Leibniz Institute of Plant Genetics and Crop Plant Research, Corrensstraße 3, 06466 Gatersleben, Germany.
Plant J. 2011 Nov;68(4):646-56. doi: 10.1111/j.1365-313X.2011.04715.x. Epub 2011 Aug 30.
To elucidate the epigenetic maintenance mechanism for functional plant centromeres, we studied transcriptional regulation of the centromere-specific histone H3 variant CENH3 in Arabidopsis thaliana. We focused on the structure and activity of the CENH3 promoter (CENH3pro) and its regulation by E2F transcription factors. Use of CENH3pro::GUS reporter gene constructs showed that CENH3pro is active in dividing tissues, and that full expression in root meristems depends on intragenic regulatory elements within the second intron. Chromatin immunoprecipitation identified CENH3 as an E2F target gene. Transient co-expression of a CENH3pro::GUS reporter gene construct with various E2F transcription factors in A. thaliana protoplasts showed that E2Fa and E2Fb (preferentially with dimerization protein DPb) activate CENH3pro. Stable over-expression of E2Fa and E2Fb increased the CENH3 transcript level in planta, whereas over-expression of E2Fc decreased the CENH3 transcript level. Surprisingly, mutation of the two E2F binding sites of CENH3pro, in particular the more upstream one (E2F2), caused an increase in CENH3pro activity, indicating E2F-dependent transcriptional repression. CENH3pro repression may be triggered by the interplay of typical and atypical E2Fs in a cell cycle-dependent manner, and/or by interaction of typical E2Fs with retinoblastoma-related (RBR) protein. We speculate that E2Fs are involved in differential transcriptional regulation of CENH3 versus H3, as H3 promoters lack E2F binding motifs. E2F binding motifs are also present in human and Drosophila CENH3pro regions, thus cell cycle-dependent transcriptional regulation of CENH3 may be highly conserved.
为了阐明功能植物着丝粒的表观遗传维持机制，我们研究了拟南芥中着丝粒特异性组蛋白 H3 变体 CENH3 的转录调控。我们专注于 CENH3 启动子（CENH3pro）的结构和活性及其由 E2F 转录因子的调节。使用 CENH3pro::GUS 报告基因构建体表明，CENH3pro 在分裂组织中具有活性，并且在根分生组织中的完全表达依赖于第二内含子内的基因内调节元件。染色质免疫沉淀鉴定 CENH3 为 E2F 靶基因。在拟南芥原生质体中瞬时共表达 CENH3pro::GUS 报告基因构建体与各种 E2F 转录因子表明，E2Fa 和 E2Fb（优先与二聚化蛋白 DPb）激活 CENH3pro。E2Fa 和 E2Fb 的稳定过表达增加了 CENH3 在植物中的转录本水平，而 E2Fc 的过表达降低了 CENH3 转录本水平。令人惊讶的是，CENH3pro 中两个 E2F 结合位点（特别是更上游的一个 E2F2）的突变导致 CENH3pro 活性增加，表明 E2F 依赖的转录抑制。CENH3pro 的抑制可能是通过典型和非典型 E2F 在细胞周期依赖性方式中的相互作用，和/或通过典型 E2F 与视网膜母细胞瘤相关（RBR）蛋白的相互作用触发的。我们推测 E2F 参与 CENH3 与 H3 之间的差异转录调节，因为 H3 启动子缺乏 E2F 结合基序。E2F 结合基序也存在于人类和果蝇 CENH3pro 区域中，因此 CENH3 的细胞周期依赖性转录调节可能高度保守。
