RESUMO
Maize Opaque-2 (ZmO2), a bZip class transcription factor has been shown to activate the transcription of a series of genes expressed in the maturation phase of endosperm development. Activation requires the presence of one or more enhancer binding sites, which confer the propensity for activation by ZmO2 on heterologous promoters and in heterologous plant cell types, such as tobacco mesophyll protoplasts. The region of ZmO2 required for conferring transcriptional activation has been localised to a stretch of acidic residues in the N-terminal portion of the ZmO2 sequence, which is conserved between O2-related bZip factor sequences. Previously we identified the maize homologues of yeast transcriptional co-activators GCN5 and ADA2 that are implicated in nucleosome modification and transcription. In the present study we have shown that transcriptional modulation by ZmO2 involves the intranuclear interaction of ZmO2 with ZmADA2 and ZmGCN5. Förster resonance energy transfer (FRET) based techniques have enabled us to estimate the intracellular site of these intermolecular interactions. As a functional readout of these intranuclear interactions, we used the ZmO2 responsive maize b-32 promoter to drive the beta-glucuronidase (GUS) in the presence and absence of ZmGCN5 and ZmADA2. Our results suggest that the likely recruitment of ZmADA2 and ZmGCN5 modulates the transactivation of b-32 promoter by ZmO2 and that there may be a competition between ZmGCN5 and ZmO2 for binding to the amino-terminal of ZmADA2. The results may be taken as a paradigm for other processes of transcriptional modulation in planta involving acidic activation domains.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas de Plantas/metabolismo , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Acetiltransferases/genética , Acetiltransferases/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação/genética , Proteínas de Ligação a DNA/genética , Transferência Ressonante de Energia de Fluorescência/métodos , Glucuronidase/genética , Glucuronidase/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Histona Acetiltransferases , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Microscopia Confocal , Dados de Sequência Molecular , Células Vegetais , Proteínas de Plantas/genética , Plantas/genética , Plantas/metabolismo , Regiões Promotoras Genéticas/genética , Ligação Proteica , Protoplastos/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transativadores/genética , Fatores de Transcrição/genética , Transcrição Gênica/genética , Ativação Transcricional/genética , TransfecçãoRESUMO
The role played by histone acetyltransferase (HAT), GCN5, in transcriptional co-activation has been analysed in detail in yeast and mammals. Here, we present the cloning and expression pattern of Zmgcn5, the maize homologue. The enzymatic activity of the recombinant ZmGCN5 was analysed with histone and nucleosome substrates. In situ hybridisation of developing maize kernels using Zmgcn5 as probe shows that the transcript is concentrated in rapidly dividing cells. To investigate the role of ZmGCN5 in the transcription of specific plant genes, direct protein-protein interactions were tested. A cDNA clone encoding a putative interacting partner in GCN5-adapter complexes, ZmADA2, was isolated and the interaction between ZmGCN5 and ZmADA2 was confirmed by a GST-spin down experiment. Co-immunoprecipitation of the plant transcriptional activator Opaque-2 and ZmADA2 in nuclear extracts suggests ADA2/GCN5-containing complexes to mediate transcriptional activation by binding of this bZIP factor. For a more general analysis of the effects of histone acetylation on plant gene expression, 2500 ESTs spotted on filters were hybridised with cDNA probes derived either from maize cell lines treated with Trichostatin A (TSA), or from a transgenic line expressing the ZmGCN5 antisense transcript. Several sequences showing marked changes in abundance were confirmed by RNA blot analysis. Inhibition of histone deacetylation with TSA is accompanied by a decrease in the abundance of ZmGCN5 acetylase protein, but by increases in mRNAs for histones H2A, H2B, H3 and H4. The elevated histone mRNA levels were not reflected in increasing histone protein concentrations, suggesting hyperacetylated histones arising from TSA treatment may be preferentially degraded and substituted by de novo synthesised histones. The ZmGCN5 antisense material showed suppression of the endogenous ZmGCN5 transcript and the profiling analysis revealed increased mRNA levels for H2A, H2B and H4. Furthermore, in the antisense line, a reduction in the amount of the RPD3-type HD1B-I histone deacetylase protein was observed. A model for linked regulation of histone acetylation and histone mRNA transcription is discussed.