Search

We report on the site specificity of two intact pea histone deacetylase complexes. HD1 deacetylates lysines 5 and 16 of H4 in the order K16 > K5, while in the case of H3 the preferred order is K4 >> K18 approximately K9. The specificity of the HD2 complex is markedly different. The preferred residues in H4 are K8 approximately K5 > K16, while in H3 deacetylation, the complex HD2 prefers sites 4 and 18. To obtain these results, we have used a novel procedure based on the SPOT technique, a method to synthesize peptides on membrane supports. Different sets of membranes with sequentially overlapping histone peptides containing acetylated lysines in the sites corresponding to all in vivo acetylatable residues were incubated with the complexes. The acetyl groups removed by the deacetylase activity were then replaced by radioactive acetate by treating the membranes with labeled acetic anhydride. The subsequent counting of the membranes allows the quantification of the acetate removal in the histone deacetylase reaction in a way that circumvents some of the inconveniences of other available procedures.

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.

We have previously reported [López-Rodas et al. (1989) J. Biol. Chem. 264, 19028-19033] that the yeast Saccharomyces cerevisiae contains four histone acetyltransferases, which can be resolved by ion-exchange chromatography, and their specificity toward yeast free histones was studied. In the present contribution we show that three of the enzymes are nuclear, type A histone acetyltransferases and they are able to acetylate nucleosome-bound histones. They differ in their histone specificity. Enzyme A1 acetylates H2A in chicken nucleosomes, although it is specific for yeast free H2B; histone acetyltransferase A2 is highly specific for H3, and histone acetyltransferase A3 preparations acetylate both H3 and H4 in nucleosomes. The fourth enzyme, which is located in the cytoplasm, does not accept nucleosomes as substrate, and it represents a canonical type B, H4-specific histone acetyltransferase. Finally, histone deacetylase activity is preferentially found in the nucleus.