This document summarizes a presentation on non-coding RNA mediated epigenetic regulation of agronomic traits in crop plants. It discusses mechanisms of epigenetic regulation including DNA methylation, histone modification, and RNA interference. It provides examples of how small interfering RNAs are involved in establishing epigenetic patterns in fungi and plants. Case studies on epigenetic regulation of rice flowering time and reproduction are described. Loops of self-reinforcing interactions between small RNAs, DNA methylation and histone modifications establish stable epigenetic states.

1. ICAR-IndianAgriculturalResearchInstitute
TO
CREDIT SEMINAR
ON
"Non-coding RNA mediated epigenetic regulation of
agronomic traits in crop plants”
Presented by :
Nilesh Joshi
Ph.D. - 2nd Year
Division of Genetics
INDIAN AGRICULTURAL RESEARCH INSITUTE
ICAR-IndianAgriculturalResearchInstitute

2. Outline of presentation
• Introduction
• Mechanisms of Epigenetic regulation
• Mechanisms of DNA methylation and histone
modification
• Molecular mechanism of RNAi
• Evidence of SiRNA involvement in epigenetics
• Case studies
• summary
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3. What is epigenetics..?ICAR-IndianAgriculturalResearchInstitute
 Epigenetics is defined as nucleotide sequence-independent changes in the
gene expression that are mitotically and/or meiotically heritable
 Literally, epigenetics means ‘above’ or ‘on top of’ genetics
 The term “epigenetic” was coined by Conrad Waddington, he is known as
father of epigenetics
 The epigenetic compound attach to DNA and modify its function, they are
said to have ‘marked’ to genome. These marks do not change the sequence
of the DNA. Rather, they change the way cell use the DNA instruction. The
marked are some time passed on cell to cell as cell divided. They also can
be passed down from one generation to next.
 Epigenetic modification is responsible for differentiation of organism
because at the cellular level all the cell contain similar genetic material

4. Mechanisms of epigeneticsICAR-IndianAgriculturalResearchInstitute
 DNA Methylation
Histone modifications
 Acetylation
 Methylation
Phosphorylation
 RNA mediated Interference
Zaidi et al., 2010

5. Epigenetic marks: DNA methylation and
histone modification
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 In plants methylate cytosine within CG, CHG, or CHH motifs.
 Histone modification like methylation, acetylation, phosphorylation, ubiquitination etc.
are commonly occur in histone containing amino acid.
Can DNA methylation and Histone modification linked to each other?

6. Epigenetic marks in transcriptionally active
versus silenced genes
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In particular, H3K27ac, H3K4me3,
H3K4me1, and H3K36me3[tri-methyl
histone H3 (Lys-36)] are associated with
the active transcription region
In contrast, H3K27me3 and H3K9me3
are distributed mainly in the inactive
gene locus
Chen et al., Annu. Rev. Biomed. Eng. 2017. 19:195–219

7. Writers, readers, and erasers of epigenetic
markers
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 The “writers” include histone
acetyltransferase (HAT), histone
methyltransferase (HMT), and protein
arginine methyltransferase (PRMT)
 whereas the “erasers” are histone
deacetylase (HDAC) and lysine
demethylase (KDM).
 The “reader” proteins containing
distinct domains (e.g., bromodomains,
chromodomains, and tudordomains)
can recognize the differentially modified
histones
Chen et al., Annu. Rev. Biomed. Eng. 2017. 19:195–219

8. Links between DNA methylation, histone
modification and chromatin remodeling
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Methyl-CpG-binding
proteins (MBD) and
histone deacetylase
(HDAC)
These models suggest that DNA methylation and histone modification are interlinked to each
other. Presence and absence of these marks decides the chromatins are either euchromatin or
heterochromatin En Li, et al., Nature; 2002, Vol-3: 662-673

9. Distribution of epigenetic marks on
chromosome
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BLUE = Gene
density
RED = Repetitive
element density
Genome-wide high-resolution mapping and functional analysis of DNA methylation in Arabidopsis
 Pericentromeric heterochromatin, repetitive sequences, and regions producing siRNAs are heavily methylated
 Over one third of expressed genes contain methylation within transcribed regions, whereas only ~ 5% of genes
show methylation within promoter regions
 Promoter-methylated genes show a greater degree of tissue specific expression
Heterochromatin region are more methylated than euchromatin region Zhang, et al., (2006) Cell 126: 1189–1201
Densely packaged
heterochromatin
Less densely
packaged, gene-rich
euchromatin
The centromere and
regions around it are
usually densely
packaged with few
protein-coding genes

