Although researchers have established that DNA methylation and active demethylation are dynamically regulated in plant cells, the molecular mechanism for the regulation of active DNA demethylation is not well understood. By using an Arabidopsis (
Arabidopsis thaliana) line expressing the
Promoter RESPONSIVE TO DEHYDRATION 29A:
LUCIFERASE (
ProRD29A:
LUC) and
Promoter cauliflower mosaic virus 35S:
NEOMYCIN PHOSPHOTRANSFERASE II (
Pro35S:
NPTII) transgenes, we isolated an
mbd7 (for
methyl-CpG-binding domain protein7) mutant. The
mbd7 mutation causes an inactivation of the
Pro35S:
NPTII transgene but does not affect the expression of the
ProRD29A:
LUC transgene. The silencing of the
Pro35S:
NPTII reporter gene is associated with DNA hypermethylation of the reporter gene. MBD7 interacts physically with REPRESSOR OF SILENCING5/INCREASED DNA METHYLATION2, a protein in the small heat shock protein family. MBD7 prefers to target the genomic loci with high densities of DNA methylation around chromocenters. The Gypsy-type long terminal repeat retrotransposons mainly distributed around chromocenters are most affected by
mbd7 in all transposons. Our results suggest that MBD7 is required for active DNA demethylation and antisilencing of the genomic loci with high densities of DNA methylation in Arabidopsis.DNA methylation is an important epigenetic marker for genome stability and the regulation of gene expression in both plants and animals (
Law and Jacobsen, 2010;
He et al., 2011). In plants, the molecular mechanisms for DNA methylation have been well characterized by the use of powerful genetic screening systems (
Bartee et al., 2001;
Lindroth et al., 2001;
Matzke et al., 2004;
He et al., 2009). A transgene or an endogenous gene may be silenced because of DNA hypermethylation in the promoter region. Screenings for mutants with release of the silenced marker genes have identified many components that are involved in RNA-directed DNA methylation (
RdDM) and in maintaining DNA methylation (
Matzke and Birchler, 2005;
Law and Jacobsen, 2009;
He et al., 2011;
Bender, 2012). DNA methylation is catalyzed by DNA methyltransferases including DNA METHYLTRANSFERASE1 (MET1) and CHROMOMETHYLASE3 (CMT3), which maintain symmetric CG and CHG methylation, respectively, during DNA replication, and DOMAINS REARRANGED METHYLASE2 (DRM2) and CMT2, which are required for establishing CHG and asymmetric CHH methylation during each cell cycle. DRM2 also catalyzes CG methylation (
Law and Jacobsen, 2010;
Haag and Pikaard, 2011;
He et al., 2011;
Zemach et al., 2013;
Stroud et al., 2014). Twenty-four-nucleotide small RNAs produced through the
RdDM pathway target genomic regions to guide the establishment of DNA methylation by DRM2 (
Cao et al., 2003).DNA methylation can be actively removed by a subfamily of bifunctional DNA glycosylases/lyases including REPRESSOR OF SILENCING1 (ROS1;
Gong et al., 2002) and its paralogs DEMETER and DEMETER-LIKE2/3 (
Gehring et al., 2006;
Ortega-Galisteo et al., 2008). DNA methylation can also be passively lost during DNA replication when DNA methylation cannot be maintained (
Zhu, 2009).
Promoter RESPONSIVE TO DEHYDRATION 29A:
LUCIFERASE (
ProRD29A:
LUC) in the
ProRD29A:
LUC/Promoter cauliflower mosaic virus 35S:
NEOMYCIN PHOSPHOTRANSFERASE II (
Pro35S:
NPTII) transgenic Arabidopsis (
Arabidopsis thaliana) line has been used as a marker to identify
ros1 and
ros3 mutants in which both
ProRD29A:
LUC and
Pro35S:
NPTII are silenced (
Gong et al., 2002;
Zheng et al., 2008). ROS3 is an RNA-binding protein that facilitates the function of ROS1 in active DNA demethylation at certain genomic loci. Using
Pro35S:
NPTII as a selection marker for kanamycin-sensitive mutants and the
35S-SUC2 transgene or a chop PCR marker for assaying DNA methylation at the 3′ region of
At1g26400 from transfer DNA (
T-DNA) insertion mutants, researchers recently identified two genes involved in active DNA demethylation:
ROS4/INCREASED DNA METHYLATION1 (
IDM1) and
ROS5/IDM2 (
Li et al., 2012;
Qian et al., 2012,
2014;
Zhao et al., 2014). ROS4/IDM1 is a plant homeodomain-finger domain-containing histone acetyltransferase that catalyzes histone H3 lysine18 (H3K18) and lysine23 (H3K23) acetylation (
Li et al., 2012;
Qian et al., 2012). ROS5/IDM2 is a member of the small heat shock protein family that interacts physically with ROS4/IDM1 for the regulation of active DNA demethylation. Genetic analysis indicates that ROS1, ROS4/IDM1, and ROS5/IDM2 are in the same genetic pathway and that ROS4/IDM1 and ROS5/IDM2 may form a protein complex for the regulation of active DNA demethylation (
Qian et al., 2014;
Zhao et al., 2014).During the genetic screening for kanamycin-sensitive mutants using the
ProRD29A:
LUC/Pro35S:
NPTII transgenic line in this study, we identified another mutant,
mbd7, where the
Pro35S:
NPTII transgene is specifically silenced. MBD7 is a methyl-CpG-binding domain (MBD) protein containing three MBD motifs that bind in vitro to methylated symmetric CG sites. MBD7 localizes to all highly CpG-methylated chromocenters in vivo (
Zemach and Grafi, 2003;
Zemach et al., 2008). Recruitment of MBD7 to chromocenters is disrupted in
decrease in DNA methylation1 (
ddm1) and
met1, two mutants with great reductions in DNA methylation, suggesting that DNA methylation is required for proper MBD7 localization (
Zemach et al., 2005). In this study, we found that MBD7 interacts physically with ROS5/IDM2 and is required for the active DNA demethylation of certain genomic loci, especially for the Gypsy-type long terminal repeat (LTR) retrotransposons with high densities of DNA methylation around chromocenters in Arabidopsis.
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