C2H2-zinc finger domains mediate targeting of REF6 on the genome of Arabidopsis

In Arabidopsis, H3K27me3 demethylase JMJ12 / RELATIVE OF EARLY FLOWERING 6 (REF6) has been shown to play important roles in transcriptional regulation and plant development via demethylation of H3K27me3 at hundreds of regions in the genome. However, the mechanism underlying REF6 targeting remains elusive. Here, we show that a tandem of C2H2-zinc finger (C2H2-ZF) domains on the C-terminal of JMJ12/REF6 are required for its biological function. The REF6 proteins without C2H2-ZF domains (REF6?ZF) failed to rescue the ref6 mutant phenotypes and resulted in an abnormal subcellular localization of REF6 protein. Genome-wide analysis revealed that REF6?ZF missed its targets in the genome and blocked the function of H3K27me3 demethylation on its target genes. Our findings indicate that C2H2-ZF domains are essential for REF6 biological function by mediating REF6 targeting, which provide a new clue to explore the targeting mechanism of JmjC-containing proteins in plant.

拟南芥中，H3K27me3去甲基化酶（H3K27me3 demethylase）JMJ12/早期开花6同源蛋白（RELATIVE OF EARLY FLOWERING 6，REF6）已被证实可通过对基因组数百个区域的H3K27me3进行去甲基化修饰，在转录调控与植物发育过程中发挥关键作用。然而，REF6的靶向识别机制至今仍不明确。本研究发现，JMJ12/REF6的C端串联C2H2型锌指（C2H2-ZF）结构域是其行使生物学功能所必需的核心元件。缺失C2H2-ZF结构域的REF6蛋白（REF6ΔZF）无法恢复ref6突变体的异常表型，同时会导致REF6蛋白出现异常的亚细胞定位。全基因组分析结果显示，REF6ΔZF无法精准识别其基因组靶位点，且会阻断其靶基因的H3K27me3去甲基化功能。本研究结果表明，C2H2-ZF结构域通过介导REF6的靶向识别，对其生物学功能至关重要，这一发现为探究植物中含JmjC结构域的蛋白质（JmjC-containing proteins）的靶向调控机制提供了全新的研究思路。