Subcellular Location of Enzymes Involved in Core Histone Acetylation

Abstract

Multiple enzyme forms of histone deacetylase and histone acetyltransferase exist in germinating maize embryos. We analyzed the association of the different enzymes to chromatin by ion exchange chromatography of subcellular fractions from different time points of embryo germination. The vast majority of histone deacetylase HD-1A was not bound to chromatin, since it was solubilized during chromatin isolation, regardless of its phosphorylation state and the phase of embryo germination. In contrast, HD-2 was chromatin bound during the entire germination pathway. Histone deacetylase HD-1B was present in a chromatin-bound and a soluble form; the ratio between these two forms changed during germination. Both nuclear histone acetyltransferases, HAT-A1 and HAT-A2, were tightly chromatin-bound and could only be released from chromatin by salt extraction. To test whether histone acetyltransferases or deacetylases are associated with the nuclear matrix, we analyzed nuclear matrix preparations from yeast, Physarum, and maize step by step for both enzyme activities. This analysis confirmed that part of the activity is chromatin bound, but no significant enzyme activity could be found in the final nuclear matrix, regardless of the preparation protocol. This result was further substantiated by detailed analysis of histone deacetylases and acetyltransferases during cellular fractionation and nuclear matrix preparation of chicken erythrocytes. Altogether our results suggest that the participation of these enzymes in different nuclear processes may partly be regulated by a distinct location to intranuclear components.

Purification of histone deacetylase HD1‐A of germinating maize embryos

“…In most experimental systems investigated so far histone acetyltransferases and deacetylases exist as multiple enzyme forms; in pea, three histone deacetylases exist [26]. In maize we could distinguish four histone deacetylases [16,17,20,22,29]. The four histone deacetylases of maize are distinct with respect to their biochemical and kinetic properties [29], to modification by phosphorylation [20], pattern of enzyme activity during the cell cycle and differentiation [16][17][18], substrate specificity [16,20,29] and subnuclear localization [22].…”

“…In maize we could distinguish four histone deacetylases [16,17,20,22,29]. The four histone deacetylases of maize are distinct with respect to their biochemical and kinetic properties [29], to modification by phosphorylation [20], pattern of enzyme activity during the cell cycle and differentiation [16][17][18], substrate specificity [16,20,29] and subnuclear localization [22]. It is puzzling that despite their different properties, all deacetylases of maize are equally inhibited by different unspecific and specific inhibitors, like butyrate, Trichostatin and cyclic tetrapeptides [21,29].…”

“…The different deacetylases are distinguished by their association with chromatin [22]. The histone deacetylase HD1-A is not associated with chromatin and is therefore readily solubilized and cofractionates with the bulk of cytoplasmic proteins at 72 h after the start of embryo germination.…”

“…The steady state of histone acetylation is maintained by two enzyme activities, histone acetyltransferase and histone deacetylase. These enzyme activities are present as multiple enzyme forms whose activities, properties, intracellular/intranuclear location and substrate specificities are regulated in a complex, cell cycle-related manner [16][17][18][19][20][21][22].…”

“…In maize four different histone deacetylases can be distinguished during embryo ger-*Corresponding author. Fax: (43) (512) 507 2866. mination [16,17,20,22,29]; these enzymes differ considerably from each other in terms of substrate specificity, intranuclear location, kinetic properties and posttranslational modification. In yeast a nuclear histone deacetylase could be isolated as a high molecular weight complex (> 350 kDa) at low ionic strength [27]; when the ionic strength was increased to 0.5 M NH4C1, the enzyme activity was present in a low molecular weight form of ~ 150 kDa.…”