Expression, Purification, and Biochemical Analysis of the LSD1/KDM1A Histone Demethylase

Methods Enzymol. 2016;573:241-59. doi: 10.1016/bs.mie.2016.02.001. Epub 2016 Mar 4.

Abstract

Posttranslational modifications (PTMs) of histones play important roles in the regulation of chromatin architecture and gene transcription. A decade ago, it was still believed that methyl groups could not be removed from histones, until the first histone demethylase LSD1 (lysine-specific demethylase 1; also known as KDM1A) was identified. This discovery initiated an era in the understanding of chromatin dynamic regulation by active histone demethylation. Since then, the repertoire of histone demethylases has expanded, and our understanding of the molecular mechanisms, structures, and macromolecular complexes of the demethylases has grown significantly. Histone demethylases have emerged as important players in developmental processes and have been linked to human diseases and cancer. Studies highlighting the functions of LSD1 have significantly increased our understanding of chromatin biology and have revealed that new facets of histone demethylases remain to be discovered. In vitro methods have been developed to assess the biochemistry, structure, and enzymology of lysine demethylases. Here, we describe the methods of expression, purification, and biochemical analysis that we have successfully used in characterizing the functions of LSD1.

Keywords: Chromatin; Demethylase; Epigenetics; Histones; LSD1; Methylation.

Publication types

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

MeSH terms

  • Animals
  • Bacteria / genetics
  • CRISPR-Cas Systems
  • Cell Line
  • Cloning, Molecular / methods
  • Enzyme Assays / methods
  • Histone Demethylases / chemistry
  • Histone Demethylases / genetics*
  • Histone Demethylases / isolation & purification
  • Histone Demethylases / metabolism*
  • Histones / metabolism
  • Humans
  • Insecta
  • Methylation
  • Protein Conformation
  • Protein Processing, Post-Translational
  • RNA Interference
  • RNA, Small Interfering / genetics
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / metabolism

Substances

  • Histones
  • RNA, Small Interfering
  • Recombinant Proteins
  • Histone Demethylases
  • KDM1A protein, human