ChIP bias as a function of cross-linking time
Open accessAbstract
The chromatin immunoprecipitation (ChIP) assay is widely used to capture interactions between chromatin and regulatory proteins in vivo. Formaldehyde cross-linking of DNA and proteins is a critical step required to trap their interactions inside the cells before immunoprecipitation and analysis. Yet insufficient attention has been given to variables that might give rise to artifacts in this procedure, such as the duration of cross-linking. We analyzed the dependence of the ChIP signal on the duration of formaldehyde cross-linking time for two proteins: DNA topoisomerase 1 (Top1) that is functionally associated with the double helix in vivo, especially with active chromatin, and green fluorescent protein (GFP) that has no known bona fide interactions with DNA. With short time of formaldehyde fixation, only Top1 immunoprecipation efficiently recovered DNA from active promoters, whereas prolonged fixation augmented non-specific recovery of GFP dramatizing the need to optimize ChIP protocols to minimize the time of cross-linking, especially for abundant nuclear proteins. Thus, ChIP is a powerful approach to study the localization of protein on the genome when care is taken to manage potential artifacts.
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The ChIP technique in general can produce false findings.
“Notably, the ChIP technique in general can produce false findings.”
From Census and evaluation of p53 target genes (2017) · cites this paper as [141]
ChIP signals vary relative to formaldehyde crosslinking time.
“and ChIP signals vary in general relative to formaldehyde crosslinking time.140, 141”
From Census and evaluation of p53 target genes (2017) · cites this paper as [141]
ChIP is not equally adept at measuring all interactions.
“While ChIP is not equally adept at measuring all interactions (Baranello et al., 2016; Teytelman et al., 2013), recent methods have overcome some of these limitations (Janssens et al., 2018; Skene and Henikoff, 2017; Skene et al., 2018; Zhu et al., 2019).”
From Unraveling Hematopoiesis through the Lens of Genomics (2020) · cites this paper as Baranello et al., 2016
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- Authors
- Baranello, L., Kouzine, F., Sanford, S., & Levens, D.
- Affiliations
- National Institutes of Health (US)
- Corresponding author
- Baranello, Laura
- Venue
- Chromosome Research
- Published
- 2015-12-21
- Volume
- 24
- Issue
- 2
- Pages
- 175-181
- ISSN
- 0967-3849 (ISSN-L)
- 1573-6849
- PMC
- PMC4860130
- OpenAlex
- W2276366744
- Cited by
- 95
- References
- 18
- Citations by year
- 2026: 7 · 2025: 16 · 2024: 3 · 2023: 10 · 2022: 8 · 2021: 10 · 2020: 11 · 2019: 10 · 2018: 10 · 2017: 8 · 2016: 2
- FWCI
- 2.89
- Type
- journal article
- Language
- en
- Open access
- green
- www.ncbi.nlm.nih.gov
- Licence
- www.springer.com/tdm (tdm)
- Locations
- Chromosome Research (publishedVersion)
- PubMed (publishedVersion)
- PubMed Central (acceptedVersion)
- Funders
- National Institutes of Health (Z99 CA999999)
- 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
- chromatin immunoprecipitation, formaldehyde crosslinking, crosslinking time, green fluorescent protein, protein-DNA interactions, protocol optimization
- MeSH terms
- Chromatin, Cross-Linking Reagents, DNA, DNA Topoisomerases, Type I, DNA-Binding Proteins, Formaldehyde, Humans, Time Factors, HCT116 Cells, Cell Line, Tumor, Chromatin Immunoprecipitation, Green Fluorescent Proteins
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