10. RNA Interference (RNAi)ICAR-IndianAgriculturalResearchInstitute
 RNA interference (RNAi) is a mechanism for RNA-guided regulation of
gene expression in which double-stranded ribonucleic acid inhibits the
expression of genes with complementary nucleotide sequences.
 Rich Jorgensen experiment (1990) in petunias. Introduced a pigment
producing (Chalcone synthase) gene under the control of a powerful
promoter for deepen purple color of these flowers. Instead of the expected
deep purple color, many of the flowers appeared variegated or even white.
 Jorgensen named the observed phenomenon "co-suppression", since the
expression of both the introduced gene and the homologous endogenous
gene was suppressed.
 Co-suppression has since been found to occur in many species of plants. It has also been observed in fungi,
and has been particularly well characterized in “Neurospora crassa” where it is known as "quelling“.
Jorgensen R. (1990) Plant Cell 2: 279-289.

11. Contd…ICAR-IndianAgriculturalResearchInstitute
Fire and Mello extensively purified the sense and antisense ssRNA
preparations, then directly compared their effects to dsRNA on
the unc-22 gene. The purified ssRNAs consistently found to be 10-
100 fold less effective than dsRNA targeting the same RNA.
The Nobel Prize in Physiology or Medicine for 2006
Awarded jointly to
Andrew Z. Fire and Craig C. Mello
for their discovery of
"RNA interference – gene silencing by double-stranded RNA
Fire and Mello., Nature 1998; 391:806-811

12. Molecular mechanism of RNAiICAR-IndianAgriculturalResearchInstitute
 The cellular origins of miRNA and siRNA are somewhat
disparate
 miRNAs are derived from the genome, whereas siRNAs may
be endogenous or arise via viral infection or other exogenous
sources
 Typically, the genesis of a miRNA occurs in the nucleus with a
transcript known as a primary miRNA (1000nt long)
 In the cytoplasm, the processing pathways converge for
endogenous miRNAs and for typically exogenous siRNAs
 Both types of RNAi precursors are trimmed down to a dsRNA
duplex of the appropriate size for loading onto an Argonaute
protein
Wilson and Jennifer, Annu. Rev. Biophys. 2013. 42:217–39

13. Amplification of SiRNAICAR-IndianAgriculturalResearchInstitute
dsRNA present in the cytoplasm of the cell are cleaved by
dicer enzyme in 21-24nt siRNA
RISC complex bind to siRNA and remove passenger
strand
 RISC assembly bind target mRNA, either degrade the
target mRNA or translational inhibition mRNA to inhibit
for production of functional protein.
Before the degradation of target mRNA, guide strand of
siRNA act as primer and using RdRP start synthesis of
second strand complimentary to target mRNA
These dsRNA again targeted by dicer enzymes and
produces more amount of siRNA for same target.
The siRNA amplified using RdRP pathway in the target organism and hence siRNA present in the organism
throughout its life cycle and in some organism it also transfer to next generation

14. Evidence of SiRNA involvement in epigeneticsICAR-IndianAgriculturalResearchInstitute
 Experiment carried out in S. pombe because it contain a
single gene each for Ago (ago1+), Dicer (dcr1+) and
RNA-dependent RNA polymerase (RdRP; rdp1+).
 Deletion of any of these genes was shown to result in
loss of heterochromatic gene silencing at
pericentromeric DNA repeat regions
 Reduction in the levels of histone H3 lysine 9 (H3K9)
methylation (conserved marker of heterochromatin).
How siRNA activate H3K9 methylation ?
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Daniel Holoch and Danesh Moazed (2015 ). Nature rev genet ,doi:10.1038/nrg3863

15. RNAi-Mediated Targeting of Heterochromatin
by the RITS Complex
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 Study was carried out to identify factors important for RNAi-
mediated targeting of heterochromatin complexes
 The Chp1 protein binds to centromeric repeats and is required
for methylation of histone H3-K9 and for localization of Swi6.
 The phenotypes of chp1Δ strains are identical to RNAi
mutants.
 To test whether Chp1 provides a physical and functional link
between RNAi and heterochromatin assembly, used a tandem
affinity purification procedure (TAP) and a TAP tag to
identify factors that interact with Chp1.
 Several protein species of about 65, 90 and 120 kD were
specifically purified from the Chp1-TAP strain.
Verdel et al. (2004 ). Science 303, 672-676.

