Rogers J C, Rogers S W
Biochemistry Department, University of Missouri, Columbia 65211.
Plant J. 1995 Feb;7(2):221-33. doi: 10.1046/j.1365-313x.1995.7020221.x.
Methylation of deoxycytosine residues in plant nuclear DNA at CpG dinucleotides is generally assumed to suppress transcription, while deoxyadenosine methylation on recombinant reporter gene constructs transiently introduced into plant cells increases expression by uncharacterized mechanisms. A particle bombardment transient expression system was used in intact barley aleurone layers to quantitate the effects of CpG and A methylation on transcription from well-characterized hormone-regulated alpha-amylase promoters. Methylation of C in all CpG pairs had little effect on transcription. In contrast, the presence of methylated A residues in the sequence GATC scattered in the reporter plasmid outside of the promoter increased transcription two- to fivefold, regardless of the strength of the promoter, and proper hormonal regulation of transcription was maintained. The methyl-dA effect was similar when the amount of reporter construct DNA used was varied over a 20-fold range, beginning with an amount that gave only a small increment of expression above background. Similar enhancing effects for methyl-dA were seen with the CaMV 35S, maize Adh1, and maize ubiquitin promoters, though the magnitude varied for each individual promoter. Methyl-dA did not detectably affect plasmid DNA stability in aleurone cells in transient expression experiments because the enhancing effect of methyl-dA on expression was the same regardless of whether the assay was performed at 12 h or 40 h. Several proteins in wheat germ nuclear extracts bound preferentially to methylated DNA as assessed by gel retardation assays; one showed preferential binding to methyl-dA rather than methyl-CpG residues. The data indicate that the presence of methyl-dA in the vicinity of active promoters enhances transcription of nuclear genes in barley in a manner independent of the strength of the promoter. This effect may be mediated by a methyl-dA-binding protein.
一般认为，植物核DNA中CpG二核苷酸处的脱氧胞嘧啶残基甲基化会抑制转录，而瞬时导入植物细胞的重组报告基因构建体上的脱氧腺苷甲基化则通过未知机制增加表达。利用粒子轰击瞬时表达系统，在完整的大麦糊粉层中定量研究CpG和A甲基化对特征明确的激素调节α-淀粉酶启动子转录的影响。所有CpG对中的C甲基化对转录影响很小。相反，报告质粒中启动子以外分散的GATC序列中存在甲基化的A残基时，无论启动子强度如何，转录都会增加2至5倍，并且转录的正常激素调节得以维持。当所用报告构建体DNA的量在20倍范围内变化时，甲基化dA的效应相似，起始量仅比背景表达量有少量增加。对于CaMV 35S、玉米Adh1和玉米泛素启动子，甲基化dA也有类似的增强作用，尽管每个启动子的增强幅度不同。在瞬时表达实验中，甲基化dA对糊粉层细胞中质粒DNA稳定性没有明显影响，因为无论在12小时还是40小时进行检测，甲基化dA对表达的增强作用都是相同的。通过凝胶阻滞试验评估，小麦胚芽核提取物中的几种蛋白质优先结合甲基化DNA；其中一种显示出优先结合甲基化dA而非甲基化CpG残基。数据表明，活性启动子附近存在甲基化dA以一种与启动子强度无关的方式增强大麦核基因的转录。这种效应可能由一种甲基化dA结合蛋白介导。
