[Epigenetics 3:3, 122-124; May/June 2008]; ©2008 Landes Bioscience
Point of View
Negative regulation of DNA methylation in plants Hidetoshi Saze,* Taku Sasaki and Tetsuji Kakutani Department of Integrated Genetics; National Institute of Genetics; Mishima, Shizuoka Japan
Key words: epigenetics, DNA methylation, DNA demethylation, histone demethylation, arabidopsis
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CHROMOMETHYLASE3 (CMT3).1,2,7 A chromatin remodeling factor, Decrease in DNA Methylation1 (DDM1), is required to maintain both CG and non-CG methylation at repetitive sequences.8 In addition, chromatin and RNAi components are involved in de novo DNA methylation at cytosines in all contexts, a phenomenon known as RNA-directed DNA methylation (RdDM).9 In plants, DNA methylation is antagonized by the DNA demethylation pathway, mediated by DNA glycosylase/lyases. Four proteins for DNA demethylation have been characterized in Arabidopsis; DEMETER (DME), REPRESSOR OF SILENCING1 (ROS1), DEMETER-LIKE2 (DML2) and DEMETER-LIKE3 (DML3).10-12 These proteins can excise a 5-methylcytosine base and introduce a nick to the DNA backbone, where the DNA repair pathway can eventually add an unmethylated cytosine nucleotide.13,14 DME is required to establish genomic imprinting of MEDEA (MEA), FWA and FIS2, which is important for endosperm development and seed viability.15 DME removes DNA methylation on the maternal alleles of MEA and FWA, resulting in maternal-specific expression of these genes in central cells.15 ROS1 was first identified as a factor maintaining the low methylation state of a transgene and a homologous endogenous sequence.11 In contrast to dme, ros1 single or ros1/dml2/dml3 triple mutants do not show immediate developmental defects.10,11 Genomewide analysis of DNA methylation in ros1/dml2/dml3 by tiling array technology identified ~180 loci targeted by the DNA demethylases, and showed that these enzymes primarily undergo demethylation at the 5' and 3' ends of the genes.10 Surprisingly, the target loci of the demethylases commonly overlap with transposon and repeat sequences.16 These regions are enriched for ~24 nt small interfering RNAs (siRNAs), indicating that the hypermethylation induced in the mutants is mediated by the RdDM pathway. Indeed, methylation of non-CG sites at some loci of ros1 is partially suppressed by mutation of factors in the RdDM pathway such as RNA-DEPENDENT RNA POLYMERASE2 (RDR2) or DRM2, while CG methylation is less affected by these mutations. Most of the CG sites at the transposon sequences are heavily methylated in the wild-type genome. However, ros1 mutation results in a further increase in DNA methylation at non-CG sites.17 Since DNA demethylation enzymes are potentially able to remove methylated cytosines at all contexts, it is unclear how CG methylation at the transposon and repeat sequences escapes their demethylation activity. One possibility is that CG methylase MET1 can overcome demethylation activity at these loci to maintain gene silencing of the sequences. Alternatively, CG methylation at the repetitive sequences might be protected by unknown mechanisms from DNA demethylation.
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Cytosine methylation of repeats and genes is important for coordination of genome stability and proper gene function. In plants, DNA methylation is regulated by DNA methyltransferases, chromatin remodeling factors and RNAi machinery. Ectopic DNA hypermethylation at genes causes transcriptional repression and silencing, and the methylation patterns often become heritable over generations. DNA methylation is antagonized by the DNA demethylation enzymes. Recently, we identified a novel jmjCdomain containing gene IBM1 (increase in bonsai methylation1) that also negatively regulates DNA methylation in Arabidopsis. The ibm1 plants show a variety of developmental phenotypes. IBM1 prevents ectopic accumulation of DNA methylation at the BNS genic region, likely through removal of heterochromatic H3K9 methylation mark. DNA and histone demethylation pathways are important for genome-wide patterning of DNA methylation and for epigenetic regulation of plant development.