16. Two RNAi complexes, RITS and RDRC, physically
interact and localize to noncoding centromeric RNAs
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 Rdp1 is associated with two conserved proteins, Hrr1 (RNA
helicase), and Cid12 (member of the polyA polymerase
family), in a complex that has RNA-directed RNA
polymerase activity (RDRC).
 RDRC physically interacts with RITS but requires the Dicer
ribonuclease (Dcr1) and the Clr4 histone H3-K9
methyltransferase.
Motamedi et al., (2004 ). Cell 119, 789-802.

17. The ‘nascent transcript’ model and a self-
reinforcing epigenetic loop in S. pombe
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 siRNA-targeted centromeric lncRNA bound to the RITS
complex becomes a template for double-stranded RNA
(dsRNA) synthesis by RDRC and generation of new
siRNAs by Dicer 1 (Dcr1).
 The Chp1 subunit of the RITS complex anchors the
complex onto nucleosomes with H3K9 methylation, and the
RITS complex recruits the CLRC, of which Clr4 is the
methyltransferase and via Rik1 and Stc1 promote and
spread of H3K9 methylation.
 The heterochromatin protein 1 (HP1) homologue Swi6
binds to methylated H3K9 and promotes RDRC
recruitment and siRNA biogenesis via the silencing factor
Ers1.
 Swi6 and Chp2, help to restrict RNA pol II access by
recruiting the Snf2–histone deacetylase repressor complex
(SHREC ).
Daniel Holoch and Danesh Moazed (2015 ). Nature rev genet ,doi:10.1038/nrg3863

18. A self-reinforcing loop linking siRNAs to DNA
and histone methylation in A. thaliana
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 RNA polymerase IV (Pol IV)
transcripts are processed by the
RNA-dependent RNA polymerase
RDR2 and the Dicer protein DCL3
into 24-nucleotide (nt) small
interfering (siRNAs)
 RDM1 associates with the Pol V–
AGO4–DRM2 complex and may
link siRNA amplification to pre-
existing DNA methylation
 CMT3, is recruited directly to
methylated histone H3 lysine 9
(H3K9).
Daniel Holoch and Danesh Moazed (2015 ). Nature rev genet ,doi:10.1038/nrg3863

19. Involvement of RNA Pol IV and V in siRNA
mediated epigenetic pathway
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 Analyses of Pol IV-defective mutants have shown that this
polymerase is responsible for producing the precursor of >90%
of 24-nucleotide
 Pol IV is recruited to a subset of its genomic targets by the Pol
IV-interacting protein SAWADEE HOMEODOMAIN
HOMOLOGUE 1 (SHH1), which binds to H3K9me and
unmethylated H3K4 through its unique tandem Tudor-like fold
 Pol IV transcribe single-stranded RNAs (ssRNAs) at its target
loci and then ssRNA is copied by the RNA-dependent RNA
polymerase RDR2, which physically associates with Pol IV, to
produce dsRNAs.
 The chromatin remodeller CLASSY 1 (CLSY1) participates at
some point in these steps, presumably to ease the passage of Pol
IV along the genomic locus.
 DCL3 processes dsRNAs to 24-nucleotide siRNAs, which are
stabilized by methylation at their 3ʹ-OH groups by HUA
ENHANCER 1 (HEN1)19 and loaded onto AGO4.
Matzkae MA and Mosher RA (2014) . Nature rev genet , doi:10.1038/nrg3683

20. Case study-1ICAR-IndianAgriculturalResearchInstitute
Epigenetic regulation of rice flowering
 Within the rice flowering pathways, the close paralogs Heading date 3a (Hd3a) and RICE
FLOWERING LOCUS T1 (RFT1) are specifically upregulated upon the inductive SD
photoperiods in leaf phloem tissue and encode small globular proteins named florigens,
which move to the shoot apex to promote flowering
 There are at least two pathways that control the Hd3a/RFT1 expression under either SD
or LD photoperiods: the Early heading date 1 (Ehd1) and the Hd1 pathways

21. Active chromatin marks are involved in rice
flowering time Regulation
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Short day
Photoperiod
Long Day
photoperiod

22. Epigenetic regulation of rice reproduction and
Seed formation
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 Important roles of sRNA (both miRNAs and siRNAs) in rice floral organ development are
also evidenced by mutants of several sRNA-pathway genes, including SHOOT
ORGANIZATION 1 (SHO1) encoding a DICER-LIKE 4 homolog, SHOOTLESS 2 (SHL2)
encoding a RDR6 homolog), and WAVY LEAF1 (WAF1) encoding a HEN1 homolog
 Genome-wide analyses in rice have revealed that sRNA expression, DNA methylation, and
histone modifications (e.g.,H3K9ac, H3K4me3, and H3K27me3) significantly differ between
hybrids and their parents

