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.     

Arabidopsis DEMETER-LIKE proteins DML2 and DML3 are required for appropriate distribution of DNA methylation marks

Cytosine DNA methylation is a stable epigenetic mark for maintenance of gene silencing across cellular divisions, but it is a reversible modification. Genetic and biochemical studies have revealed that the Arabidopsis DNA glycosylase domain-containing proteins ROS1 (REPRESSOR OF SILENCING 1) and DME (DEMETER) initiate erasure of 5-methylcytosine through a base excision repair process. The Arabidopsis genome encodes two paralogs of ROS1 and DME, referred to as DEMETER-LIKE proteins DML2 and DML3. We have found that DML2 and DML3 are 5-methylcytosine DNA glycosylases that are expressed in a wide range of plant organs. We analyzed the distribution of methylation marks at two methylated loci in wild-type and dml mutant plants. Mutations in DML2 and/or DML3 lead to hypermethylation of cytosine residues that are unmethylated or weakly methylated in wild-type plants. In contrast, sites that are heavily methylated in wild-type plants are hypomethylated in mutants. These results suggest that DML2 and DML3 are required not only for removing DNA methylation marks from improperly-methylated cytosines, but also for maintenance of high methylation levels in properly targeted sites.     

Genetic interactions between DNA demethylation and methylation in Arabidopsis

DNA demethylation in Arabidopsis (Arabidopsis thaliana) is mediated by DNA glycosylases of the DEMETER family. Three DEMETER-LIKE (DML) proteins, REPRESSOR OF SILENCING1 (ROS1), DML2, and DML3, function to protect genes from potentially deleterious methylation. In Arabidopsis, much of the DNA methylation is directed by RNA interference (RNAi) pathways and used to defend the genome from transposable elements and their remnants, repetitive sequences. Here, we investigated the relationship between DML demethylation and RNAi-mediated DNA methylation. We found that genic regions demethylated by DML enzymes are enriched for small interfering RNAs and generally contain sequence repeats, transposons, or both. The most common class of small interfering RNAs was 24 nucleotides long, suggesting a role for an RNAi pathway that depends on RNA-DEPENDENT RNA POLYMERASE2 (RDR2). We show that ROS1 removes methylation that has multiple, independent origins, including de novo methylation directed by RDR2-dependent and -independent RNAi pathways. Interestingly, in rdr2 and drm2 mutant plants, we found that genes demethylated by ROS1 accumulate CG methylation, and we propose that this hypermethylation is due to the ROS1 down-regulation that occurs in these mutant backgrounds. Our observations support the hypothesis that DNA demethylation by DML enzymes is one mechanism by which Arabidopsis genes are protected from genome defense pathways.     

Methylation-Sensitive Expression of a DNA Demethylase Gene Serves As an Epigenetic Rheostat

Genomes must balance active suppression of transposable elements (TEs) with the need to maintain gene expression. In Arabidopsis, euchromatic TEs are targeted by RNA-directed DNA methylation (RdDM). Conversely, active DNA demethylation prevents accumulation of methylation at genes proximal to these TEs. It is unknown how a cellular balance between methylation and demethylation activities is achieved. Here we show that both RdDM and DNA demethylation are highly active at a TE proximal to the major DNA demethylase gene ROS1. Unexpectedly, and in contrast to most other genomic targets, expression of ROS1 is promoted by DNA methylation and antagonized by DNA demethylation. We demonstrate that inducing methylation in the ROS1 proximal region is sufficient to restore ROS1 expression in an RdDM mutant. Additionally, methylation-sensitive expression of ROS1 is conserved in other species, suggesting it is adaptive. We propose that the ROS1 locus functions as an epigenetic rheostat, tuning the level of demethylase activity in response to methylation alterations, thus ensuring epigenomic stability.     

An AP Endonuclease Functions in Active DNA Demethylation and Gene Imprinting in Arabidopsis

Active DNA demethylation in plants occurs through base excision repair, beginning with removal of methylated cytosine by the ROS1/DME subfamily of 5-methylcytosine DNA glycosylases. Active DNA demethylation in animals requires the DNA glycosylase TDG or MBD4, which functions after oxidation or deamination of 5-methylcytosine, respectively. However, little is known about the steps following DNA glycosylase action in the active DNA demethylation pathways in plants and animals. We show here that the Arabidopsis APE1L protein has apurinic/apyrimidinic endonuclease activities and functions downstream of ROS1 and DME. APE1L and ROS1 interact in vitro and co-localize in vivo. Whole genome bisulfite sequencing of ape1l mutant plants revealed widespread alterations in DNA methylation. We show that the ape1l/zdp double mutant displays embryonic lethality. Notably, the ape1l^+/−zdp^−/− mutant shows a maternal-effect lethality phenotype. APE1L and the DNA phosphatase ZDP are required for FWA and MEA gene imprinting in the endosperm and are important for seed development. Thus, APE1L is a new component of the active DNA demethylation pathway and, together with ZDP, regulates gene imprinting in Arabidopsis.     

