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.     

AP endonucleases process 5-methylcytosine excision intermediates during active DNA demethylation in Arabidopsis

DNA methylation is a primary epigenetic modification regulating gene expression and chromatin structure in many eukaryotes. Plants have a unique DNA demethylation system in that 5-methylcytosine (5mC) is directly removed by DNA demethylases, such as DME/ROS1 family proteins, but little is known about the downstream events. During 5mC excision, DME produces 3′-phosphor-α, β-unsaturated aldehyde and 3′-phosphate by successive β- and δ-eliminations, respectively. The kinetic studies revealed that these 3′-blocking lesions persist for a significant amount of time and at least two different enzyme activities are required to immediately process them. We demonstrate that Arabidopsis AP endonucleases APE1L, APE2 and ARP have distinct functions to process such harmful lesions to allow nucleotide extension. DME expression is toxic to E. coli due to excessive 5mC excision, but expression of APE1L or ARP significantly reduces DME-induced cytotoxicity. Finally, we propose a model of base excision repair and DNA demethylation pathway unique to plants.     

Valproate induces replication-independent active DNA demethylation

In this report, we demonstrate that valproic acid (VPA), a drug that has been used for decades in the treatment of epilepsy and as a mood stabilizer, triggers replication-independent active demethylation of DNA. Thus, this drug can potentially reverse DNA methylation patterns and erase stable methylation imprints on DNA in non-dividing cells. Recent discoveries support a role for VPA in the regulation of methylated genes; however, the mechanism has been unclear because it is difficult to dissociate active demethylation from the absence of DNA methylation during DNA synthesis. We therefore took advantage of an assay that measures active DNA demethylation independently from other DNA methylation and DNA replication activities in human embryonal kidney 293 cells. We show that VPA induces histone acetylation, DNA demethylation, and expression of an ectopically methylated CMV-GFP plasmid in a dose-dependent manner. In contrast, valpromide, an analogue of VPA that does not induce histone acetylation, does not induce demethylation or expression of CMV-GFP. Furthermore, we illustrate that methylated DNA-binding protein 2/DNA demethylase (MBD2/dMTase) participates in this reaction since antisense knockdown of MBD2/dMTase attenuates VPA-induced demethylation. Taken together, our data support a new mechanism of action for VPA as enhancing intracellular demethylase activity through its effects on histone acetylation and raises the possibility that DNA methylation is reversible independent of DNA replication by commonly prescribed drugs.     

Involvement of MEM1 in DNA demethylation in Arabidopsis

Plant Molecular Biology - MEM1 participates in ROS1-mediated DNA demethylation pathway, and acts functionally as ROS3 to counteract the effects of RdDM pathway. mem1 mutation leads to large numbers...     

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.     

The colorful history of active DNA demethylation

Patterns of DNA cytosine methylation are subject to mitotic inheritance in both plants and vertebrates. Plants use 5-methylcytosine glycosylases and the base excision repair pathway to remove excess cytosine methylation. In mammals, active demethylation has been proposed to operate via several very different mechanisms. Two recent reports in Nature now claim that the demethylation process is initiated by the same enzymes that establish the methylation mark, the DNA methyltransferases DNMT3A and DNMT3B (Kangaspeska et al., 2008; Métivier et al., 2008).     

Gemcitabine functions epigenetically by inhibiting repair mediated DNA demethylation

Gemcitabine is a cytotoxic cytidine analog, which is widely used in anti-cancer therapy. One mechanism by which gemcitabine acts is by inhibiting nucleotide excision repair (NER). Recently NER was implicated in Gadd45 mediated DNA demethylation and epigenetic gene activation. Here we analyzed the effect of gemcitabine on DNA demethylation. We find that gemcitabine inhibits specifically Gadd45a mediated reporter gene activation and DNA demethylation, similar to the topoisomerase I inhibitor camptothecin, which also inhibits NER. In contrast, base excision repair inhibitors had no effect on DNA demethylation. In Xenopus oocytes, gemcitabine inhibits DNA repair synthesis accompanying demethylation of oct4. In mammalian cells, gemcitabine induces DNA hypermethylation and silencing of MLH1. The results indicate that gemcitabine induces epigenetic gene silencing by inhibiting repair mediated DNA demethylation. Thus, gemcitabine can function epigenetically and provides a tool to manipulate DNA methylation.     

The mechanism and function of active DNA demethylation in plants

DNA methylation is a conserved and important epigenetic mark in both mammals and plants.DNA methylation can be dynamically established,maintained,and removed through different pathways.In plants,active DNA demethylation is initiated by the RELEASE OF SILENCING 1(ROS1)family of bifunctional DNA glycosylases/lyases.Accumulating evidence suggests that DNA demethylation is important in many processes in plants.In this review,we summarize recent studies on the enzymes and regulatory factors that have been identified in the DNA demethylation pathway.We also review the functions of active DNA demethylation in plant development as well as biotic and abiotic stress responses.Finally,we highlight those aspects of DNA demethylation that require additional research.     