23. Conclusion of the experimentICAR-IndianAgriculturalResearchInstitute
 In particular, H3K27me3 is recognized as a crucial epigenetic mark associated with gene
transcriptional repression, and the classical model proposes a sequential mode of action of the two
Polycomb complexes: PRC2 is responsible H3K27me3 establishment, and PRC1 recognizes the
H3K27me3 mark and further catalyzed downstream H2A mono-ubiquitination.
 Utilization of advanced technologies in proteomics, deep sequencing, and gene knockdown will
facilitate future studies in functional characterization of interesting genes, investigation of protein
complex composition and function, and gene networks controlling rice flowering and reproduction.

24. Case study-2ICAR-IndianAgriculturalResearchInstitute
Epi-sp, a gain-of-function epiallele of the rice ESP (Epigenetic Short Panicle, Os01g0356951),
which encodes a putative long noncoding RNA. The Epi-sp plants show a dense and short
panicle phenotype, an agronomically important phenotypes that is inherited in a semi-
dominant manner

25. Characterization of a semi-dominant rice
mutant with short panicle
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 The heterozygous Epi-sp (+/−) mutant showed a
significant reduction in plant height (97.4±3.4cm),
compared to the wild-type plants
 For the homozygous mutant, it (Epi-sp) exhibited a
dwarf phenotype (24.8±2.8cm) with a defect in shoot
apical meristem (SAM) and could not set seeds in
paddy field

26. Characterization of a semi-dominant rice
mutant with short panicle
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 Expression analysis demonstrated that the transcript level of Os01g0356951 was dramatically elevated in
Epi-sp plant
 The higher expression in homozygous Epi-sp plants than in heterozygous lines
 The ESP transcript was not detected in normal panicles of these same plants

27. DNA methylation analysis of the ESP locusICAR-IndianAgriculturalResearchInstitute
 Higher CG and CHG has been found, but not CHH
DNA methylation in the downstream transcriptional
termination region of ESP gene in the wild type
compared with the Epi-sp mutant
 This region is hypermethylated in the wild type but
is demethylated in the Epi-sp mutant, spanning 26
CG sites and 13 CHG sites
 Transcript levels of ESP were measured in 7-d-old
seedlings with or without 5-aza-dC treatment and
found that treatment with 5-aza-dC up-regulated
ESP expression

28. Effect of DNA methylation on ESP expression
is conserved in cultivated rice
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The ESP is conserved in the AA-genome
species including cultivated rice genomes but is
absent in O. brachyantha and Oryza punctata
Compared with the high expression of ESP
observed in Epi-sp, no ESP expression in
seedlings in all the cultivated rice strains tested

29. Conclusion of the experimentICAR-IndianAgriculturalResearchInstitute
 In this study, Epi-sp mutant shows low levels of DNA CG and CHG methylation in the TTR of ESP
gene causing ectopic ESP expression and a dense and short panicle architecture in rice.
 It is important to point out that, although the mechanism for the spontaneous hypomethylation of ESP
TTR remains unknown, the TTR of ESP gene displays the characteristic of CpG island
 Since there is no CHH methylation in the TTR of ESP, CG and CHG hypermethylation in this region
is likely not established by the RNA-directed DNA methylation (RdDM) pathway
 Consequently, hypomethylation of this region in the Epi-sp mutant is probably not due to loss of small
interfering RNAs (siRNAs)
 Therefore, it is revealed that epiallele is probably induced by some sort of aberrant active DNA
demethylation activity

30. SummaryICAR-IndianAgriculturalResearchInstitute
 DNA methylation and histone modification are responsible for epigenetic changes of an organism in
response to various environmental factor
 Gene silencing involved small 21 to 24nt dsRNA molecule
 dsRNAs either processed from endogenously originated from single RNA molecules that include an
imperfect stem loop secondary structure (miRNA) or endogenously/exogenously originated long
dsRNAs (siRNA)
 The siRNA amplified using RdRP pathway in the target organism and hence present in the organism
throughout its life cycle and in some organism it also transfer to next generation
 Epigenetic modification is responsible for differentiation of organism because at the cellular level all
cells contain similar genetic material
 DNA methylation and histone modification are interlinked to each other
 Epigenetic modification play an important role, in regulation of various agronomic traits, such as
flowering, reproduction, seed development and panicle architecture in rice

31. ICAR-IndianAgriculturalResearchInstitute