DNA methylation repels targeting of Arabidopsis REF6

Qi Qiu,Jinbiao Ma,Falong Lu,Xiaofeng Cao,Baixing Wu,Qingqing Yao,Kaixuan He,Hailiang Mei,Jixiang Zhang,Xian Deng

doi:10.1038/s41467-019-10026-1

Abstract

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.

Highlights

RELATIVE OF EARLY FLOWERING 6 (REF6/JMJ12), a Jumonji C (JmjC)-domain-containing H3K27me[3] histone demethylase, finds its target loci in Arabidopsis genome by directly recognizing the CTCTGYTY motif via its zinc-finger (ZnF) domains
Because REF6-bound regions are depleted in heterochromatin regions marked by H3K9me[27], which is strongly associated with DNA methylation[17,18,19,20], and REF6-binding motifs contain non-CG sequence context (CHG and CHH), we hypothesized that DNA methylation affects REF6 binding
24,786 CTCTGYTY motif-containing regions not bound by REF6 (REF6−) were evenly distributed throughout the genome, sites of REF6 occupancy (REF6+) were mainly located on chromosome arms and were negatively correlated with hypermethylated regions of constitutive heterochromatin (Fig. 1a and Supplementary Fig. 1a), regardless of CG or non-CG sequence context (Mann–Whitney U-test, P-value < 2.2e−16) (Fig. 1b, c)

Summary

Introduction

RELATIVE OF EARLY FLOWERING 6 (REF6/JMJ12), a Jumonji C (JmjC)-domain-containing H3K27me[3] histone demethylase, finds its target loci in Arabidopsis genome by directly recognizing the CTCTGYTY motif via its zinc-finger (ZnF) domains. REF6 has intrinsic DNA-binding ability and recognizes its target sequence (CTCTGYTY motif, Y = T or C) via tandem zinc-finger (ZnF) domains located at its C-terminus[7,8]. CTCTGYTY motifs are not sufficient for REF6 recruitment, and only ~15% of such sequences in the Arabidopsis genome are bound by the enzyme[7], suggesting that an additional layer of regulation is involved in targeting REF6 in order to precisely control the level of H3K27me[3] at developmentally important loci. In short transposable elements (TEs), the 24nt-siRNA targeting DOMAINS REAR-

Methods

Results

Conclusion