The bZIP-type transcriptional activator opaque2 (o2) has been shown by a number of groups to be a major regulator of storage protein expression in maize endosperm. The availability of a heterologous protoplast trans-activation assay enabled us to map the region of Opaque-2 which functions as a major activation domain, i.e., is needed for Opaque-2 protein to activate expression from a target promoter. Similarities between the acidic activation domain sequence of Opaque-2, and several other plant transcription factors with that of the yeast bZIP factor GCN-4 led us to speculate that the mechanism of acidic domain-mediated activation in yeast and maize would be similar (current models for transcriptional activation invoke adaptor complexes which mediate between transcription factors binding to upstream activating sequences and the RNA-polII located at the transcription start). To get more insight into the detailed mechanism of transcriptional activation in the developing endosperm, we have begun to isolate central components of the yeast GCN5/ADA2 complex from maize. The histone acetyltransferase (HAT) clone zmGCN5 was isolated by 2-Hybrid screening of a maize cDNA library using a GAL4-ADA2 fusion protein as bait construct. The bait ADA2-sequence information was derived from heterologous plant EST information. The full length zmGCN5 encodes a 515 amino acid protein which possesses 59% similarity and 49% identity to yeast GCN5 over the C-terminal 2/3 of the sequence, including the catalytic domain, ADA2-interaction domain and a bromodomain. A single gene copy of zmGCN5 is present in the maize genome. A 2.2 kb mRNA is detected in the endosperm at all stages of maize karyopsis development analyzed so far, and elsewhere in the plant, predominantly in mRNA from actively dividing cells. The protein was expressed as a GST-fusion in E. coli, and the E coli-derived zmGCN5 was shown to have HAT activity on core histones in vitro. HAT enzymes are found as components of multiprotein complexes in yeast and other eukaryotes. In the best characterised system, yeast, at least 4 different complexes have been identified, the simplest of which contains, in addition to GCN5, ADA2 and ADA3. Although histone acetyltransferase activity is retained by isolated HAT enzymes in vitro with core histones as a substrate, the supplementary proteins are required for the acetylation of histones in nucleosomes in vitro, and putatively, in vivo too. Recently, we have isolated a putative partner of zmGCN5, zmADA2, from a maize cDNA library by heterologous probe screening. However, we have yet to find an ADA3 homologue. Approaches to unravel the role of both proteins in transcriptional activation and attempts to isolate further components of adaptor complexes are in progress.
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