Histone deacetylase from HeLa cells: properties of the high molecular weight complex

Abstract

In previous work [Hay, C. W., & Candido, E. P. M. (1983) J. Biol. Chem. 258, 3726-3734], we have shown that the histone deacetylase from HeLa cell nuclei is associated with a high molecular weight, nuclease-resistant complex. This complex was found to contain a variety of non-histone proteins, and indirect evidence for the importance of protein-protein interactions in the maintenance of its structure was obtained. In the present report, we examine the effects of beta-mercaptoethanol and neocuproine on the deacetylase complex and present data on the level of histone acetylation and the presence of satellite DNA sequences in this material. HeLa cell histone deacetylase complex partially dissociates in 10 mM beta-mercaptoethanol, resulting in a loss of non-histone proteins. The presence of 10 mM beta-mercaptoethanol during the micrococcal nuclease digestion of HeLa cell nuclei results in a greatly reduced yield of histone deacetylase complex, with a correspondingly large increase in the production of small oligonucleosomes and mononucleosomes. Histone deacetylase activity on endogenous labeled histone within the complex is strongly inhibited by either 1 or 10 mM beta-mercaptoethanol or 3 mM neocuproine. This loss of histone deacetylase activity does not seem to be due to an inactivation of the enzyme but appears to be a consequence of the disruption of the structure of the deacetylase complex itself. Histone H4 in the deacetylase complex prepared from HeLa cell nuclei by micrococcal nuclease digestion was more highly acetylated than H4 in bulk nucleosomes. Restriction enzyme analysis of the DNA associated with the histone deacetylase complex revealed neither an enrichment nor a depletion of major satellite sequences in this material.

Properties of chicken erythrocyte histone deacetylase associated with the nuclear matrix

“…Gelexclusion chromatography of the nuclear matrix-and nuclearsolubilized HD activities revealed several HD forms, with the high-molecular-mass (at least 400 kDa) HD form predominating. These results are similar to those obtained by Hay and Candido [29,30] who found that HD from HeLa cells exists as a highmolecular-mass complex with a protein composition analogous to that of nuclear-matrix preparations.…”

Nonisotopic substrate for assaying both human zinc and NAD+-dependent histone deacetylases

“…One of the drawbacks of our substrate MAL is a limited rate of conversion by HeLa nuclear extract which is a standard source of human HDAC activity [16]. Here, we report an improved substrate with better acceptance by HeLa deacetylase activity.…”

Alteration of Nucleosome Structure as a Mechanism of Transcriptional Regulation

“…The importance of histone deacetylase enzymes (HDs) in the turnover of the acetate modification became clear from early studies that illustrated that inhibition of HD activity led to an accumulation of acetylated histones in vivo (207). Multiple forms of HDs have been characterized in several organisms (139,140,(208)(209)(210), and like HATs, they appear to be found in high molecular weight complexes (211)(212)(213)(214). As discussed below, some histone deacetylases have been linked to RNA polymerase II transcription; however, there are likely to be additional HDs that perform other functions in chromatin.…”