Epigenetics: Any Potentially Stable and Heritable Change in Gene Expression That Occurs Without A Change in DNA Sequence

Epigenetics
Any potentially stable and

heritable change in gene
expression that occurs without a
change in DNA sequence
© 2016 American Society of Plant Biologists

What does “epigenetics” mean?
Literally, epigenetics
means above, or on top of, Transcription
genetics.
Usually this means

information coded beyond Epigenetic
Silencing
the DNA sequence, such as
in covalent modifications to
the DNA or modifications to
the chromatin structure.

What does “epigenetics” mean?
Practically, epigenetics describes phenomena in which
genetically identical cells or organisms express their
genomes differently, causing phenotypic differences.
Different epigenetic
modifications leading to
different expression patterns
Genetically Different
identical cells or phenotypes
individuals

X chromosome inactivation involves
epigenetic silencing
X
X X
X
X
X
X X
XX
X
X X X
X X
X X X X
X X
X
X X X
X X X X
In female mammals, one copy of

the X chromosome in each cell is
epigenetically inactivated.
Fur color in cats is determined in part by

orange, an X-linked gene. A female cat that is
heterozygous for the orange gene shows
black and orange patches, corresponding to
which X chromosome is active.
Photo credit: DrL

Epigenetic programming in plants
helps silence transposons and
maintain centromere function
Photo by Damon Lisch, in Gross, L. (2006) Transposon silencing keeps jumping genes in their place. PLoS Biology 4(10): e353.
Photos courtesy of the Barbara McClintock Papers, American Philosophical Society. Zhang, W., et al. (2008) Plant Cell 20: 25-34..

Epigenetic programming in plants
helps control developmental
transitions
Embryonic to Vegetative to
vegetative reproductive
transition transition
Embryonic Vegetative Reproductive

development development development

Lecture Outline
Epigenetic marks and their maintenance
• DNA methylation
• Histone modifications
Epigenetic genome regulation in plants
• Transposon silencing
• Control of flowering time
• Developmental switches and stress responses
• Control of imprinted genes
• Gene silencing in trans; paramutation
• Resetting the epigenome

Epigenetic marks and their
maintenance
Transcription
Epigenetic
Silencing

Eukaryotic DNA is packaged in
nucleosomes
Approximately 147 base

pairs of DNA wrapped
around a histone octamer
8 histones:
+ 2 each H2A
H2B
~ 147 bp DNA H3
H4

Chromosomes consist of
heterochromatin and euchromatin
Densely packaged
heterochromatin
CENTROMERE
DNA around the Less densely

centromere is usually packaged
packaged as euchromatin
heterochromatin.

Heterochromatin and euchromatin
form distinct domains in nuclei
Euchromatin DAPI (DNA stain) Merged
Centromeric
heterochromatin
Deal, R.B., Topp, C.N., McKinney, E.C., and Meagher, R.B. (2007) Repression of flowering in Arabidopsis
requires activation of FLOWERING LOCUS C expression by the histone variant H2A.Z. Plant Cell 19: 74-83.

Epigenetic modifications affect
chromatin structure
Epigenetic modifications include:
Cytosine methylation of DNA
Histone modifications Collectively, these
changes contribute to the
distribution of DNA into
silent, heterochromatin
and active euchromatin
euchromatin heterochromatin
Deal, R.B., Topp, C.N., McKinney, E.C., and Meagher, R.B. (2007) Repression of flowering in Arabidopsis
requires activation of FLOWERING LOCUS C expression by the histone variant H2A.Z. Plant Cell 19: 74-83.

DNA methylation
e
r as
n sfe
tra
thy
l DNA can be covalently
Me
modified by cytosine
methylation.
cytosine 5-methylcytosine
Methyl-
cytosine TTCGCCGACTAA

DNA methyltransferases in
Arabidopsis
MET1 (METHYLTRANSFERASE1)
• 5'-CG-3' sites
• Silencing of transposons, repetitive elements, some
imprinted genes
CMT3 (CHROMOMETHYLASE3)
• 5'-CHG-3' sites
• (H= A, C or T)
• Interacts with histone mark
DRM 1, DRM 2 (DOMAINS REARRANGED 1 and 2)
• 5'-CHH-3' sites
• Primarily targets repetitive elements
• Requires the active targeting of siRNAs

