State Key Laboratory of Plant Genomics and National Center for Plant Gene Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, 100101, China.
University of Chinese Academy of Sciences, Beijing, 100049, China.
Nat Commun. 2019 May 2;10(1):2063. doi: 10.1038/s41467-019-10026-1.
RELATIVE OF EARLY FLOWERING 6 (REF6/JMJ12), a Jumonji C (JmjC)-domain-containing H3K27me3 histone demethylase, finds its target loci in Arabidopsis genome by directly recognizing the CTCTGYTY motif via its zinc-finger (ZnF) domains. REF6 tends to bind motifs located in active chromatin states that are depleted for heterochromatic modifications. However, the underlying mechanism remains unknown. Here, we show that REF6 preferentially bind to hypo-methylated CTCTGYTY motifs in vivo, and that CHG methylation decreases REF6 DNA binding affinity in vitro. In addition, crystal structures of ZnF-clusters in complex with DNA oligonucleotides reveal that 5-methylcytosine is unfavorable for REF6 binding. In drm1 drm2 cmt2 cmt3 (ddcc) quadruple mutants, in which non-CG methylation is significantly reduced, REF6 can ectopically bind a small number of new target loci, most of which are located in or neighbored with short TEs in euchromatic regions. Collectively, our findings reveal that DNA methylation, likely acting in combination with other epigenetic modifications, may partially explain why REF6 binding is depleted in heterochromatic loci.
早开花 6 号（REF6/JMJ12）是一种含有 JmjC 结构域的 H3K27me3 组蛋白去甲基酶，它通过锌指（ZnF）结构域直接识别 CTCTGYTY 基序，从而找到其在拟南芥基因组中的靶标位点。REF6 倾向于与富含组蛋白 H3 第 27 位赖氨酸三甲基化（H3K27me3）的异染色质修饰缺失的活性染色质状态的基序结合。然而，其潜在的机制仍然未知。在这里，我们表明 REF6 在体内优先与低甲基化的 CTCTGYTY 基序结合，并且 CHG 甲基化降低了 REF6 在体外的 DNA 结合亲和力。此外，锌指簇与 DNA 寡核苷酸复合物的晶体结构表明，5-甲基胞嘧啶不利于 REF6 的结合。在非 CG 甲基化显著降低的 drm1 drm2 cmt2 cmt3（ddcc）四重突变体中，REF6 可以异位结合少数新的靶标位点，这些靶标位点大多位于常染色质区域内或附近的短转座元件中。总的来说，我们的研究结果表明，DNA 甲基化可能与其他表观遗传修饰一起，部分解释了为什么 REF6 结合在异染色质位点上会被耗尽。