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In higher eukaryote genomes, cytosine residues are modified by methylation. DNA methylation in plants is mediated by DNA methyltransferases that target cytosines in all contexts (CG, CNG and CNN), preferentially at repeats and transposon sequences.1,2 However, recent studies of whole genome profiling of DNA methylation in the model plant Arabidopsis revealed the presence of DNA methylation at approximately 20~30% of genic sequences.3-5 Since DNA methylation is generally associated with transcriptional repression of genes,6 control of the genic methylation is crucial for proper gene function. In contrast to the mammalian genome, where DNA methylation patterns are erased and reprogrammed in every generation, the patterns in plants had long been considered to be static. However, recent findings of DNA and histone demethylation pathways have provided new insights into the dynamic regulation of DNA methylation in plants. The Arabidopsis genome encodes DNA methyltransferases that have a preference for specific target sequences. Methylation at CG sites is maintained by METHYLTRANSFERASE1 (MET1), whereas non-CG methylation is redundantly maintained by DOMAINS REARRANGED METHYLTRANSFERASE2 (DRM2) and *Correspondence to: Hidetoshi Saze; Department of Integrated Genetics; National Institute of Genetics; Mishima, Shizuoka 411-8540 Japan; Tel.: +81.55.981.6805; Fax: +81.55.981.6804; Email:
[email protected] Submitted: 03/04/08; Accepted: 05/27/08 Previously published online as an Epigenetics E-publication: http://www.landesbioscience.com/journals/epigenetics/article/6355 122
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DNA demethylation and histone demethylation in plants
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Figure 1. A model for control of heterochromatin distribution by IBM1 and DDM1. DDM1 is required for maintenance of H3K9 methylation and non-CG methylation at repeat sequences. H3K9 methylation and non-CG methylation are controlled by KYP and CMT3, respectively. On the other hand, in some low copy loci including the BNS locus, accumulation of H3K9 methylation is repressed by IBM1, possibly through its demethylase activity. DDM1 is also required for repression of DNA methylation at some low copy loci. The gray box shows heterochromatin formation in the low copy region and the resulting developmental defects, which are induced in the ibm1 mutant background.
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show accumulation of H3K9me concomitant with non-CG methylation at the BNS locus. Furthermore, mutation of histone H3K9 methylase KRYPTONITE (KYP)/SUVH4 or CMT3 suppresses both the ibm1-induced BNS hypermethylation and the developmental abnormalities. The primal targets of IBM1 seem to be genic regions rather than heterochromatic loci since DNA and histone methylation of repetitive sequences are largely unchanged in ibm1 plants (Fig. 1). Although the substrate specificity of IBM1 is yet to be determined, these data suggest that IBM1 is required for removal of H3K9me, which eventually prevents ectopic accumulation of non-CG methylation at low copy genic regions (Fig. 1). Mutation of IBM1 and DDM1 both induced hypermethylation at BNS, and the BNS methylation and developmental phenotypes are further enhanced in ibm1 ddm1 double mutants (Saze H, unpublished observations).26 This might be due to the ectopic deposition of H3K9me marks in genic regions in the ddm1 background, which is normally compensated for by IBM1 function (Fig. 1). Although JHDM2A in mammals shows hormone-dependent recruitment to and activation of androgen-receptor target genes,29 it is not clear whether similar pathways are present in Arabidopsis that recruit IBM1 to the target sequence. DNA methylation is a versatile epigenetic mark that is important for silencing repeats and transposons as well as for transcriptional repression of genes. However, at the same time, genes need to be protected from DNA methylation to ensure their proper expression. To cope with the problem, plants seem to have evolved two distinct pathways, DNA demethylation and histone demethylation, for regulation of DNA methylation. Interaction of these pathways, i.e., whether the targets of histone demethylation overlap with that of DNA demethylation pathway, is of particular interest. Another important question that should be addressed is the mechanism that directs the demethylation factors to specific targets, which might require DNA binding proteins, histone modifiers, small RNAs or RNAi machinery. In humans, local DNA hypermethylation causes silencing of tumor suppressor genes in many types of cancer
