Abstract
Histone post-translational modifications (PTMs) are important genomic regulators often studied by chromatin immunoprecipitation (ChIP), whereby their locations and relative abundance are inferred by antibody capture of nucleosomes and associated DNA. However, the specificity of antibodies within these experiments has not been systematically studied. Here, we use histone peptide arrays and internally calibrated ChIP (ICeChIP) to characterize 52 commercial antibodies purported to distinguish the H3K4 methylforms (me1, me2, and me3, with each ascribed distinct biological functions). We find that many widely used antibodies poorly distinguish the methylforms and that high- and low-specificity reagents can yield dramatically different biological interpretations, resulting in substantial divergence from the literature for numerous H3K4 methylform paradigms. Using ICeChIP, we also discern quantitative relationships between enhancer H3K4 methylation and promoter transcriptional output and can measure global PTM abundance changes. Our results illustrate how poor antibody specificity contributes to the “reproducibility crisis,” demonstrating the need for rigorous, platform-appropriate validation. Shah et al. evaluate 54 antibodies for specific H3K4 methylforms by internally calibrated chromatin immunoprecipitation (ICeChIP) and peptide arrays. Many of the most commonly used antibodies poorly distinguish between methylforms, and high-specificity ICeChIP datasets show deviation from literature paradigms of H3K4 methylation biology based on ChIP datasets generated with low-specificity antibodies.
Original language | English |
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Pages (from-to) | 162-177.e7 |
Journal | Molecular Cell |
Volume | 72 |
Issue number | 1 |
DOIs | |
State | Published - Oct 4 2018 |
Externally published | Yes |
Funding
We wish to thank P. Faber and H. Whitehurst in the University of Chicago Functional Genomics Facility for Illumina sequencing. We also thank the following for donating reagents used in this study: Abclonal (six antibodies), Active Motif (six antibodies), Cell Signaling Technology (three antibodies), and Diagenode (three antibodies). We also thank Dr. Chuck Epstein of the Broad Institute for his generous gift of five antibodies used by the ENCODE Consortium. We further thank Dr. Joanna Wysocka of Stanford University for her generous gift of mESC cell lines. We also wish to thank Claire Kokontis and Lindsay Stolzenburg for their helpful comments on this manuscript. This study was supported by the NIH under award numbers R44-HG008907 to Z.-W.S. and M.-C.K., R00-CA181343 to S.B.R., R35-GM124736 to S.B.R., and R01-GM115945 to A.J.R.
Keywords
- H3 lysine 4 methylation
- antibody
- chromatin
- chromatin immunoprecipitation
- histone
- molecular biology
- peptide array
- transcription
- transcriptional enhancer