植物组蛋白H3K27me3去甲基酶的结构生物学研究

Epigenetic modification is a crucial regulation of gene expression, especially histone modification.The dynamic production and erasure of H3K27me3 modification is of vital importance for plant growth and development. AtREF6 and AtELF6 are two important H3K27me3/2 demethylase, and their mutants show late flowering and early flowering, respectively. Nevertheless, the recognition mechanism of how the demethylase AtREF6, AtELF6 specifically recognize and erase H3K27me3 modification is still unclear. In this research, the structure of AtREF6(catalytic domain)-H3K27me3 and AtELF6(catalytic domain)-H3K27me3 complex will be determined by X-ray crystallography, and critical contacts at the interface will be confirmed by mutagenesis, providing a structural basis for AtREF6, AtELF6 and H3K27me3 interaction. The specific recognition mechanism between AtREF6, AtELF6 and H3K27me3 will be determined by enzymatic activity assay in vitro, ITC, ChIP-seq and other methods. Whereafter the substrate recognition pattern of Arabidopsis H3K27me3 demethylase will be investigated and whether there is a potential pattern that single domain recognize multiple modification marks. The completion of this study will elucidate the substrate recognition mechanism of plant H3K27me3 demethylase and deepen our understanding about the role of histone modifications in epigenetic regulation networks.

表观修饰是调控基因表达的重要方式，其中组蛋白修饰发挥了重要的调控功能。H3K27me3修饰的产生与擦除是一个动态过程，对于植物的生长发育至关重要。AtREF6、AtELF6是拟南芥中两个重要的H3K27me3/2去甲基酶，其突变体分别表现为晚花和早花。它们如何特异识别并擦除H3K27me3修饰的具体机制尚不清楚。本研究拟利用X射线晶体学解析AtREF6、AtELF6催化结构域与H3K27me3的复合物结构，精确定位参与识别的关键氨基酸。利用体外酶活测定、ITC、ChIP-seq等方法探究拟南芥H3K27me3去甲基酶的底物识别模式并进一步探寻其催化结构域识别多个表观修饰的潜在调控模式。本研究的完成将揭示植物H3K27me3去甲基酶的底物识别机制，拓展我们对植物表观调控网络的认知。

组蛋白的N端尾巴被大量的翻译后修饰占据，是一种重要而又多样化的表观调控机制。组蛋白甲基化在调控拟南芥的染色质状态和基因表达方面起着核心作用，并参与了各种生理和发育过程。组蛋白H3K27me3是由PcG抑制复合体PRC2催化，富集在基因的转录5′区，沉默基因表达。拟南芥中REF6、ELF6、JMJ13通过擦除H3K27me3修饰参与调控植物的花期。本项目主要开展了H3K27me3去甲基酶REF6和ELF6的结构功能研究，揭示其特异识别K27me3的工作机制。（1）解析了REF6-NOG-H3K27me3 多肽的三元复合物结构，分辨率为2.6 Å。（2）结合生化实验阐释了REF6催化结构域识别H3K27me3的工作机制。（3）通过生物信息学分析和体外酶活实验，我们发现REF6与ELF6采用相同的底物识别模式，与JMJ13不同。（4）我们将H3K27me3去甲基酶KDM4家族的REF6、JMJ13以及动物KDM6家族的UTX进行结构叠加，尽管序列相似性低却共有相似的三维结构，但又因为序列差异导致了不同的底物识别模式。这些研究成果揭示了拟南芥H3K27me3去甲基酶识别组蛋白靶标的分子机制，阐述了组蛋白修饰的动态调控对植物发育的重要意义。