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Ectopic DNA hypermethylation at genes causes transcriptional repression or silencing, and once established, the methylation patterns often become heritable over generations. This heritable DNA hypermethylation mimics traditional mutations in both animals and plants.18 In Arabidopsis, several heritable developmental abnormalities caused by DNA hypermethylation have been identified. Notably, ectopic DNA hypermethylation occurs frequently in a global hypomethylation background. For instance, overall reduction of DNA methylation in the genome due to mutation of MET1 or DDM1 induces local hypermethylation at SUPERMAN (SUP), AGAMOUS (AG) and BONSAI (BNS) loci.19,20 sup and ag epigenetic mutants exhibit homeotic changes in flower structure due to silencing of the genes. The BNS gene encodes a putative subunit of the anaphase-promoting complex, and silencing of BNS results in a reduction of plant height and disruption of phyllotaxis.20 The BNS gene is flanked by a non-LTR type retrotransposon (LINE) sequence, and repeated self-pollination of ddm1 plants induces hypomethylation of the LINE sequence and hypermethylation of the BNS gene. The precise mechanism of the hypermethylation at these loci in a global hypomethylation background remains unclear. An Arabidopsis natural accession that lacks the LINE insertion at the BNS locus does not show induction of methylation by the ddm1,20 suggesting that the hypermethylation is mediated by the flanking LINE sequence. However, SUP and AG apparently lack transposons near the genes, and therefore the hypermethylation at SUP and AG may be directed by distinct mechanisms. Recently, it was shown that loss of CG methylation by met1 triggers genome-wide de novo non-CG methylation due to activation and/or misdirection of the RdDM pathway.21,22 In parallel, met1 causes transcriptional repression of DNA demethylases including DME and ROS1, which further contribute to form an aberrant DNA methylation pattern in the genome.21 Mutation of components of the RdDM pathway such as DRM2, RDR2, DEFECTIVE IN RNA-DIRECTED DNA METHYLATION1 (DRD1), DICER-LIKE3 (DCL3) and NRPD1a also downregulate ROS1 gene expression.16,21,23 Thus, although it is not yet known how DNA methylation regulates expression of DNA demethylase genes, global hypomethylation of the genome triggers both inhibition of DNA demethylation and de novo methylation by the RdDM pathway, which leads to local hypermethylation at a number of loci. However, local hypermethylation of genes has been observed not only in hypomethylation laboratory strains but also in naturally propagating plant populations.24,25 Further studies are needed to understand the underlying mechanisms of DNA hypermethylation that target specific loci. Besides DNA demethylation enzymes, Arabidopsis appears to have other factors, histone demethylases, for negative regulation of DNA methylation. A screen for mutants that cause ectopic DNA methylation at the BNS locus identified a gene named INCREASE IN BONSAI METHYLATION1 (IBM1).26 ibm1 mutation causes hypermethylation at the BNS gene, particularly in non-CG contexts, and the mutant plants show various developmental abnormalities including leaf deformation and sterility. IBM1 encodes a jmjC (jumonji C) domain containing protein similar to the mammalian JHDM2 family, which is constituted of demethylases of histone H3 lysine 9 methylation (H3K9me).27 In Arabidopsis, methylation of histone H3K9 and K27 (H3K27me) guides CMT3-mediated non-CG methylation at heterochromatic loci.28 Indeed, ibm1 plants www.landesbioscience.com
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cells, and reversal of the gene activities by treatment with DNA methyltransferase inhibitors has been tested in clinical trials.30 Further studies of the mechanisms of DNA demethylation have great importance for understanding the epigenetic regulation of plant development and genome integrity, and could have broad implications for the dynamic regulation of DNA methylation in other organisms.
29. Yamane K, Toumazou C, Tsukada Y, Erdjument-Bromage H, Tempst P, Wong J, Zhang Y. JHDM2A, a JmjC-containing H3K9 demethylase, facilitates transcription activation by androgen receptor. Cell 2006; 125:483-95. 30. Lyko F, Brown R. DNA methyltransferase inhibitors and the development of epigenetic cancer therapies. J Natl Cancer Inst 2005; 97:1498-506.
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