The involvement of 5-hydroxymethylcytosine in active DNA demethylation in mice

Active DNA demethylation occurs after a sperm enters an egg. However, the mechanisms for the active DNA demethylation remain poorly understood. Ten-eleven translocation enzymes were recently shown to catalyze the conversion of 5-methylcytosine to 5-hydroxymethylcytosine (5hmC). Thus, we decided to investigate the role of 5hmC in active demethylation. We analyzed the methylation and hydroxymethylation status in metaphase II oocytes as well as 1-cell stage and cleavage stage embryos. In zygotes, 5hmC was mainly detected in the paternal pronucleus and it increased from the pronuclear-2 (PN2) to PN5 stages, an indication that 5hmC was involved in paternal genomic DNA demethylation. Bisulfite-sequencing PCR and qGluMS-PCR (DNA glucosylation and digestion before quantitative PCR) results showed that a large reduction of methylcytosine and hydroxymethylcytosine in LINE1 (long interspersed nuclear element 1) occurred between the 4- and 8-cell stages, which indicates that demethylation potentially occurred after the 4-cell stage. We then microinjected mouse zygote with plasmids that were methylated in vitro by SssI methylase and analyzed for the hydroxymethylation status of the plasmids promoter region. We found that the rapid onset of expression of the unmethylated plasmids in mouse embryos happened in <12 h, but the expression of methylated plasmids was delayed until 50 h when most embryos were at the 8-cell stage. Quantitative GluMS-PCR results suggested that 5hmC was present in the plasmid's promoter region at the MspI site where the active demethylation occurred. Our results demonstrate that 5hmC is involved in active demethylation in mice.     

A conserved 3'----5' exonuclease active site in prokaryotic and eukaryotic DNA polymerases

The 3'----5' exonuclease active site of E. coli DNA polymerase I is predicted to be conserved for both prokaryotic and eukaryotic DNA polymerases based on amino acid sequence homology. Three amino acid regions containing the critical residues in the E. coli DNA polymerase I involved in metal binding, single-stranded DNA binding, and catalysis of the exonuclease reaction are located in the amino-terminal half and in the same linear arrangement in several prokaryotic and eukaryotic DNA polymerases. Site-directed mutagenesis at the predicted exonuclease active site of the phi 29 DNA polymerase, a model enzyme for prokaryotic and eukaryotic alpha-like DNA polymerases, specifically inactivated the 3'----5' exonuclease activity of the enzyme. These results reflect a high evolutionary conservation of this catalytic domain. Based on structural and functional data, a modular organization of enzymatic activities in prokaryotic and eukaryotic DNA polymerases is also proposed.     

DNA excision repair proteins and Gadd45 as molecular players for active DNA demethylation

DNA cytosine methylation represents an intrinsic modification signal of the genome that plays important roles in heritable gene silencing, heterochromatin formation and certain transgenerational epigenetic inheritance. In contrast to the process of DNA methylation that is catalyzed by specific classes of methyltransferases, molecular players underlying active DNA demethylation have long been elusive. Emerging biochemical and functional evidence suggests that active DNA demethylation in vertebrates can be mediated through DNA excision repair enzymes, similar to the well-known repair-based DNA demethylation mechanism in Arabidopsis. As key regulators, non-enzymatic Gadd45 proteins function to recruit enzymatic machineries and promote coupling of deamination, base and nucleotide-excision repair in the process of DNA demethylation. In this article, we review recent findings and discuss functional and evolutionary implications of such mechanisms underlying active DNA demethylation.     

Thymine DNA glycosylase is essential for active DNA demethylation by linked deamination-base excision repair

DNA methylation is a major epigenetic mechanism for gene silencing. Whereas methyltransferases mediate cytosine methylation, it is less clear how unmethylated regions in mammalian genomes are protected from de novo methylation and whether an active demethylating activity is involved. Here, we show that either knockout or catalytic inactivation of the DNA repair enzyme thymine DNA glycosylase (TDG) leads to embryonic lethality in mice. TDG is necessary for recruiting p300 to retinoic acid (RA)-regulated promoters, protection of CpG islands from hypermethylation, and active demethylation of tissue-specific developmentally and hormonally regulated promoters and enhancers. TDG interacts with the deaminase AID and the damage response protein GADD45a. These findings highlight a dual role for TDG in promoting proper epigenetic states during development and suggest a two-step mechanism for DNA demethylation in mammals, whereby 5-methylcytosine and 5-hydroxymethylcytosine are first deaminated by AID to thymine and 5-hydroxymethyluracil, respectively, followed by TDG-mediated thymine and 5-hydroxymethyluracil excision repair.     

Genome-wide DNA demethylation in mammals

The cytidine deaminase AID and elongator-complex proteins contribute to the extensive removal of DNA methylation in mammalian primordial germ cells and in the paternal pronucleus of the zygote.     

HMG domain containing SSRP1 is required for DNA demethylation and genomic imprinting in Arabidopsis

In Arabidopsis, DEMETER (DME) DNA demethylase contributes to reprogramming of the epigenetic state of the genome in the central cell. However, other aspects of the active DNA demethylation processes remain elusive. Here we show that Arabidopsis SSRP1, known as an HMG domain-containing component of FACT histone chaperone, is required for DNA demethylation and for activation and repression of many parentally imprinted genes in the central cell. Although loss of DNA methylation releases silencing of the imprinted FWA-GFP, double ssrp1-3;met1-3 mutants surprisingly showed limited activation of maternal FWA-GFP in the central cell, and only became fully active after several nuclear divisions in the endosperm. This behavior was in contrast to the dme-1;met1 double mutant in which hypomethylation of FWA-GFP by met1 suppressed the DNA demethylation defect of dme-1. We propose that active DNA demethylation by DME requires SSRP1 function through a distinctly different process from direct DNA methylation control.