Active promoters give rise to false positive ‘Phantom Peaks’ in ChIP-seq experiments
Open accessAbstract
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.
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It remains unknown what proportion of candidate enhancers identified solely by biochemical marks are technical false positives or have enhancer-like features but no meaningful impact on expression of cis-encoded genes.
“Although unquestionably useful, it remains unknown what proportion of candidate enhancers identified solely by biochemical marks are technical false positives130-132 or a product of having enhancer-like biochemical features but no meaningful impact on the expression of cis encoded genes133.”
From Towards a comprehensive catalogue of validated and target-linked human enhancers (2020) · cites this paper as [132]
The Discussion reports that some ChIP-seq enrichments may be false-positive Phantom Peaks and says it cannot determine which overlapping signals represent genuine binding. The cited paper does not assess candidate enhancers identified solely by biochemical marks or estimate what proportion of them are technical false positives. The separate claim about enhancer-like features without an effect on cis-gene expression is attached to citation 133, not this source. (LLM, conf=0.84)
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- Authors
- Jain, D., Baldi, S., Zabel, A., Straub, T., & Becker, P. B.
- Affiliations
- Center for Integrated Protein Science Munich (DE)
- Corresponding author
- Becker, Peter B.
- Venue
- Nucleic Acids Research
- Publisher
- Oxford University Press (OUP)
- Published
- 2015-06-27
- Volume
- 43
- Issue
- 14
- Pages
- 6959-6968
- ISSN
- 0305-1048 (ISSN-L)
- 1362-4962
- PMC
- PMC4538825
- OpenAlex
- W1610193264
- Cited by
- 156
- References
- 65
- Citations by year
- 2026: 6 · 2025: 12 · 2024: 13 · 2023: 10 · 2022: 8 · 2021: 16 · 2020: 13 · 2019: 20 · 2018: 19 · 2017: 15 · 2016: 22 · 2015: 2
- FWCI
- 8.40
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Locations
- Nucleic Acids Research (publishedVersion, cc-by)
- Europe PMC (PubMed Central) (publishedVersion, cc-by)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- Open access LMU (Ludwid Maxmilian's Universitat Munchen) (submittedVersion)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Genomics and Chromatin Dynamics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Chromatin Remodeling and Cancer
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Epigenetics and DNA Methylation
- Keywords
- ACF, Drosophila embryo, chromatin immunoprecipitation, modENCODE
- MeSH terms
- RNA Splicing Factors, Animals, Binding Sites, Drosophila, Promoter Regions, Genetic, Repressor Proteins, Transcription Factors, Artifacts, RNA-Binding Proteins, Sequence Analysis, DNA, Drosophila Proteins, Databases, Genetic, Chromatin Immunoprecipitation, Chromosomes, Insect, High-Throughput Nucleotide Sequencing
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