Posttranslational acetylation of core histones: Plant histone deacetylases

Research project P 14528 Posttranslational acetylation of core histones: Plant histone deacetylases Peter LOIDL 09.10.2000 The DNA of eukaryotic cells is associated with basic proteins, the histones, which have been highly conserved during evolution. The repeating basic subunit of this protein-DNA complex is the nucleosome core particle which consists of two molecules each of H2A, 1-1213, H3, and H4. The core histones contain N-terminal extensions which are characterized by conserved residues that are subject of posttranslational modification. The most important is acetylation of epsilon-amino groups of lysines. Acetylation is established by histone acetyltransferases and reverted by histone deacetylases. Until recently, the function of this modification was totally unclear. The identification of histone acetyltransferases and deacetylases as transcriptional regulators was a major breakthrough in the chromatin field. We have concentrated our effort on the characterization, purification and molecular cloning of histone deacetylases in maize. Germinating embryos of maize contain at least 3 distinct histone deacetylases: HD1-B which exists in two forms was identified as an Rpd3-type deacetylase, whereas we could show that HD1 - A and HD2 are members of novel classes of deacetylases, that were not defined before in eukaryotic organisms. Maize therefore at present represents the bestcharacterized system for these enzymes. The major aim of the proposed project is to complete our knowledge on plant histone deacetylases and to investigate the expression of al I deacetylase types. I n particular it deals with 1 ) the molecular clon ing of maize H D 1 A, which is regulated by phosphorylation, and an additional deacetylase homologous to yeast Hdal p; 2) the identification and characterization of an NAD-dependent deacetylase activity with homology to yeast Sir, which is essential for silencing; 3) the analysis of interaction partners of the nucleolar histone deacetylase HD2 of maize and Arabidopsis thaliana, as well as the nature of complex formation of the different HD2 forms; 4) the regulated expression of all types of deacetylases during maize embryo germination and in different plant tissues or organs; and 5) the effect of HC toxin or in vivo infection with Cochliobolus carbonum (a maize specific pathogenic fungus) on the expression of histone deacetylases and pathogenesis related proteins. The results of this project will not only increase our understanding of the complex interplay of different deacetylases in plants but will also have important implications for the identification of novel classes of deacetylases in non-plant organism.

The genome contains all the information needed to build an entire organism. However, during differentiation and development, additional epigenetic information determines the functional state of cells and tissues. This epigenetic information can be be introduced by DNA methylation and by marking nucleosomal histones. Therefore the term "epigenetic histone code" in contrast to the genetic DNA code has been introduced. Posttranslational modifications of core histones represent essential elements of this "histone code". The most extensively investigated modification is acetylation, catalyzed by histone acetyltransferases and histone deacetylases. Our laboratory has purified and identified members of three types of histone deacetylases: the plant specific HD2 family, the classical Rpd3-type enzymes and recently an HDA-related enzyme, ZmHDA1. The latter one is remarkable because it displays a novel level of regulation, namely that the enzymatic activity of ZmHDA1 is regulated by limited proteolysis. For all three types of histone deacetylases we could show that each is able to repress transcription efficiently in reporter gene assays. A further type of histone deacetylase activity, an NAD-dependent sirtuine, is also present in maize embryos, but contributes less than 5% of the total deacetylase activity, regardless of the germination stage; this enzyme turned out not to deacetylate histones in vitro, but two distinct high molecular weight proteins of unknown identity. ZmHDA1 is synthesized as an enzymatically inactive protein with an apparent MW of 84.000 which is converted to an enzymatically active 48 kD HDAC by proteolytic removal of the C-terminal part, presumably by the aid of a 65 kD intermediate protein. The enzymatically inactive 84 kD precursor protein is part of a 300 kD complex of unknown composition and function. Only the processed 48 kD protein as a monomer had deacetylase activity and was able to repress transcription efficiently in a reporter gene assay; deacetylase activity of the 48 kD protein was dependent on phosphorylation. Interestingly, Arabidopsis not only contains an Hda1 homologous gene, but also a distinct gene encoding a small protein of 252 amino acids; this small protein is highly related to the C-terminal proteolytic cleavage product of ZmHDA1. The function of this protein is unknown. The regulation of maize HDA1 by limited proteolysis is likely to represent a unique, plant specific level of HDAC regulation, connecting for the first time nuclear chromatin acetylation and proteolytic pathways.