INSIGHT REVIEW NATURE Vol 447 24 May 2007 In contrast to the independently segregating epialleles that arise in DNA glycosylase-lyase DEMETER(DME), which can directly excise backcrossing drmI drm2 cmt3 triple mutants to wild-type plants or differentiating extra-embryonic tissue, this mechanism does not neces- introducing either DRM2 or CMT3 by transformation immediately state remethylation of FWA. This is in contrast to mammals, in which rescues these morphological phenotypes". This finding suggests that demethylation of imprinted genes occurs in primordial germ cells(the non-CG methylation can be more easily re-established, possibly allowing cells that ultimately generate the germ line) and is followed by germline- flexible regulation of genes. However, it is unclear how commonly this specific remethylation and silencing(see page 425). Other imprinted type of regulation is used, because few examples of DNA-methylation- genes such as MEA and FERTILIZATION-INDEPENDENT SEED 2 also regulated plant genes have been described. have cytosine-methylated regions in their promoters that are associated with maternally restricted expression.However, only for FWA has Silencing through time and development it been shown that differential methylation of particular sequences is The life cycles of plants differ from those of animals in that the prod- required for the regulation of imprinting ss. ucts of meiosis undergo mitotic proliferation to form multicellular Cytosine demethylation is also likely to have an important role in gametophytes(that is, the embryo sac and the pollen in flowering the control of silencing in situations other than gametophytic genera The embryo sac(female) contains an egg cell, which is haploid, tion and imprinting. DMe belongs to a small A. thaliana gene family is fertilized by a sperm nucleus, which is also haploid, to form a that includes the somatically expressed gene REPRESSOR OF SILEN- embryo. A second sperm nucleus fertilizes the central cell, which CING I(ROSI)..Mutations in ROSI l en shown to increase is diploid, to form triploid endosperm, an extra-embryonic tissue that RNA-directed DNA methylation, and ROSI has been shown to func- endosperm show parent-of-origin-dependent monoallelic expression, ies have defined a long-sought cytosine demethylation pathway, and or imprinting, which is important for proper seed development. For they raise many interesting questions. For example, to what extent are example, in A thaliana, the tandem repeats of maternal FWA alleles are genomic methylation patterns balanced by the targeting of de novo specifically demethylated in the central cell and the endosperm, lead DNA methyltransferases and DNA glycosylases? Furthermore, there ng to expression of FWA in these tissues. Demethylation and activa- are indications of a similar mechanism for cytosine demethylation in tion of FWA depend on maternal expression of the gene encoding the vertebrates Adult plant Vegetative c Flowering Ovary Anther Germination Flower FLC Embryo sac Mitosis Pollen Figure 4 I PeG-protein-mediated silencing throughout the A thaliana be induced by other cues. d, During flower development, the anthers cycle. The activation state of the PcG protein target FLC is illustrated d ovaries are sites of meiotic differentiation, giving rise to haploid throughout the plant life cycle. a, FLC is transcriptionally active in seeds cells known as microspores and megaspores, respectively. e, These and seedlings, preventing the plant from flowering and prolonging meiotic products undergo mitotic proliferation to form the multicellular vegetative development. b, Exposure to a long period of cold(that embryo sac and pollen gametophytes. f, PcG-protein-mediated vernalization)results in the expression of VIN3(red), which initiates repression at FLC is removed during an undefined resetting proce repression of FLC transcription, and the binding of the PcG protein VRN2, g, Then, the pollen contributes sperm nuclei to the embr ac, and these well as VRNI and LHPl(blue). In this process, chromatin at FLC is fertilize the haploid egg cell and diploid central cell (not shown), formi pigenetically modified by the trimethylation of H3K27. c, After warmer nbr n anew seed. in which flc is temperatures return, FLC repression is maintained, allowing flowering to re-expressed @2007 Nature Publishing GroupIn contrast to the independently segregating epialleles that arise in met1 mutants (as a result of the stable loss of CG methylation)39,40,51, backcrossing drm1 drm2 cmt3 triple mutants to wild-type plants or reintroducing either DRM2 or CMT3 by transformation immediately rescues these