Maize histone deacetylase HDA1 complexes

Posttranslational acetylation of core histones represents an essential element of the "epigenetic histone code". The most extensively investigated modification is acetylation, catalyzed by histone acetyltransferases and histone deacetylases.Our laboratory has recently reported a novel level of regulation of histone deacetylases in maize, namely that the histone deacetylase activity of an ZmHda1 homolog is regulated by limited proteolysis (Pipal et al., 2003). 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 chromatin acetylation and protolytic pathways. Based on these findings the project proposal addresses this novel interconnection by characterization, purification and identification of ZmHDA1 forms and associated complexes. In particular it will focus on: -the purification and identification of proteins associated with ZmHda1-p84 in high molecular weight complexes, -the purification and identification of the protease responsible for conversion of enzymatically inactive ZmHda1-p84 into enzymatically active ZmHda1-p48, -the characterization of the proteolytic cleavage product (p36) and its complex, -the modifications of ZmHDA1-p84, p48 and p36, and -the localization of the relevant proteins and protein complexes in different maize tissues.

Posttranslational acetylation of core histones represents an essential element of the "epigenetic histone code". The most extensively investigated modification is acetylation, catalyzed by histone acetyltransferases and histone deacetylases.Our laboratory has recently reported a novel level of regulation of histone deacetylases in maize, namely that the histone deacetylase activity of an ZmHda1 homolog is regulated by limited proteolysis (Pipal et al., 2003). 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 chromatin acetylation and protolytic pathways. Based on these findings the project proposal addresses this novel interconnection by characterization, purification and identification of ZmHDA1 forms and associated complexes. In particular it will focus on: the purification and identification of proteins associated with ZmHda1-p84 in high molecular weight complexes, the purification and identification of the protease responsible for conversion of enzymatically inactive ZmHda1-p84 into enzymatically active ZmHda1-p48, the characterization of the proteolytic cleavage product (p36) and its complex, the modifications of ZmHDA1-p84, p48 and p36, and the localization of the relevant proteins and protein complexes in different maize tissues.