RNA-directed DNA methylation (RdDM) is an epigenetic pathway in plants and fungi where small RNAs direct DNA methylation and transcriptional gene silencing. The process involves RNA polymerase IV transcribing RNA from the locus to be silenced. This RNA is then copied to double stranded RNA by RNA-dependent RNA polymerase 2 and processed into 24 nucleotide small interfering RNAs by Dicer-like 3. Argonaute 4 incorporates these siRNAs and guides DNA methyltransferases like Domains Rearranged Methyltransferase 2 to introduce methyl groups at cytosines in DNA, leading to transcriptional gene silencing of the locus. RdDM is an important genome defense mechanism in plants against viruses.

1. RNA-directed DNA
methylation
(RdDM)
PRESENTED BY
AYUSH JAIN (PALB 7286)
BASAVARAJ BIDARAKUNDI
(PALB 7287)
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2. CONTENTS
What is DNA methylation??
What is the Significance of DNA methylation ??
DNA Methylation and transcription??
What is RdDM??
How RdDM is done ??
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3. WHAT IS DNA METHYLATION ??
 DNA methylation is
an epigenetic mechanism that
occurs by the addition of a methyl
(CH3) group to DNA, thereby
often modifying the function of the
genes and affecting gene
expression.
 The most widely characterized
DNA methylation process is the
covalent addition of the methyl
group at the 5-carbon of the
cytosine ring
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4. What is the significance DNA methylation??
All the cell in our
body have same
genomic DNA
sequence, But all
cells don’t need all
genes to be
expressed as they
constitute different
tissue which differ
in structure and
function.
Chemical modification of
chromosomal DNA(like
methylation and acetylation)
that changes pattern of gene
expression without altering
the DNA sequence. These
are called epigenetic
modifications Ex: Muscle
cells will have genes turned
on that are required by
muscle cell and turn off
genes(silencing) that liver
cell requires.
Silencing of tissue
specific genes are
important to
prevent them from
being expressed in
wrong tissues. For
this the cell need to
methylate some
genes, so that they
cannot be expressed
RNA-directed DNA
methylation is used
as a genome
defense mechanism
in plants
(Particularly during
viral infections)
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5. DNA methylation and transcription
 Methylation block
transcription to stop
expression of certain
genes.
 Due to methylation
activator protein and
transcription factors are
no longer be able bind
to enhancer element of
transcription site.
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6. WHAT IS RdDM??
RNA-directed DNA
methylation (RdDM) is an
small RNA-mediated
epigenetic pathway in
plants,fungi (both are si-
RNAs mediated)and
metazoans(piRNAs).
In the nucleus, small RNAs
elicit transcriptional gene
silencing (TGS) by directing
repressive epigenetic
modifications, such as DNA
cytosine methylation and
histone methylation, to
homologous regions of the
genome.
DNA methyltransferase act
to mediate de novo
methylation of cytosine's in
all classes of sequence
contexts(that is, CG, CHG
and CHH, where H
represents A,C or T).
de novo methylation
is the Methylation of a
previously unmodified
DNA sequence.
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7. How it is Done???
 RNA triggers for DNA methylation can come from different
sources, including invasive viral, transgene or transposon
sequences, and in some cases are derived from single-
stranded RNA precursors by RNA-dependent RNA
polymerases.
 The mechanism by which RNA signals are translated into
DNA methylation imprints are not clear but certain models
have been proposed.
 The model is based on studies done in Arabidopsis thaliana
and is particularly common with plants.
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8. Transcription fork model for
RNA-directed DNA methylation
(RdDM)
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9. RNA polymerase IV (Pol IV)-dependent small interfering RNA (siRNA)
biogenesis
1. Pol IV transcribes a single-stranded RNA (ssRNA).
2. Which is copied into a double-stranded RNA (dsRNA) by RNA-
DEPENDENT RNA POLYMERASE 2 (RDR2) with the assistance of the
chromatin remodeller CLASSY 1 (CLSY1).
3. The dsRNA is processed by DICER-LIKE 3 (DCL3) into 24-nucleotide
siRNAs.
4. Which are methylated at their 3ʹ ends by HUA ENHANCER 1 (HEN1)
5. And are incorporated into ARGONAUTE 4 (AGO4).
6. Then the SAWADEE HOMEODOMAIN HOMOLOGUE 1 (SHH1),
which binds to histone H3 methylated at lysine 9 (H3K9me), interacts with
Pol IV and recruits it to some target loci.
Courtesy :REF. 148, Cold Spring Harbor Laboratory Press.
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10. All mechanisms
dfiscussed are based
on adrabidopsis
thaliana
Pol V-mediated de novo methylation
1. Pol V transcribes a scaffold RNA
(in green) that base-pairs with
AGO4-bound siRNAs.
2. Then the AGO4 is recruited
through interactions with the AGO
hook regions in the carboxy-
terminal domain of the largest
subunit of Pol V and with KOW
DOMAIN-CONTAINING
TRANSCRIPTION FACTOR 1
(KTF1).
3. RNA-DIRECTED DNA
METHYLATION 1 (RDM1) links
AGO4 and DOMAINS
REARRANGED
METHYLTRANSFERASE 2
(DRM2), which catalyses de novo
methylation of DNA.
4. Pol V transcription may be enabled by the duplex unwinding activity of the chromatin remodeller
DEFECTIVE IN RNA-DIRECTED DNA METHYLATION 1 (DRD1)
5. The single-stranded DNA-binding activity of RDM1 and the putative cohesin-like roles of
DEFECTIVE IN MERISTEM SILENCING 3 (DMS3) and MICRORCHIDIA 6 (MORC6) may help to
generate and stabilize the unwound state.
6. Pol V recruitment is potentially aided by SUVH2 or SUVH9, both of which bind to methylated DNA.
Courtesy :REF. 148, Cold Spring Harbor Laboratory Press.
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11. Nucleosome positioning
1. Adjustments are done by the SWI/SNF
complex, which interacts with the IDN2
(INVOLVED IN DE NOVO 2)–IDP
(IDN2 PARALOGUE) complex that
binds to Pol V scaffold RNAs.
2. Deposition of repressive histone
modifications — such as H3K9me by
SUVH4, SUVH5 and SUVH6.
3. This is also facilitated by removal of
active marks by HISTONE
DEACETYLASE 6 (HDA6), JUMONJI
14 (JMJ14) and UBIQUITIN-SPECIFIC
PROTEASE 26 (UBP26).
Courtesy :REF. 148, Cold Spring Harbor Laboratory Press.
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12. QUESTIONS ???
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13. BIBLIOGRAPHY
VEDIO REFERENCES
• iBiology.com
• Shomu’s Biology
• Khan academy
Textual references
• Genes XI by Lewin
PUBLICATIONS
• RNA-directed DNA methylation: an epigenetic pathway of increasing complexity by
Marjori A. Matzke and Rebecca A. Mosher Nature genetics June 2014|vol 15 |page
394-408
• RNA-directed DNA methylation:Olivier Mathieu, Judith Bender Journal of Cell
Science 2004
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14. Thankyou
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