MET18 Connects the Cytosolic Iron-Sulfur Cluster Assembly Pathway to Active DNA Demethylation in Arabidopsis.

Cheng-Guo Duan,Chuan-Chih Hsu,Kai Tang,Huiming Zhang,Chunguo Wang,Yueh-Ju Hou,Jian-Kang Zhu,Mingguang Lei,W Andy Tao,Satendra K Mangrauthia,Xingang Wang,Yan Li,Tetsuji Kakutani

doi:10.1371/journal.pgen.1005559

Abstract

DNA demethylation mediated by the DNA glycosylase ROS1 helps determine genomic DNA methylation patterns and protects active genes from being silenced. However, little is known about the mechanism of regulation of ROS1 enzymatic activity. Using a forward genetic screen, we identified an anti-silencing (ASI) factor, ASI3, the dysfunction of which causes transgene promoter hyper-methylation and silencing. Map-based cloning identified ASI3 as MET18, a component of the cytosolic iron-sulfur cluster assembly (CIA) pathway. Mutation in MET18 leads to hyper-methylation at thousands of genomic loci, the majority of which overlap with hypermethylated loci identified in ros1 and ros1dml2dml3 mutants. Affinity purification followed by mass spectrometry indicated that ROS1 physically associates with MET18 and other CIA components. Yeast two-hybrid and split luciferase assays showed that ROS1 can directly interact with MET18 and another CIA component, AE7. Site-directed mutagenesis of ROS1 indicated that the conserved iron-sulfur motif is indispensable for ROS1 enzymatic activity. Our results suggest that ROS1-mediated active DNA demethylation requires MET18-dependent transfer of the iron-sulfur cluster, highlighting an important role of the CIA pathway in epigenetic regulation.

Highlights

DNA 5-cytosine methylation (5mC) is an epigenetic mark that is critical for maintaining genome integrity, regulating gene expression and responding to environmental stress in many higher eukaryotes [1,2,3,4,5,6]
In the model plant species Arabidopsis thaliana, active DNA demethylation depends on a family of 5-methylcytosine DNA glycosylases/demethylases including ROS1, DME, and others
Our results suggested that MET18 affects DNA demethylation by influencing ROS1 enzymatic activity via direct interaction with the iron-sulfur-binding motif of ROS1, highlighting a direct connection between iron-sulfur cluster assembly and active DNA demethylation

Summary

Introduction

DNA 5-cytosine methylation (5mC) is an epigenetic mark that is critical for maintaining genome integrity, regulating gene expression and responding to environmental stress in many higher eukaryotes [1,2,3,4,5,6]. DNA demethylation by DME in the central cell contributes to parental specific expression of some imprinted genes in the endosperm, whereas ROS1 and other family members mainly affect the DNA methylation status in vegetative tissues [2]. ROS3, which is an RNA-binding protein, has been suggested to mediate ROS1 recruitment at some loci because its mutation disrupts the subnuclear localization pattern of ROS1 and increases DNA methylation [19]. It is unclear, how the enzymatic activity of ROS1 may be regulated

Methods

Results

Discussion

Conclusion