The cytosolic Fe-S cluster assembly component MET18 is required for the full enzymatic activity of ROS1 in active DNA demethylation.

Xiaokang Wang,Weiqiang Qian,Wei Yuan,Suresh Kumar,Qi Li,Zhendong Cao,Yan Li,Bei Qi

doi:10.1038/srep26443

Abstract

DNA methylation patterns in plants are dynamically regulated by DNA methylation and active DNA demethylation in response to both environmental changes and development of plant. Beginning with the removal of methylated cytosine by ROS1/DME family of 5-methylcytosine DNA glycosylases, active DNA demethylation in plants occurs through base excision repair. So far, many components involved in active DNA demethylation remain undiscovered. Through a forward genetic screening of Arabidopsis mutants showing DNA hypermethylation at the EPF2 promoter region, we identified the conserved iron-sulfur cluster assembly protein MET18. MET18 dysfunction caused DNA hypermethylation at more than 1000 loci as well as the silencing of reporter genes and some endogenous genes. MET18 can directly interact with ROS1 in vitro and in vivo. ROS1 activity was reduced in the met18 mutant plants and point mutation in the conserved Fe-S cluster binding motif of ROS1 disrupted its biological function. Interestingly, a large number of DNA hypomethylated loci, especially in the CHH context, were identified from the met18 mutants and most of the hypo-DMRs were from TE regions. Our results suggest that MET18 can regulate both active DNA demethylation and DNA methylation pathways in Arabidopsis.

Highlights

(DML3)[10,11]
Screening of the Arabidopsis T-DNA insertion mutants library, which includes mutants of chromatin-related genes, DNA or RNA binding protein-encoding genes and genes co-expressed with ROS1 or ROS3 et al (Supplementary Table 1), using this chop-PCR marker resulted in the identification of two mutants showing DNA hypermethylation at the EPF2 promoter region
Active DNA demethylation is initiated by the ROS1/DME family of 5mC DNA glycosylases/lyases, and it is presumably continued through a base excision repair (BER) pathway[3,17]

Summary

Introduction

(DML3)[10,11] All of these enzymes are bifunctional 5-methylcytosine-DNA glycosylases/lyases that remove the 5-methylated cytosine, and cleave phosphodiester backbone at the abasic site[10,12,13,14] resulting in a gap with 3′phosphate or 3′dRP (3′α, β-unsaturated aldehyde) termini. Active DNA demethylation induced by DME in the central cell is essential for the expression of imprinting genes, including FWA and MEA11. We report MET18 as a component of the active DNA demethylation pathway in plants. Identified through a forward genetic screening, the met[18] mutant plants exhibited DNA hypermethylation at hundreds of genetic loci and conferred transcriptional silencing of reporter genes and some endogenous targets. Our results revealed an epigenetic role of MET18 in the regulation of gene expression in Arabidopsis

Methods

Results

Conclusion