The DNA Repair Protein XRCC1 Functions in the Plant DNA Demethylation Pathway by Stimulating Cytosine Methylation (5-meC) Excision,Gap Tailoring,and DNA Ligation

DNA methylation patterns are the dynamic outcome of antagonist methylation and demethylation mechanisms, but the latter are still poorly understood. Active DNA demethylation in plants is mediated by a family of DNA glycosylases typified by Arabidopsis ROS1 (repressor of silencing 1). ROS1 and its homologs remove 5-methylcytosine and incise the sugar backbone at the abasic site, thus initiating a base excision repair pathway that finally inserts an unmethylated cytosine. The DNA 3′-phosphatase ZDP processes some of the incision products generated by ROS1, allowing subsequent DNA polymerization and ligation steps. In this work, we examined the possible role of plant XRCC1 (x-ray cross-complementing group protein 1) in DNA demethylation. We found that XRCC1 interacts in vitro with ROS1 and ZDP and stimulates the enzymatic activity of both proteins. Furthermore, extracts from xrcc1 mutant plants exhibit a reduced capacity to complete DNA demethylation initiated by ROS1. An anti-XRCC1 antibody inhibits removal of the blocking 3′-phosphate in the single-nucleotide gap generated during demethylation and reduces the capacity of Arabidopsis cell extracts to ligate a nicked DNA intermediate. Our results suggest that XRCC1 is a component of plant base excision repair and functions at several stages during active DNA demethylation in Arabidopsis.     

A novel protein complex that regulates active DNA demethylation in Arabidopsis

Active DNA demethylation is critical for altering DNA methylation patterns and regulating gene expression. The 5‐methylcytosine DNA glycosylase/lyase ROS1 initiates a base‐excision repair pathway for active DNA demethylation and is required for the prevention of DNA hypermethylation at 1 000s of genomic regions in Arabidopsis. How ROS1 is regulated and targeted to specific genomic regions is not well understood. Here, we report the discovery of an Arabidopsis protein complex that contains ROS1, regulates ROS1 gene expression, and likely targets the ROS1 protein to specific genomic regions. ROS1 physically interacts with a WD40 domain protein (RWD40), which in turn interacts with a methyl‐DNA binding protein (RMB1) as well as with a zinc finger and homeobox domain protein (RHD1). RMB1 binds to DNA that is methylated in any sequence context, and this binding is necessary for its function in vivo. Loss‐of‐function mutations in RWD40, RMB1, or RHD1 cause DNA hypermethylation at several tested genomic regions independently of the known ROS1 regulator IDM1. Because the hypermethylated genomic regions include the DNA methylation monitoring sequence in the ROS1 promoter, plants mutated in RWD40, RMB1, or RHD1 show increased ROS1 expression. Importantly, ROS1 binding to the ROS1 promoter requires RWD40, RMB1, and RHD1, suggesting that this complex dictates ROS1 targeting to this locus. Our results demonstrate that ROS1 forms a protein complex with RWD40, RMB1, and RHD1, and that this novel complex regulates active DNA demethylation at several endogenous loci in Arabidopsis.     

Methyl-CpG-Binding Domain Protein MBD7 Is Required for Active DNA Demethylation in Arabidopsis

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.     

A null mutation of ROS1a for DNA demethylation in rice is not transmittable to progeny

Genes that promote DNA methylation and demethylation in plants have been characterized mainly in Arabidopsis. Arabidopsis DNA demethylation is mediated by bi-functional DNA enzymes with glycosylase activity that removes 5-methylcytosine and lyase activity that nicks double-stranded DNA at an abasic site. Homologous recombination-promoted knock-in targeting of the ROS1a gene, the longest of six putative DNA demethylase genes in the rice genome, by fusing its endogenous promoter to the GUS reporter gene, led to reproducibly disrupted ROS1a in primary (T(0)) transgenic plants in the heterozygous condition. These T(0) plants exhibited no overt morphological phenotypes during the vegetative phase, and GUS staining showed ROS1a expression in pollen, unfertilized ovules and meristematic cells. Interestingly, neither the maternal nor paternal knock-in null allele, ros1a-GUS1, was virtually detected in the progeny; such an intransmittable null mutation is difficult to isolate by conventional mutagenesis techniques that are usually used to identify and isolate mutants in the progeny population. Even in the presence of the wild-type paternal ROS1a allele, the maternal ros1a-GUS1 allele caused failure of early-stage endosperm development, resulting in incomplete embryo development, with embryogenesis producing irregular but viable embryos that failed to complete seed dormancy, implying non-equivalent maternal and paternal contribution of ROS1a in endosperm development. The paternal ros1a-GUS1 allele was not transmitted to progeny, presumably because of a male gametophytic defect(s) prior to fertilization. Thus, ROS1a is indispensable in both male and female gametophytes, and DNA demethylation must plays important roles in both gametophytes.     

DNA demethylation dynamics

The discovery of cytosine hydroxymethylation (5hmC) suggested a simple means of demethylating DNA and activating genes. Further experiments, however, unearthed an unexpectedly complex process, entailing both passive and active mechanisms of DNA demethylation by the ten-eleven translocation (TET) and AID/APOBEC families of enzymes. The consensus emerging from these studies is that removal of cytosine methylation in mammalian cells can occur by DNA repair. These reports highlight that in certain contexts, DNA methylation is not fixed but dynamic, requiring continuous regulation.