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PLoS Genet. 2015 Oct 22;11(10):e1005559. doi: 10.1371/journal.pgen.1005559. eCollection 2015 Oct.

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

Author information

1
Department of Horticulture and Landscape Architecture, Purdue University, West Lafayette, Indiana, United States of America.
2
Shanghai Center for Plant Stress Biology, Shanghai Institute of Biological Sciences, Chinese Academy of Sciences, China.
3
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.
4
Department of Biochemistry, Purdue University, West Lafayette, Indiana, United States of America.
5
Department of Horticulture and Landscape Architecture, Purdue University, West Lafayette, Indiana, United States of America; College of Life Sciences Nankai University, Tianjin, China.
6
Ecological Security and Protection Key laboratory of Sichuan Province, Mianyang Normal University, Mianyang, Sichuan, China.
7
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.

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.

PMID:
26492035
PMCID:
PMC4619598
DOI:
10.1371/journal.pgen.1005559
[Indexed for MEDLINE]
Free PMC Article

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