No abstract

Posttranslational core histone acetylation is established and maintained by histone acetyltransferases and deacetylases. Both have been identified as important transcriptional regulators in various eukaryotic systems. In contrast to nonplant systems where only RPD3-related histone deacetylases (HD) have been characterized so far, maize embryos contain three unrelated families of deacetylases (HD1A, HD1B, and HD2). Purification, cDNA cloning, and immunological studies identified the two maize histone deacetylase HD1B forms as close homologues of the RPD3-type deacetylase HDAC1. Unlike the other maize deacetylases, HD1A and nucleolar HD2, HD1B copurified as a complex with a protein related to the retinoblastoma-associated protein, Rbap46. Two HD1B mRNA species could be detected on RNA blots, encoding proteins of 58 kDa (HD1B-I) and 51 kDa (HD1B-II). HD1B-I (zmRpd3) represents the major enzyme form as judged from RNA and immunoblots. Levels of expression of HD1B-I and -II mRNA differ during early embryo germination; HD1B-I mRNA and protein are present during the entire germination pathway, even in the quiescent embryo, whereas HD1B-II expression starts when meristematic cells enter S-phase of the cell cycle. In line with previous results, HD1B exists as soluble and chromatin-bound enzyme forms. In vivo treatment of meristematic tissue with the deacetylase inhibitor HC toxin does not affect the expression of the three maize histone deacetylases, whereas it causes downregulation of histone acetyltransferase B.

The dynamic state of core histone acetylation is maintained by histone acetyltransferases and deacetylases. In germinating maize embryos, four nuclear histone deacetylases can be distinguished. From a chromatin fraction prepared at 72 h after start of embryo germination, we have purified the nuclear histone deacetylase HD2 to homogeneity. Using a sequence of chromatographic steps, we achieved the purification of an enzymatically active high molecular weight protein complex with an apparent molecular mass of 400 kDa, as determined by gel filtration chromatography. The purified enzyme was characterized in terms of enzymatic and kinetic properties, and sensitivity to several histone deacetylase inhibitors. In SDS-polyacrylamide gels, HD2 split into three polypeptides of 45, 42, and 39 kDa, suggesting that the native enzyme is a multimer-protein complex. Electrophoresis under nondenaturing conditions in combination with second dimension SDS-gel electrophoresis indicated that all three protein components of the HD2 complex were enzymatically active. Polyclonal antibodies against each of the three polypeptides were raised in rabbits. Each antiserum reacted with all three polypeptides on Western blots, suggesting that p45, p42, and p39 are highly homologous. This homology was confirmed by amino acid sequencing of peptides generated from each of the three HD2 components.

The upstream binding factor UBF, an activator of RNA polymerase I transcription, is posttranslationally modified by phosphorylation and acetylation. We found that in NIH3T3 cells, UBF is acetylated in S-phase but not in G1-phase. To assess the role of acetylation in regulation of UBF activity, we have established an NIH3T3 cell line that inducibly overexpresses HDAC1. Both in vivo and in vitro, HDAC1 efficiently hypoacetylates UBF. Immunoprecipitation with antibodies against the Pol I-associated factor PAF53 co-precipitated UBF in mock cells but not in cells overexpressing HDAC1. Pull-down experiments showed that acetylation of UBF augments the interaction with Pol I. Consistent with acetylation of UBF being important for association of PAF53 and recruitment of Pol I, the level of Pol I associated with rDNA and pre-rRNA synthesis were reduced in cells overexpressing HDAC1. The results suggest that acetylation and deacetylation of UBF regulate rRNA synthesis during cell cycle progression.

