Bhat Riyaz A, Riehl Marcus, Santandrea Geraldina, Velasco Riccardo, Slocombe Stephen, Donn Günter, Steinbiss Hans-Henning, Thompson Richard D, Becker Heinz-Albert
Max-Planck-Institut für Züchtungsforschung, Carl-von-Linné Weg 10, D-50829 Köln, Germany.
Plant J. 2003 Feb;33(3):455-69. doi: 10.1046/j.1365-313x.2003.01642.x.
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.
组蛋白乙酰转移酶（HAT）GCN5在转录共激活中所起的作用已在酵母和哺乳动物中得到详细分析。在此，我们展示了玉米同源物Zmgcn5的克隆及表达模式。用组蛋白和核小体底物分析了重组ZmGCN5的酶活性。以Zmgcn5为探针，对发育中的玉米籽粒进行原位杂交，结果显示转录本集中在快速分裂的细胞中。为研究ZmGCN5在特定植物基因转录中的作用，我们检测了直接的蛋白质-蛋白质相互作用。分离出一个编码GCN5-衔接子复合物中假定相互作用伙伴的cDNA克隆ZmADA2，并通过GST沉降实验证实了ZmGCN5与ZmADA2之间的相互作用。植物转录激活因子不透明-2（Opaque-2）与核提取物中的ZmADA2进行共免疫沉淀，表明含ADA2/GCN5的复合物通过该bZIP因子的结合介导转录激活。为更全面地分析组蛋白乙酰化对植物基因表达的影响，将点样在滤膜上的2500个EST与来源于用曲古抑菌素A（TSA）处理的玉米细胞系或表达ZmGCN5反义转录本的转基因系的cDNA探针进行杂交。通过RNA印迹分析证实了几个丰度有显著变化的序列。用TSA抑制组蛋白去乙酰化伴随着ZmGCN5乙酰化酶蛋白丰度的降低，但组蛋白H2A、H2B、H3和H4的mRNA水平升高。组蛋白mRNA水平的升高并未反映在组蛋白蛋白浓度的增加上，这表明TSA处理产生的高度乙酰化组蛋白可能被优先降解，并被重新合成的组蛋白所取代。ZmGCN5反义材料显示内源性ZmGCN5转录本受到抑制，谱分析显示H2A、H2B和H4的mRNA水平升高。此外，在反义系中，观察到RPD3型HD1B-I组蛋白去乙酰化酶蛋白的量减少。本文讨论了组蛋白乙酰化与组蛋白mRNA转录的关联调控模型。
