Abstract
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.
Similar content being viewed by others
Abbreviations
- ChIP:
-
chromatin immunoprecipitation
- GFP:
-
green fluorescent protein
- NLS:
-
nuclear localization signal
- PCR:
-
polymerase chain reaction
- qPCR:
-
quantitative PCR
- Top1:
-
DNA topoisomerase 1
- RNAPII:
-
RNA polymerase II
References
Baranello L, Kouzine F, Levens D (2013) DNA topoisomerases beyond the standard role. Transcription 4(5):232–237
Baranello L et al (2014) DNA break mapping reveals topoisomerase II activity genome-wide. Int J Mol Sci 15(7):13111–13122
Barski A et al (2007) High-resolution profiling of histone methylations in the human genome. Cell 129(4):823–837
Beneke S et al (2012) Chromatin composition is changed by poly(ADP-ribosyl)ation during chromatin immunoprecipitation. PLoS ONE 7(3):e32914
Champoux JJ (2001) DNA topoisomerases: structure, function, and mechanism. Annu Rev Biochem 70:369–413
Fan X, Struhl K (2009) Where does mediator bind in vivo? PLoS ONE 4(4):e5029
Gilmour DS et al (1986) Topoisomerase I interacts with transcribed regions in Drosophila cells. Cell 44(3):401–407
Gueron M, Kochoyan M, Leroy JL (1987) A single mode of DNA base-pair opening drives imino proton exchange. Nature 328(6125):89–92
Kouzine F et al (2013) Transcription-dependent dynamic supercoiling is a short-range genomic force. Nat Struct Mol Biol 20(3):396–403
Marinov GK et al. (2014) Large-scale quality analysis of published ChIP-seq data. G3 (Bethesda). 4(2): p. 209–23
Milne TA, Zhao K, Hess JL (2009) Chromatin immunoprecipitation (ChIP) for analysis of histone modifications and chromatin-associated proteins. Methods Mol Biol 538:409–423
O'Neill LP, Turner BM (2003) Immunoprecipitation of native chromatin: NChIP. Methods 31(1):76–82
Park D et al (2013) Widespread misinterpretable ChIP-seq bias in yeast. PLoS ONE 8(12):e83506
Poorey K et al (2013) Measuring chromatin interaction dynamics on the second time scale at single-copy genes. Science 342(6156):369–372
Puc J et al (2015) Ligand-dependent enhancer activation regulated by topoisomerase-I activity. Cell 160(3):367–380
Solomon MJ, Larsen PL, Varshavsky A (1988) Mapping protein-DNA interactions in vivo with formaldehyde: evidence that histone H4 is retained on a highly transcribed gene. Cell 53(6):937–947
Teytelman L et al (2013) Highly expressed loci are vulnerable to misleading ChIP localization of multiple unrelated proteins. Proc Natl Acad Sci U S A 110(46):18602–18607
Tsompana M, Buck MJ (2014) Chromatin accessibility: a window into the genome. Epigenetics Chromatin 7(1):33
Acknowledgments
Our research is supported by the Intramural Research Program of the US National Institutes of Health, Center for Cancer Research of the National Cancer Institute.
Author information
Authors and Affiliations
Corresponding author
Additional information
Responsible Editor: Beth A Sullivan
Rights and permissions
About this article
Cite this article
Baranello, L., Kouzine, F., Sanford, S. et al. ChIP bias as a function of cross-linking time. Chromosome Res 24, 175–181 (2016). https://doi.org/10.1007/s10577-015-9509-1
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10577-015-9509-1