Substrate Specificity Profiling of Histone-Modifying Enzymes by Peptide Microarray

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

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Enzyme Assays / instrumentation
  • Enzyme Assays / methods*
  • Equipment Design
  • High-Throughput Screening Assays / instrumentation
  • High-Throughput Screening Assays / methods
  • Histone Code
  • Histone Demethylases / metabolism*
  • Histone-Lysine N-Methyltransferase / metabolism*
  • Histones / chemistry
  • Histones / metabolism*
  • Humans
  • Peptides / chemistry
  • Peptides / metabolism*
  • Protein Array Analysis / instrumentation
  • Protein Array Analysis / methods*
  • Protein Processing, Post-Translational*
  • Substrate Specificity

Substances

  • Histones
  • Peptides
  • Histone Demethylases
  • Histone-Lysine N-Methyltransferase