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111109_paszkowski - letter Maintenance of CpG methylation...

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letter nature genetics • volume 34 • may 2003 65 Maintenance of CpG methylation is essential for epigenetic inheritance during plant gametogenesis Hidetoshi Saze, Ortrun Mittelsten Scheid & Jerzy Paszkowski Friedrich Miescher Institute for Biomedical Research, P.O. Box 2543, CH-4002 Basel, Switzerland. Correspondence should be addressed to H.S. (e-mail: [email protected]). In mammals, the DNA methyltransferase 1 (Dnmt1) faithfully copies the pattern of cytosine methylation at CpG sites to the newly synthesized strand, and this is essential for epigenetic inheritance 1 . In Arabidopsis thaliana , several DNA methyltrans- ferases or chromatin modifiers coupled to methylation changes have been characterized, and mutations that cause loss of their function are recessive 2–7 . This is surprising because plant gameto- genesis includes postmeiotic DNA replication in haploid nuclei before fertilization. Therefore, the recessive character of the muta- tions excludes the affected components from a regulatory role in postmeiotic maintenance or modification of epigenetic states. Here we show, however, that depletion of A. thaliana MET1, a homolog of mammalian Dnmt1 (ref. 8), results in immense epige- netic diversification of gametes. This diversity seems to be a con- sequence of passive postmeiotic demethylation, leading to gametes with fully demethylated and hemidemethylated DNA, followed by remethylation of hemimethylated templates once MET1 is again supplied in a zygote. In a screen for insertion mutants impaired in transcriptional gene silencing (TGS), we identified mutant met1-3 with T-DNA integrated into the gene encoding DNA cytosine methyltrans- ferase1 (MET1). Another insertion mutant allele ( met1-4 ) was Published online 31 March 2003; doi:10.1038/ng1138 Fig. 1 Characterization of met1 mutants. a , Positions of T-DNA insertions (not drawn to scale) in MET1 . The conserved methyltransferase domains according to ref. 10 are marked in gray. wt, wild-type. b , RT–PCR detection of MET1 transcript and ACT2 as a control (lower panel). wt, wild-type. c , Genotyping of segregat- ing met1-3 and met1-4 alleles on Southern blots hybridized with probe A (indicated in a ). DNA from independent T2 plants was digested with Eco R Ι ( met1-3 ) or Cla Ι ( met1-4 ). Col, wild-type Columbia ecotype. d , DNA of individual T2 plants (as in c ) digested with Hpa ΙΙ , which is sensitive to methylation. Controls with hypomethylated genomic DNA included ddm1 (mutant ddm1-5 ( som8 ); ref. 14) and AsMET1 (a transgenic line expressing MET1 antisense RNA; refs. 12,30). The blot was hybridized with a probe representing centromeric 180-bp repeats methylated in wild-type plants. e , Northern blot with 10 µ g total RNA isolated from T2 plants (as in c ) hybridized with a TSI-specific probe (ref. 16; upper panel) and gel image (lower panel). Control for TSI transcripts was mom1 RNA 16 . Col, wild- type Columbia ecotype.
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