Biochemical Methods for Analysis of Histone Deacetylases Doris Ko ¨lle, Gerald Brosch, Thomas Lechner, Alexandra Lusser, and Peter Loidl 1 Department of Microbiology, Medical School, University of Innsbruck, Fritz-Pregl-Strasse 3, A-6020 Innsbruck, Austria Specific lysine residues in the N-terminal extensions of core histones can be posttranslationally modified by acetylation of the -amino group. The dynamic equilibrium of core histone acetylation is established and maintained by histone acetyl- transferases and deacetylases. Both enzymes exist as multiple enzyme forms. Histone acetyltransferases and deacetylases have recently been identified as transcriptional regulators as well as nucleolar phosphoproteins, and have therefore at- tracted considerable research interest. Analysis of the func- tional significance of histone deacetylases for nuclear pro- cesses in certain cases demands the separation and biochemical analysis of different members of the histone deacetylase families. We have characterized three different histones deacetylases in maize embryos and subsequently purified these enzymes to homogeneity. Here we describe methods for extraction, enzymatic assay, chromatographic and electrophoretic separation, and purification of deacety- lases. A novel one-step procedure for large-scale preparation of individual histones and their acetylated isoforms for the analysis of substrate and site specificity of the enzymes is presented. © 1998 Academic Press Since the early discovery of posttranslational core histone acetylation by Allfrey and co-workers (1), this modification has been correlated with chroma- tin assembly and transcription, but also with DNA repair and recombination events (2–7). Postsyn- thetic acetylation of core histones is a ubiquitous modification found in all eukaryotic species so far examined. There are 26 –28 N-terminal lysine sites per nucleosome being subject to reversible acetyla- tion, thus giving rise to a remarkable heterogeneity of nucleosomes with respect to the degree and pat- tern of modification (3). The enzyme histone acetyl- transferase (HAT) introduces an acetyl group from acetyl-CoA into the -amino group of lysine. Each acetate introduced reduces the net positive charge of the histone. The removal of acetate is catalyzed by the activity of histone deacetylase (HD). The specific acetylation state of a histone molecule in the nucleo- some is generated and maintained by these two en- zyme activities. All histones, except H1, can be mod- ified by posttranslational acetylation in vivo. Histone acetylation and hyperacetylation have long been correlated with transcriptionally active chromatin. The basic idea was that the highly charged N-terminal extensions interact with DNA when nonacetylated; after acetylation and the re- sulting charge reduction, this interaction would be weakened and therefore nucleosome structure loos- ened, so that transcription factors could gain access to the DNA. Although this view was conceptually supported by the identification of HATs as transcrip- tional regulators (7), the recent 2.8-Å resolution of the nucleosome core particle structure (8) argued against such a simplistic model, because regions of acetylation are not tightly bound to DNA and are disordered and fully accessible to other proteins. Although the effects of core histone acetylation are usually interpreted in terms of accessibility of pro- moter regions, it is possible that core histone acety- lation rather acts in the destabilization of higher- order structures of chromatin, but also as a specific flag or signal for regulatory proteins or enzymes that 1 To whom correspondence should be addressed. Fax: +43– 512–5072866. E-mail: Peter.Loidl@uibk.ac.at. METHODS: A Companion to Methods in Enzymology 15, 323–331 (1998) Article No. ME980636 323 1046-2023/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.