morphological phenotypes27. This finding suggests that non-CG methylation can be more easily re-established, possibly allowing flexible regulation of genes. However, it is unclear how commonly this type of regulation is used, because few examples of DNA-methylation￾regulated plant genes have been described. Silencing through time and development The life cycles of plants differ from those of animals in that the prod￾ucts of meiosis undergo mitotic proliferation to form multicellular gametophytes (that is, the embryo sac and the pollen in flowering plants). The embryo sac (female) contains an egg cell, which is haploid, and this is fertilized by a sperm nucleus, which is also haploid, to form a diploid embryo. A second sperm nucleus fertilizes the central cell, which is diploid, to form triploid endosperm, an extra-embryonic tissue that has a supportive role during embryogenesis. The central cell and the endosperm show parent-of-origin-dependent monoallelic expression, or imprinting, which is important for proper seed development52. For example, in A. thaliana, the tandem repeats of maternal FWA alleles are specifically demethylated in the central cell and the endosperm, lead￾ing to expression of FWA in these tissues53. Demethylation and activa￾tion of FWA depend on maternal expression of the gene encoding the DNA glycosylase–lyase DEMETER (DME), which can directly excise the base 5-methylcytosine54–56. Because the endosperm is a terminally differentiating extra-embryonic tissue, this mechanism does not neces￾sitate remethylation of FWA53. This is in contrast to mammals, in which demethylation of imprinted genes occurs in primordial germ cells (the cells that ultimately generate the germ line) and is followed by germline￾specific remethylation and silencing (see page 425). Other imprinted genes such as MEA and FERTILIZATION-INDEPENDENT SEED 2 also have cytosine-methylated regions in their promoters that are associated with maternally restricted expression55,57. However, only for FWA has it been shown that differential methylation of particular sequences is required for the regulation of imprinting53,58. Cytosine demethylation is also likely to have an important role in the control of silencing in situations other than gametophytic genera￾tion and imprinting. DME belongs to a small A. thaliana gene family that includes the somatically expressed gene REPRESSOR OF SILEN￾CING 1 (ROS1) 54,59. Mutations in ROS1 have been shown to increase RNA-directed DNA methylation, and ROS1 has been shown to func￾tion as a cytosine demethylase56,59,60. Together, these exciting discover￾ies have defined a long-sought cytosine demethylation pathway, and they raise many interesting questions. For example, to what extent are genomic methylation patterns balanced by the targeting of de novo DNA methyltransferases and DNA glycosylases? Furthermore, there are indications of a similar mechanism for cytosine demethylation in vertebrates61,62. Flowering Flower Vegetative development Adult plant Germination Fertilization Seed Seedling Megaspore Embryo sac Pollen Microspore Resetting Meiosis Mitosis Mitosis Anther FLC VIN3 FLC FLC Ovary Vernalization FLC FLC × FLC × × a b c d e f g VRN2 VRN2 LHP1 LHP1 VRN1 VRN1 LHP1 VRN1 VRN2 Figure 4 | PcG-protein-mediated silencing throughout the A. thaliana life cycle. The activation state of the PcG protein target FLC is illustrated throughout the plant life cycle. a, FLC is transcriptionally active in seeds and seedlings, preventing the plant from flowering and prolonging vegetative development. b, Exposure to a long period of cold (that is, vernalization) results in the expression of VIN3 (red), which initiates repression of FLC transcription, and the binding of the PcG protein VRN2, as well as VRN1 and LHP1 (blue). In this process, chromatin at FLC is epigenetically modified by the trimethylation of H3K27. c, After warmer temperatures return, FLC repression is maintained, allowing flowering to be induced by other cues. d, During flower development, the anthers and ovaries are sites of meiotic differentiation, giving rise to haploid cells known as microspores and megaspores, respectively. e, These meiotic products undergo mitotic proliferation to form the multicellular embryo sac and pollen gametophytes. f, PcG-protein-mediated repression at FLC is removed during an undefined resetting process. g, Then, the pollen contributes sperm nuclei to the embryo sac, and these fertilize the haploid egg cell and diploid central cell (not shown), forming the embryo and endosperm (respectively) in a new seed, in which FLC is re-expressed. 422 INSIGHT REVIEW NATURE|Vol 447|24 May 2007 ￾