F-actin-dependent insolubility of chromatin-modifying components.
Many complexes involved in chromatin modification are difficult to isolate and commonly found associated with nuclear matrix preparations. In this study, we examine the elution properties of chromatin-modifying components under different extraction conditions. We find that most, but not all, histone acetyltransferases and histone deacetylases predominantly partition with the nuclear pellet during intermediate salt extraction. In attempts to identify a biological basis for the observed insolubility, we demonstrate that depolymerizing cellular actin, but not cellular tubulin, mobilizes a significant proportion of the insoluble pool into the intermediate salt-soluble nuclear extract. The disruption of cellular F-actin releases a specific subset of high molecular weight, active, nuclear histone deacetylase complexes that are not found under normal conditions. This study demonstrates that actin polymerization, a physiologically relevant process, is responsible for the observed insolubility of these components during nuclear extract preparation and establishes a simple strategy for isolating subsets of chromatin-modifying complexes that are otherwise depleted or absent under the same extraction conditions.
Andrin
Christi
C
Department of Oncology, University of Alberta, Edmonton, Alberta T6G 1Z2, Canada.
Hendzel
Michael J
MJ
D000123:Acetyltransferases / Q000737:chemistry*; D000199:Actins / Q000502:physiology*; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000494:pharmacology; D002843:Chromatin / Q000378:metabolism; D051548:Histone Acetyltransferases; D006655:Histone Deacetylases / Q000737:chemistry*; D015739:Nocodazole / Q000494:pharmacology; D009687:Nuclear Proteins / Q000737:chemistry; D012995:Solubility; D013844:Thiazoles / Q000494:pharmacology; D053778:Thiazolidines
D000199:Actins; D019086:Bridged Bicyclo Compounds, Heterocyclic; D002843:Chromatin; D009687:Nuclear Proteins; D013844:Thiazoles; D053778:Thiazolidines; D000123:Acetyltransferases; D051548:Histone Acetyltransferases; D006655:Histone Deacetylases; C037068:latrunculin B; D015739:Nocodazole