We have characterized and purified histone acetyltransAbstract From a soluble cellular fraction of maize embryos we purified to apparent homogeneity a cytoplasmic histone acetylferases and deacetylases in germinating maize embryos, a transferase, which matches all criteria for a B-type enzyme, source particularly rich in these enzymes. Histone acetyltransUsing 8 chromatographic steps, we achieved a 6700-fold ferases can be classified into nuclear A-type enzymes (specific purification of an enzymatically active protein with a molecular for core histones assembled in nucleosomes) and cytoplasmic weight of ,~ 90 kDa. Under denaturing conditions the protein B-type activities (specific for free core histones, mainly newly split into 2 components which migrated at 45 and 50 kDa in synthesized H4). Histone acetyltransferases have been studied SDS-PAGE, suggesting that the native enzyme is a heterodimer, and partially purified from a variety of cells and organismsThe purified enzyme was characterized in terms of physicochem-(e.g. [14,[18][19][20][21][22][23][24][25][26][27][28][29][30][31][32][33][34]). However, results are difficult to compare ical and kinetic properties, and substrate specificity. It was since the reports differ with respect to isolation of enzymes, specific for histone H4, leading to acetylation of non-acetylated H4 subspecies into the di-acetylated state in vitro. Its activity assay conditions, intracellular localization, and therefore claswas coincident with the intensity of DNA replication in sification into A-or B-type enzymes. meristematic cells during embryo germination. We establishedAfter characterization of maize histone acetyltransferase an electrophoretic system under non-denaturing conditions for forms [14][15][16] we purified the cytoplasmic histone acetyltransdetection of enzyme activity within the gel matrix; in combinaferase B of germinating embryos by combining cellular fraction with second dimension SDS-PAGE the procedure allowed tionation, multiple chromatographic steps, and an electrothe unambiguous identification of histone acetyltransferase, even phoretic activity gel assay. We achieved a 6700-fold in crude enzyme preparations, purification of the enzyme, a 90 kDa protein, that is specific for histone H4 and leads to di-acetylation of purified non-

We have purified the soluble nuclear histone deacetylase HD1-A of germinating maize embryos. By a combination of 6 chromatographic steps we achieved a 77 000-fold purification of an enzymatically active protein. Gel filtration chromatography revealed a molecular weight of 45 kDa of the native enzyme and electrophoretic analysis of the purified enzyme by SDS-PAGE resulted in a single band at a molecular weight of 48 kDa, indicating that the enzyme is a monomer protein. When fractions with enzyme activity of different stages of chromatographic purification were subjected to isoelectric focusing, enzyme activity focused at a pH of around 6.4 as measured in an activity gel assay; second dimension SDS-PAGE again revealed a protein spot at a molecular weight of 48 kDa.

A maize histone deacetylase gene was identified as a homolog of yeast Hda1. The predicted protein corresponds to a previously purified maize deacetylase that is active as a protein monomer with a molecular weight of 48,000 and is expressed in all tissues of germinating embryos. Hda1 is synthesized as an enzymatically inactive protein with an apparent molecular weight of 84,000 that is processed to the active 48-kD form by proteolytic removal of the C-terminal part, presumably via a 65-kD intermediate. The enzymatically inactive 84-kD protein also is part of a 300-kD protein complex of unknown function. The proteolytic cleavage of ZmHda1 is regulated during maize embryo germination in vivo. Expression of the recombinant full-length protein and the 48-kD form confirmed that only the smaller enzyme form is active as a histone deacetylase. In line with this finding, we show that the 48-kD protein is able to repress transcription efficiently in a reporter gene assay, whereas the full-length protein, including the C-terminal part, lacks full repression activity. This report on the processing of Hda1-p84 to enzymatically active Hda1-p48 demonstrates that proteolytic cleavage is a mechanism to regulate the function of Rpd3/Hda1-type histone deacetylases.

In mammalian cells the product of the human retinoblastoma tumour suppressor gene (pRb) can recruit Rpd3-like histone deacetylases to repress transcription. In this study, we investigated whether this mechanism might also be relevant in plants and found both conserved and distinct features. The expression profiles of the Zea mays Rpd3-type histone deacetylase (ZmRpd3I) and the retinoblastoma-related (ZmRBR1) homologues were analysed during endosperm development. GST pull-down and immunoprecipitation experiments showed a physical interaction between ZmRBRI and ZmRpd3I. Because ZmRpd3I lacks a LXCXE motif, conserved in several pRb-interacting proteins, we have mapped the amino acid domains involved in the ZmRBR1/ZmRpd3I interaction. Furthermore, we observed that ZmRbAp1, a maize member of the MSI/RbAp family, facilitated this protein interaction. Co-transformations of tobacco protoplasts with plasmids expressing ZmRBRI and ZmRpd3I showed that the two proteins cooperate in repressing gene transcription. Our findings represent the first indication that in plants a regulator of important biological processes, ZmRBRI, can recruit a histone deacetylase, ZmRpd3I, to control gene transcription.