Journal
INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES
Volume 24, Issue 11, Pages -Publisher
MDPI
DOI: 10.3390/ijms24119271
Keywords
chromatin immunoprecipitation; controls; DNA-binding protein; genome; replication origins
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Chromatin ImmunoPrecipitation (ChIP) is a widely used method for studying protein-DNA interactions, but it suffers from false-positive signal enrichment. We developed a new approach to control for non-specific enrichment in ChIP by co-expressing a non-genome-binding protein with the experimental target protein and using the shared epitope tags. This method improves data quality by normalizing the experimental data with the ChIP signal of the non-specific protein, and has been successfully validated with known binding sites of multiple proteins.
Chromatin ImmunoPrecipitation (ChIP) is a widely used method for the analysis of protein-DNA interactions in vivo; however, ChIP has pitfalls, particularly false-positive signal enrichment that permeates the data. We have developed a new approach to control for non-specific enrichment in ChIP that involves the expression of a non-genome-binding protein targeted in the IP alongside the experimental target protein due to the sharing of epitope tags. ChIP of the protein provides a sensor for non-specific enrichment that can be used for the normalization of the experimental data, thereby correcting for non-specific signals and improving data quality as validated against known binding sites for several proteins that we tested, including Fkh1, Orc1, Mcm4, and Sir2. We also tested a DNA-binding mutant approach and showed that, when feasible, ChIP of a site-specific DNA-binding mutant of the target protein is likely an ideal control. These methods vastly improve our ChIP-seq results in S. cerevisiae and should be applicable in other systems.
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