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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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“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]
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Metadata
- 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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