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Methods Enzymol. 2016;574:31-52. doi: 10.1016/bs.mie.2016.01.008. Epub 2016 Feb 16.

Substrate Specificity Profiling of Histone-Modifying Enzymes by Peptide Microarray.

Author information

1
Center for Epigenetics, Van Andel Research Institute, Grand Rapids, MI, United States.
2
University of Michigan, Ann Arbor, MI, United States.
3
University of North Carolina at Chapel Hill, Chapel Hill, NC, United States.
4
Center for Epigenetics, Van Andel Research Institute, Grand Rapids, MI, United States. Electronic address: scott.rothbart@vai.org.

Abstract

The dynamic addition and removal of covalent posttranslational modifications (PTMs) on histone proteins serves as a major mechanism regulating chromatin-templated biological processes in eukaryotic genomes. Histone PTMs and their combinations function by directly altering the physical structure of chromatin and as rheostats for effector protein interactions. In this chapter, we detail microarray-based methods for analyzing the substrate specificity of lysine methyltransferase and demethylase enzymes on immobilized synthetic histone peptides. Consistent with the "histone code" hypothesis, we reveal a strong influence of adjacent and, surprisingly, distant histone PTMs on the ability of histone-modifying enzymes to methylate or demethylate their substrates. This platform will greatly facilitate future investigations into histone substrate specificity and mechanisms of PTM signaling that regulate the catalytic properties of histone-modifying enzymes.

KEYWORDS:

Chromatin; Demethylases; Epigenetics; Erasers; Histone code; Histones; Methyltransferases; Peptide microarray; Posttranslational modifications; Writers

PMID:
27423856
PMCID:
PMC5322744
DOI:
10.1016/bs.mie.2016.01.008
[Indexed for MEDLINE]
Free PMC Article

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