State Key Laboratory of Genetic Engineering, Collaborative Innovation Center for Genetics and Development, International Associated Laboratory of CNRS-Fudan-HUNAU on Plant Epigenome Research, Department of Biochemistry, Institute of Plant Biology, School of Life Sciences, Fudan University, Shanghai 200438, China.
State Key Laboratory of Genetic Engineering and Ministry of Education Key Laboratory of Contemporary Anthropology, Collaborative Innovation Center for Genetics and Development, School of Life Sciences; Institutes of Biomedical Sciences of Shanghai Medical College, Fudan University, Shanghai, China.
Nucleic Acids Res. 2020 Jan 10;48(1):460-471. doi: 10.1093/nar/gkz1081.
As the largest group of MYB family transcription factors, R2R3-MYB proteins play essential roles during plant growth and development. However, the structural basis underlying how R2R3-MYBs recognize the target DNA remains elusive. Here, we report the crystal structure of Arabidopsis WEREWOLF (WER), an R2R3-MYB protein, in complex with its target DNA. Structural analysis showed that the third α-helices in both the R2 and R3 repeats of WER fit in the major groove of the DNA, specifically recognizing the DNA motif 5'-AACNGC-3'. In combination with mutagenesis, in vitro binding and in vivo luciferase assays, we showed that K55, N106, K109 and N110 are critical for the function of WER. Although L59 of WER is not involved in DNA binding in the structure, ITC analysis suggested that L59 plays an important role in sensing DNA methylation at the fifth position of cytosine (5mC). Like 5mC, methylation at the sixth position of adenine (6mA) in the AAC element also inhibits the interaction between WER and its target DNA. Our study not only unravels the molecular basis of how WER recognizes its target DNA, but also suggests that 5mC and 6mA modifications may block the interaction between R2R3-MYB transcription factors and their target genes.
作为 MYB 家族转录因子中最大的一组，R2R3-MYB 蛋白在植物生长和发育过程中发挥着重要作用。然而，R2R3-MYB 识别靶 DNA 的结构基础仍然难以捉摸。在这里，我们报告了拟南芥 WEREWOLF（WER）的晶体结构，这是一种 R2R3-MYB 蛋白，与它的靶 DNA 复合物。结构分析表明，WER 的 R2 和 R3 重复中的第三个α-螺旋适合于 DNA 的大沟，特异性识别 DNA 基序 5'-AACNGC-3'。结合突变、体外结合和体内荧光素酶测定，我们表明 K55、N106、K109 和 N110 对于 WER 的功能至关重要。尽管 WER 的 L59 在结构中不参与 DNA 结合，但 ITC 分析表明 L59在感知 DNA 第五位胞嘧啶（5mC）甲基化方面发挥着重要作用。与 5mC 类似，AAC 元件中腺嘌呤（6mA）的第六位甲基化也抑制了 WER 与其靶 DNA 之间的相互作用。我们的研究不仅揭示了 WER 识别其靶 DNA 的分子基础，还表明 5mC 和 6mA 修饰可能阻止 R2R3-MYB 转录因子与其靶基因之间的相互作用。
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