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Mol Cell. 2018 Oct 4;72(1):162-177.e7. doi: 10.1016/j.molcel.2018.08.015. Epub 2018 Sep 20.

Examining the Roles of H3K4 Methylation States with Systematically Characterized Antibodies.

Author information

1
Department of Molecular Genetics and Cell Biology, The University of Chicago, Chicago, IL 60637, USA; Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, IL 60637, USA.
2
Department of Molecular Genetics and Cell Biology, The University of Chicago, Chicago, IL 60637, USA.
3
Center for Epigenetics, Van Andel Research Institute, Grand Rapids, MI 49503, USA.
4
Epicypher, Inc., Research Triangle Park, NC 27713, USA.
5
Department of Chemical and Systems Biology, Stanford University School of Medicine, Stanford, CA 94305, USA.
6
Center for Epigenetics, Van Andel Research Institute, Grand Rapids, MI 49503, USA. Electronic address: scott.rothbart@vai.org.
7
Epicypher, Inc., Research Triangle Park, NC 27713, USA. Electronic address: michael@epicypher.com.
8
Department of Molecular Genetics and Cell Biology, The University of Chicago, Chicago, IL 60637, USA; Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, IL 60637, USA. Electronic address: aruthenburg@uchicago.edu.

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.

KEYWORDS:

H3 lysine 4 methylation; antibody; chromatin; chromatin immunoprecipitation; histone; molecular biology; peptide array; transcription; transcriptional enhancer

PMID:
30244833
PMCID:
PMC6173622
DOI:
10.1016/j.molcel.2018.08.015
[Indexed for MEDLINE]
Free PMC Article

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