Identification of a putative histone deacetylase RPD3-like gene from maize that complements a yeast rpd3-null mutant
--Rossi, V, Hartings, H, Motto, M

One important mechanism for the dynamic alteration of chromatin structure is the acetylation and deacetylation of histones, which is affected by two enzyme activities, histone acetyltransferase (HAT) and histone deacetylase (HD). Biochemical studies have revealed a correlation between the level of histone acetylation and deacetylation with transcriptional activity and repression respectively. It is thought that the acetylation of nucleosomal histones induces an open chromatin conformation, which allows the transcription machinery access to promoters. Particularly, the central role of the RPD3 yeast-homologous histone deacetylase in many transcriptional systems was described in a wide variety of fungi and animals. These data provide evidence that histone acetylation and deacetylation are fundamental regulatory mechanisms governing cell proliferation and differentiation (for reviews see Wade and Wolffe, Curr. Biol. 7:82-84, 1997; Wolffe, Nature 387:16-17, 1997). In the germinating embryo of Zea mays three HATs and four Hds have been identified (Lechner et al., Biochim. Biophys. Acta 1296:181-188, 1997); in addition, a cDNA encoding for a HD2 chromatin-bound deacetylase with no RPD3 homology was described (Lusser et al., Science 277:88-91 1997).

We have recently reported the isolation and characterisation of a maize cDNA encoding a RPD3-homologous histone deacetylase (zmRPD3), the first to be cloned and analysed from a plant. This cDNA was identified, for the ability to weakly restore the His+ phenotype in a gcn4 - bas1 yeast strain screened with a maize 10 DAP endosperm cDNA library. þ galactosidase assays were performed showing that the zmRPD3 can transactivate the yeast HIS4 promoter in a yeast expression vector. Database analysis of this 2047 bp insert revealed an open translational reading frame of 1539 bases, extending from the first ATG located at nucleotide position 141 until a TAG stop codon at position 1679. The cDNA encodes a protein of 513 amino acid residues with a predicted molecular weight of 57.6 kDa. Analysis of the deduced amino acid sequence revealed that the protein is very similar to the yeast RPD3 protein (55.4% identity; 74.3% similarity) and other related sequences identified in a variety of organisms.

Because the amino acid sequence homology of the maize RPD3-encoded protein suggests that this protein belongs to the RPD3-family, we have examined whether the maize gene is functionally homologous to the yeast gene by expressing the maize RPD3-like protein in a yeast strain carrying a mutation in the RPD3 gene. The restoration of resistance at sublethal doses of cycloheximide has been observed. Moreover, a zmRPD3 transformed rpd3 - trk1 yeast strain failed to grow in a low salt medium as expected for a RPD3 - trk1 phenotype (Vidal and Gaber, Mol. Cell. Biol. 11:6317-6327, 1991). Hence, zmRPD3 can functionally complement a null rpd3 yeast mutation for at least two different phenotypic traits.

Analyses of the expression of the zmRPD3 transcript showed that the gene probe hybridised with the mRNA extracted from leaf, coleoptile, root, and endosperm maize tissues. The results of genomic Southern analyses revealed a moderately complex hybridisation pattern including both strongly and more weakly hybridising bands. This suggests the presence of more that one copy of the zmRPD3 sequence and/or other closely related sequences in the maize genome. These copies may possess less sequence homology at the N-terminus than at the C-terminal region. These data are in agreement with earlier results indicating the presence in maize of at least four different proteins having histone deacetylase activity (Lechner et al., Biochim. Biophys. Acta 1296:181-188, 1997). The 5'-terminal probe was employed in Southern blotting experiments using a segregating population from the cross B73xA7. Analysis of polymorphic DNA fragments obtained with BamHI and scoring in 107 F3 lines enabled us to localise one copy of the maize RPD3-like gene to the short arm of chromosome 5 near the centromeric region between the umc1 and bnl5.71 molecular markers, with relative genetic distances of 21.1 and 14.1 cM, respectively.

Phylogenetic analysis, using RPD3 homologous nucleotide sequences present in sequence databanks, clustered the RPD3-like mammalian sequences HDAC1 and HDAC2 (Taunton et al., Science 272:408-411, 1996; Yang et al., Proc. Natl. Acad. Sci. USA 93:12845-12850) in two evolutionarily different groups. The RPD3-like sequence of Zea mays as well as the RPD3-homologous sequences of other species, clustered in the HDAC1 group. The only exception was the RPD3 like sequence of X. laevis that cannot be included in any of these two groups. Analysis of evolutionary rate using relative rate tests suggest that the two groups likely evolved at different rates. The relative rate results reflect the distribution of RPD3-like sequences into two clusters as pointed out by Neighbour-joining cluster analysis. However, the HD sequence of D. melanogaster, included in HDAC1 group, showed a higher evolutionary rate. Molecular and phylogenetic data gathered in this work support the hypothesis that polymorphic forms of RPD3-homologous sequences with significant evolutionary differences are present in maize.

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