Preferential Up-Regulation of G2/M Phase-Specific Genes by Overexpression of the Hyperactive Form of NtmybA2 Lacking Its Negative Regulation Domain in Tobacco BY-2 Cells
Preferential Up-Regulation of G2/M Phase-Specific Genes by Overexpression of the Hyperactive Form of NtmybA2 Lacking Its Negative Regulation Domain in Tobacco BY-2 Cells
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DOI：
10.1104/pp.109.135582
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发表时间：
2009-04-01
期刊：
影响因子：
7.4
通讯作者：
Ito, Masaki
中科院分区：
文献类型：
--
作者：
Kato, Kiichi;Galis, Ivan;Ito, Masaki
植物中许多G2/M期特异性基因都含有有丝分裂特异性激活因子（MSA）元件，MSA元件作为G2/M期特异性增强子与R1 R2 R3-Myb转录因子结合。在这里，我们研究了烟草（Nicotiana tabacum）中R1 R2 R3-Myb转录因子之一NtmybA 2过表达的全基因组效应。我们使用定制的16-K cDNA微阵列进行过表达NtmybA 2或其截短的过度活跃形式的转基因烟草BY-2细胞系的比较转录组分析。该微阵列还用于确定BY-2细胞同步培养物中细胞周期期间的转录谱。从转基因株系和同步化细胞的组合微阵列数据显示，NtmybA 2的截短的高活性形式的过表达，而不是其全长形式，优先上调BY-2细胞中的许多G2/M期特异性基因。我们确定了几个这样的上调基因的启动子序列，并表明所有包含MSA样图案在其启动子的近端区域。其中一个上调基因NtE 2C编码细胞周期蛋白特异性泛素载体蛋白，含有一个单一的功能性MSA样基序，它特异性地控制BY-2细胞G2/M期报告基因的表达。此外，基因组足迹实验表明，在NtE 2C启动子中的MSA元件与核蛋白在体内相互作用。因此，我们认为烟草中许多G2/M期特异性基因的转录受NtmybA 2的正调控，在大多数情况下是通过与MSA元件直接结合。
Many G2/M phase-specific genes in plants contain mitosis-specific activator (MSA) elements, which act as G2/M phase-specific enhancers and bind with R1R2R3-Myb transcription factors. Here, we examined the genome-wide effects of NtmybA2 overexpression, one of the R1R2R3-Myb transcription factors in tobacco (Nicotiana tabacum). We used a custom-made 16-K cDNA microarray for comparative transcriptome analysis of transgenic tobacco BY-2 cell lines that overexpress NtmybA2 or its truncated hyperactive form. The microarray was also used to determine the transcript profile during the cell cycle in synchronized cultures of BY-2 cells. Combined microarray data from transgenic lines and synchronized cells revealed that overexpression of the truncated hyperactive form of NtmybA2, but not its full-length form, preferentially up-regulated many G2/M phase-specific genes in BY-2 cells. We determined promoter sequences of several such up-regulated genes and showed that all contain MSA-like motifs in the proximal regions of their promoters. One of the up-regulated genes, NtE2C, encoding for cyclin-specific ubiquitin carrier proteins, contained a single functional MSA-like motif, which specifically controlled the expression of a reporter gene in the G2/M phase in BY-2 cells. Furthermore, a genomic footprint experiment showed that the MSA element in the NtE2C promoter interacted with nuclear proteins in vivo. Therefore, we propose that the transcription of many G2/M phase-specific genes in tobacco is positively regulated by NtmybA2, in most cases through direct binding to the MSA elements.