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Sequential Salt Extractions for the Analysis of Bulk Chromatin Binding Properties of Chromatin Modifying Complexes
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Preparation and Analysis of Native Chromatin-Modifying Complexes.

Y Doyon1, J Côté2

  • 1Centre Hospitalier Universitaire de Québec Research Center and Faculty of Medicine, Laval University, Quebec City, QC, Canada.

Methods in Enzymology
|July 4, 2016
PubMed
Summary

This study presents new methods for purifying native histone acetyltransferase (HAT) and methyltransferase (HMT) complexes. These techniques use advanced gene editing tools to enable detailed biochemical analysis of epigenetic modifiers.

Keywords:
CRISPR/Cas9EZH2Genome editingHistone acetyltransferaseHistone methyltransferaseNuA4PRC2Protein complexesTAL effector nucleasesTIP60Tandem affinity purificationZinc-finger nucleases

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Area of Science:

  • Epigenetics and Molecular Biology
  • Chromatin Biology
  • Biochemistry

Background:

  • Nucleosomes, the fundamental units of chromatin, undergo posttranslational modifications (PTMs) by chromatin modifiers.
  • These enzymes operate within stable protein complexes, crucial for their genomic targeting, substrate specificity, and regulation.
  • PTMs influence nuclear processes and are central to epigenetic mechanisms, necessitating the study of these complexes.

Purpose of the Study:

  • To develop and describe protocols for purifying native histone acetyltransferase (HAT) and methyltransferase (HMT) complexes.
  • To enable detailed biochemical characterization of these essential epigenetic machinery components.
  • To preserve natural transcriptional regulation and minimize artifacts from ectopic expression.

Main Methods:

  • Tandem affinity purification (TAP) of native HAT and HMT complexes.
  • Utilizing human cells engineered with zinc-finger nucleases (ZFNs), TAL effector nucleases (TALENs), and CRISPR/Cas9 systems.
  • Expression of bait proteins from genomic safe harbors or endogenous loci.

Main Results:

  • Obtained near-homogenous preparations of native histone-modifying complexes.
  • Achieved sufficient quantities of purified complexes for biochemical assays.
  • Validated approaches that minimize biochemical artifacts and preserve natural regulation.

Conclusions:

  • The described protocols provide robust methods for isolating native HAT and HMT complexes.
  • These purified complexes are suitable for comprehensive biochemical characterization.
  • The study facilitates a deeper understanding of epigenetic regulation through the analysis of chromatin-modifying complexes.