Munich, Germany.
authors.forename: Angelika
authors.initials: A
authors.lastname: Zabel
authors.affiliation: Biomedical Center and Center for Integrated Protein Science Munich, Ludwig-Maximilians-University, Munich, Germany.
authors.forename: Tobias
authors.initials: T
authors.lastname: Straub
authors.affiliation: Biomedical Center and Center for Integrated Protein Science Munich, Ludwig-Maximilians-University, Munich, Germany pbecker@med.uni-muenchen.de.
authors.forename: Peter B
authors.initials: PB
authors.lastname: Becker
mesh_terms: D000818:Animals; D016477:Artifacts; D001665:Binding Sites; D047369:Chromatin Immunoprecipitation / Q000379:methods*; D059006:Chromosomes, Insect / Q000378:metabolism; D030541:Databases, Genetic; D004330:Drosophila / Q000196:embryology / Q000235:genetics / Q000378:metabolism; D029721:Drosophila Proteins / Q000378:metabolism; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D011401:Promoter Regions, Genetic; D000072260:RNA Splicing Factors; D016601:RNA-Binding Proteins / Q000378:metabolism; D012097:Repressor Proteins / Q000378:metabolism; D017422:Sequence Analysis, DNA / Q000379:methods*; D014157:Transcription Factors / Q000378:metabolism
chemical_list: C120419:Acf protein, Drosophila; D029721:Drosophila Proteins; D000072260:RNA Splicing Factors; D016601:RNA-Binding Proteins; C120397:RSF1 protein, Drosophila; D012097:Repressor Proteins; D014157:Transcription Factors