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Cited by 169Genomics and Chromatin DynamicsChromatin Remodeling and CancerEpigenetics and DNA Methylation

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

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Jain, Dhawal, Baldi, Sandro, Zabel, Angelika, Straub, Tobias, Becker, Peter B.·2015·Nucleic Acids Research
DOI 10.1093/nar/gkv637PMC PMC4538825
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Abstract

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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Figures

Figure 1. ChIP-Seq profiles obtained for ACF1 and RSF-1 in wild-type and mutant embryos show strong overlap. (A) Smoothed and background-subtracted tag density profiles are displayed over a representative region of chromosome 2L. The profiles were obtained with antibodies directed against ACF1 or RSF-1 by ChIP from chromatin of wild-type (WT) and mutant embryos as indicated to the right. The positions of Phantom Peaks are indicated by black boxes in the center track and gray-shaded rectangles across all profiles. (B, C) Venn diagrams illustrating the overlap between the peak regions of (B) ACF1 in WT and acf17 mutant embryos and (C) RSF-1 in WT and rsf-13602 mutant embryos. The peaks overlapping in (B) or (C) were termed ‘ACF1 common’ or ‘RSF-1 common’, respectively. (D) Venn diagram illustrating the overlap between ‘ACF1 common’ and ‘RSF-1 common’ peaks. The union of those peaks yields a set of 3090 loci, which we term ‘Phantom Peaks’.
Figure 1. ChIP-Seq profiles obtained for ACF1 and RSF-1 in wild-type and mutant embryos show strong overlap. (A) Smoothed and background-subtracted tag density profiles are displayed over a representative region of chromosome 2L. The profiles were obtained with antibodies directed against ACF1 or RSF-1 by ChIP from chromatin of wild-type (WT) and mutant embryos as indicated to the right. The positions of Phantom Peaks are indicated by black boxes in the center track and gray-shaded rectangles across all profiles. (B, C) Venn diagrams illustrating the overlap between the peak regions of (B) ACF1 in WT and acf17 mutant embryos and (C) RSF-1 in WT and rsf-13602 mutant embryos. The peaks overlapping in (B) or (C) were termed ‘ACF1 common’ or ‘RSF-1 common’, respectively. (D) Venn diagram illustrating the overlap between ‘ACF1 common’ and ‘RSF-1 common’ peaks. The union of those peaks yields a set of 3090 loci, which we term ‘Phantom Peaks’.CC BY · PubMed Central
Figure 2. Characterization of the Phantom Peaks. (A) Distribution of the 3090 Phantom Peaks between promoters, introns, transcription termination sites (TTS) and intergenic regions. Localization to coding regions or UTRs was negligible. (B) Prevalent sequence motifs within Phantom Peak regions. For each motif the sequence logo, its P-value of enrichment, the fraction of regions with a motif and the best matching motif in the JASPAR database are indicated. (C) Annotation of Phantom Peak localization according to the ‘Nine States’ of chromatin (35). (D) The prevalence of Phantom Peaks correlates with promoter strength. Promoters were binned into five equally-sized groups based on the 10–12 h old WT embryo expression data (36). For each group the gene expression levels and the fraction of promoters containing a Phantom Peak are displayed. (E) Phantom Peaks tend to map to clusters of transcription factor binding sites. Sites containing the indicated number of transcription factor binding events were derived from modENCODE (37). Sites with more than 8 transcription factor binding events are termed ‘HOT regions’. The graph shows the fractions of Phantom Peaks that harbor the given number of transcription factor binding sites.
Figure 2. Characterization of the Phantom Peaks. (A) Distribution of the 3090 Phantom Peaks between promoters, introns, transcription termination sites (TTS) and intergenic regions. Localization to coding regions or UTRs was negligible. (B) Prevalent sequence motifs within Phantom Peak regions. For each motif the sequence logo, its P-value of enrichment, the fraction of regions with a motif and the best matching motif in the JASPAR database are indicated. (C) Annotation of Phantom Peak localization according to the ‘Nine States’ of chromatin (35). (D) The prevalence of Phantom Peaks correlates with promoter strength. Promoters were binned into five equally-sized groups based on the 10–12 h old WT embryo expression data (36). For each group the gene expression levels and the fraction of promoters containing a Phantom Peak are displayed. (E) Phantom Peaks tend to map to clusters of transcription factor binding sites. Sites containing the indicated number of transcription factor binding events were derived from modENCODE (37). Sites with more than 8 transcription factor binding events are termed ‘HOT regions’. The graph shows the fractions of Phantom Peaks that harbor the given number of transcription factor binding sites.CC BY · PubMed Central
Figure 3. Phantom Peaks coincide with peaks of several modENCODE profiles. (A) Peaks called by modENCODE on 153 different non-histone chromatin factors were tested for overlap with Phantom Peak regions. For each profile the scatterplot graph depicts the number of peaks defined by modENCODE and the fraction of these peaks overlapping with Phantom Peaks (P-value <0.05). The P-values indicate the significance of proximity of the modENCODE profile peaks to the Phantom Peaks. (B) Smoothed and background-subtracted tag density profiles of selected modENCODE profiles are shown in a representative genomic region on chromosome X. The positions of Phantom Peaks are indicated by black boxes and gray-shaded rectangles across all profiles.
Figure 3. Phantom Peaks coincide with peaks of several modENCODE profiles. (A) Peaks called by modENCODE on 153 different non-histone chromatin factors were tested for overlap with Phantom Peak regions. For each profile the scatterplot graph depicts the number of peaks defined by modENCODE and the fraction of these peaks overlapping with Phantom Peaks (P-value <0.05). The P-values indicate the significance of proximity of the modENCODE profile peaks to the Phantom Peaks. (B) Smoothed and background-subtracted tag density profiles of selected modENCODE profiles are shown in a representative genomic region on chromosome X. The positions of Phantom Peaks are indicated by black boxes and gray-shaded rectangles across all profiles.CC BY · PubMed Central

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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.
ORCID
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
DOI
10.1093/nar/gkv637
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
Licence
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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