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Unformatted text preview: ishing Group http://www.nature.com/naturegenetics letter Fig. 2 Segregating phenotypes in T2 progeny. Wild-type segregants (MET1+/+;
+/+) were identical to wild-type Columbia plants, heterozygous (MET1+/–; +/–)
plants showed high variation in ﬂowering time and homozygous met1
mutants (MET1–/–; –/–) were consistently delayed in ﬂowering. Scale bar = 2 cm. obtained from the Syngenta Arabidopsis Insertion Library. The TDNA inserts in the met1-3 and met1-4 loci are of 7.1 kb and 4.7
kb and disrupt the conserved motif region of DNA methyltransferases8–10 and the ﬁrst exon, respectively (Fig. 1a). RT–PCR
analysis did not detect MET1 transcripts spanning the catalytic
domain in strains homozygous with respect to either mutation
(Fig. 1b), suggesting that each mutated allele is null. The insertions created new restriction sites in the met1 loci, allowing for
genotyping by Southern-blot analysis (Fig. 1c). A. thaliana
mutants deﬁcient in DNA methylation usually have reduced levels of cytosine methylation at centromeric repeats, transposonrelated sequences and silent transgenic loci11–15. Bisulﬁte analysis
of cytosine methylation at a 180-bp centromeric repeat showed
that CpG methylation at the locus was almost completely erased
in homozygous met1-3 plants.
Notably, we also observed hypomethylation in heterozygous
met1 plants in the T2 population (Fig. 1d). Once the A. thaliana
genome has been exposed to mutations that reduce DNA methylation, remethylation is extremely slow despite the presence of
wild-type alleles2,11,12,14. But our T2 plants that were heterozygous with respect to the met1 mutated alleles were derived from
mutagenized plants that were never homozygous with respect to
the mutation, excluding the possibility that hypomethylation in
heterozygotes was carried over from homozygous ancestors.
To examine whether demethylation reﬂects release of transcriptional suppression in heterozygotes, we hybridized RNA
from these plants with a probe for transcriptionally silent information (TSI) that was derived from pericentromeric repeat
sequences. These sequences are transcriptionally silent in wildtype A. thaliana strains but reactivated in TGS mutants16, including strains compromised in MET1 gene expression owing to
transgenic production of MET1 antisense transcripts (AsMET1;
refs. 12,16). TSI was reactivated in both homozygous and heterozygous met1-3 and met1-4 plants (Fig. 1e).
Hypomethylation is often accompanied by characteristic
developmental abnormalities17. In particular, it correlates well
with late-flowering phenotypes caused by ectopic expression of
the gene FWA, a regulator of flowering time18. Indeed, lateflowering plants were frequently observed among plants heterozygous with respect to both met1 alleles (Fig. 2). Therefore,
the appearance of this particular set of phenotypes
(hypomethylation of repetitive DNA, TSI reactivation and
66 delayed flowering) in T2 heterozygotes, without passage
through a homozygous geno...
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- Fall '09