MET18 Connects the Cytosolic Iron-Sulfur Cluster Assembly Pathway to Active DNA Demethylation in Arabidopsis.
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.
Department of Horticulture and Landscape Architecture, Purdue University, West Lafayette, Indiana, United States of America.
Cheng-Guo
CG
Duan
Department of Horticulture and Landscape Architecture, Purdue University, West Lafayette, Indiana, United States of America.
Xingang
X
Wang
Department of Horticulture and Landscape Architecture, Purdue University, West Lafayette, Indiana, United States of America.
Kai
K
Tang
Shanghai Center for Plant Stress Biology, Shanghai Institute of Biological Sciences, Chinese Academy of Sciences, China.
Huiming
H
Zhang
Department of Horticulture and Landscape Architecture, Purdue University, West Lafayette, Indiana, United States of America; Biotechnology Section, Indian Institute of Rice Research (IIRR), Hyderabad, India.
Satendra K
SK
Mangrauthia
Department of Horticulture and Landscape Architecture, Purdue University, West Lafayette, Indiana, United States of America.
Mingguang
M
Lei
Department of Biochemistry, Purdue University, West Lafayette, Indiana, United States of America.
Chuan-Chih
CC
Hsu
Department of Horticulture and Landscape Architecture, Purdue University, West Lafayette, Indiana, United States of America.
Yueh-Ju
YJ
Hou
Department of Horticulture and Landscape Architecture, Purdue University, West Lafayette, Indiana, United States of America; College of Life Sciences Nankai University, Tianjin, China.
Chunguo
C
Wang
Ecological Security and Protection Key laboratory of Sichuan Province, Mianyang Normal University, Mianyang, Sichuan, China.
Yan
Y
Li
Department of Biochemistry, Purdue University, West Lafayette, Indiana, United States of America.
W Andy
WA
Tao
Department of Horticulture and Landscape Architecture, Purdue University, West Lafayette, Indiana, United States of America; Shanghai Center for Plant Stress Biology, Shanghai Institute of Biological Sciences, Chinese Academy of Sciences, China.
Jian-Kang
JK
Zhu
D017360:Arabidopsis; D029681:Arabidopsis Proteins / Q000235:genetics* / Q000378:metabolism; D002352:Carrier Proteins / Q000235:genetics*; D003600:Cytosol / Q000378:metabolism; D019175:DNA Methylation / Q000235:genetics*; D044127:Epigenesis, Genetic; D018506:Gene Expression Regulation, Plant; D007506:Iron-Sulfur Proteins / Q000235:genetics*; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009687:Nuclear Proteins / Q000235:genetics* / Q000378:metabolism; D011401:Promoter Regions, Genetic
D029681:Arabidopsis Proteins; D002352:Carrier Proteins; D007506:Iron-Sulfur Proteins; C000607990:MET18 protein, Arabidopsis; D009687:Nuclear Proteins; C471271:ROS1 protein, Arabidopsis