Plant Molecular Biology 43: 203–220, 2000.
M.A. Matzke and A.J.M. Matzke (Eds.), Plant Gene Silencing.
© 2000 Kluwer Academic Publishers. Printed in the Netherlands.
RNA-directed DNA methylation
FhG Molekulare Biotechnologie, Am Klopferspitz 18A, 82152 Martinsried, Germany (e-mail
Key words: 5-methylcytosine, RNA-directed DNA methylation, RNA-DNA hybrid, RNA-directed RNA poly-
RNA-DNA interactions can serve as a signal that triggers de novo DNA methylation in plants. As yet, this RNA-
directed DNA methylation mechanism merely targets transgenes, but it appears likely that methylation of some
endogenoussequencesis also directed by RNA. RNA-directed methylationof cytosine residues speciﬁcally occurs
along the DNA regions that are complementary to the directing RNA pointing to the formation of a putative RNA-
DNA duplex. Dense methylation patterns and the methylation of cytosine residues at symmetric and asymmetric
sitesaredetectableon bothDNA strandswithinthese regions.Methylationprogressivelydecreasesinthesequences
adjacentto theputativeRNA-DNA duplex.Theextremesensitivity of RNA-directedDNA methylationwasdemon-
strated by analysing a short 30 bp DNA region that was complementary to the targeting RNA. Association of
RNA-directed DNA methylation with homology-dependentgene silencing indicated that the methylation-directing
RNA molecules may be double-stranded or may contain double-stranded regions. Whereas the function of DNA
methylation in transcriptional gene silencing is nearly understood, its role in post-transcriptional gene silencing
is still under discussion. In mammals, X-chromosome inactivation and genomic imprinting are associated with
DNA methylation but how methylation is initiated is unclear. The observation of a correlation between speciﬁc
antisense RNAs and transcriptional and post-transcriptional gene silencing may indicate that RNA-directed DNA
methylation is involved in epigenetic gene regulation throughout eukaryotes.
DNA methylation has been implicated in both gene
regulation and transgene silencing in plants, in addi-
tion to its known role in genomic imprinting and the
control of parasitic elements. Understandingtransgene
silencing is a challenge that needs to be overcome for
the successful exploitation of transgenic plants as a
recombinant protein production system. It is clear that
understanding the part that DNA methylation plays in
silencing will bring this goal much closer.
In plants, the co-ordination of DNA methylation
and gene regulation was ﬁrst detected by analysing
transgenes where primary transcription was affected
and the genes were only expressed at low levels
(Amasino et al., 1984). In contrast to expressed
transgenes, the promoter sequences of transcription-
ally silenced genes were methylated. Although DNA
methylation plays an essential role in tagging genes
for inactivation, promoter methylation alone does not
block transcription in most cases (Kass et al., 1997).
To achieve gene inactivation in mammals, methy-
lated DNA sequences are proposed to recruit repressor
complex via methyl-cytosine-binding proteins, such
as MeCP2 (Lewis et al., 1992). Binding of these
complexes then induces chromatin condensation, and
could prevent access of the transcription machinery to
the DNA (Li, 1999).
In contrast to transcriptional gene silencing (TGS),
post-transcriptional gene silencing (PTGS) is based on
a cytosolic RNA degradation process. Nevertheless,
in plants, PTGS can be associated with speciﬁc de
novo methylation of transgene coding regions, and it
is apparent that nuclear processes could also have an