Active promoters give rise to false positive 'Phantom Peaks' in ChIP seq experiments
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.
Nucleic acids research