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

Nucleic Acids Res. 2015 Aug 18;43(14):6959-68. doi: 10.1093/nar/gkv637. Epub 2015 Jun 27.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Artifacts*
  • Binding Sites
  • Chromatin Immunoprecipitation / methods*
  • Chromosomes, Insect / metabolism
  • Databases, Genetic
  • Drosophila / embryology
  • Drosophila / genetics
  • Drosophila / metabolism
  • Drosophila Proteins / metabolism
  • High-Throughput Nucleotide Sequencing / methods*
  • Promoter Regions, Genetic*
  • RNA Splicing Factors
  • RNA-Binding Proteins / metabolism
  • Repressor Proteins / metabolism
  • Sequence Analysis, DNA / methods*
  • Transcription Factors / metabolism


  • Acf protein, Drosophila
  • Drosophila Proteins
  • RNA Splicing Factors
  • RNA-Binding Proteins
  • RSF1 protein, Drosophila
  • Repressor Proteins
  • Transcription Factors