Active promoters give rise to false positive 'Phantom Peaks' in ChIP-seq experiments

• Published in: Nucleic acids research Vol. 43; no. 14; pp. 6959 - 6968
• Main Authors: Jain, Dhawal, Baldi, Sandro, Zabel, Angelika, Straub, Tobias, Becker, Peter B
• Format: Journal Article
• Language: English
• Published: England Oxford University Press 18.08.2015
• Subjects: Animals; Binding Sites; Chromatin Immunoprecipitation - methods; Chromosomes, Insect - metabolism; Databases, Genetic; Drosophila - embryology; Drosophila - genetics; Drosophila - metabolism; Drosophila Proteins - metabolism; Genomics; High-Throughput Nucleotide Sequencing - methods; Promoter Regions, Genetic; Repressor Proteins - metabolism; RNA Splicing Factors; RNA-Binding Proteins - metabolism; Sequence Analysis, DNA - methods; Transcription Factors - metabolism
• ISSN: 0305-1048; 1362-4962
• DOI: 10.1093/nar/gkv637

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.