CG methylation can be propagated
during DNA replication
ON
A T G C G T A C T A T G C G T A C T
TI
CA
T A C G C A T G A T A C G C A T G A
I
PL
MET1
RE
5’ A T G C G T A C T
3’ T A C G C A T G A
“MAINTENANCE”
METHYLATION
A T G C G T A C T A T G C G T A C T
T A C G C A T G A T A C G C A T G A

Asymmetric methylation sites
require additional information
ON
A T G C A A A C T
TI
CA
T A C G T T T G A
I
PL
RE
5’ A T G C A A A C T
3’ T A C G T T T G A
Requires
A T G C A A A C T
T A C G T T T G A } additional
information to
remethylate
Asymmetric methylation sites are maintained (and initiated) by information
on associated histones, and an RNA-based mechanism, RNA-directed DNA
Methylation (RdDM), that directs DNA methylases to these sites.

Some sites are maintained by small
interfering RNAs (siRNAs)
ON
A T G C A A A C T
TI
CA
T A C G T T T G A
LI
P
RE
5’ A T G C A A A C T
3’ T A C G T T T G A
A T G C A A A C T A T G C A A A C T
T A C G T T T G A T A C G T T T G A
siRNA DRM1/2

Different DNA methylases act on
different sites
In the met1
mutant, CG
methylation
is abolished,
revealing
that MET1 is
solely
responsible
for CG
methylation
Lister, R., O’Malley, R.C., Tonti-Filippini, J., Gregory, B.D., Berry, C.C., Millar, A.H., and Ecker, J.R. (2008).
Highly integrated single-base resolution maps of the epigenome in Arabidopsis. Cell 133: 523–536.

Different DNA methylases act on
different sites
By contrast, in
the triple
mutant of drm1,
drm2 and cmt3,
(ddc) nearly all
non-CG
methylation is
abolished,
revealing the
roles of these
genes in non-
CG methylation.
Lister, R., O’Malley, R.C., Tonti-Filippini, J., Gregory, B.D., Berry, C.C., Millar, A.H., and Ecker, J.R. (2008).
Highly integrated single-base resolution maps of the epigenome in Arabidopsis. Cell 133: 523–536.

Heterochromatin DNA is highly
methylated
BLUE = Gene
density
RED = Repetitive
element density
GREEN = DNA methylation is

Methylated DNA densest at the repetitive
BROWN = elements around the
Methylated DNA in
a met1 mutant
centromere.
Reprinted from Zhang, X., et al. (2006) Genome-wide high-resolution mapping and functional analysis of DNA methylation in Arabidopsis. Cell 126: 1189–1201 with
permission from Elsevier.

Heterochromatin DNA is highly
methylated
BLUE = Gene
density
RED = Repetitive
element density
Although CG methylation
is more abundant in
pericentromeric regions,
a higher proportion of
CHH methylation is
found there.
Reprinted from Zhang, X., Yazaki, J., Sundaresan, A., Cokus, S., Chan, S.W.-L., Chen, H., Henderson, I.R., Shinn, P., Pellegrini, M., Jacobsen, S.E., and Ecker., J.R.
(2006) Genome-wide high-resolution mapping and functional analysis of DNA methylation in Arabidopsis. Cell 126: 1189–1201 with permission from Elsevier.;
Cokus et al., (2008) Shotgun bisulphite sequencing of the Arabidopsis genome reveals DNA methylation patterning Nature 452: 215-219 .

Multiple genes contribute to DNA
methylation at transposons
Methylase Accessory protein
mutants mutants
CG methylation
MET1 is required
for CG DDM1 is for all
methylation three methylases
to access
transposons
within
heterochromatin
CMT3 is required
CHG methylation
for CHG
methylation
CMT2 and DRM2

CHH methylation
are required for

CHH methylation
Reprinted from Zemach, A., Kim, M.Y., Hsieh, P.-H., Coleman-Derr, D., Eshed-Williams, L., Thao, K., Harmer, Stacey L. and Zilberman, D. The Arabidopsis
nucleosome remodeler DDM1 allows DNA methyltransferases to access H1-containing heterochromatin. Cell. 153: 193-205 with permission from Elsevier.

Introns and transposable elements
are generally hypermethylated in
relation to exons
(CMT3)
(RdRP)
(RdRP)
(functions
unknown)
In plants, the DNA of introns and TEs

present higher methylation levels than
exonic DNA.
Reprinted from Li et al. (2014) Genetic perturbation of the maize methylome. Plant Cell 26: 4602-4616

5-methylcytosine can be excised
and replaced with unmethylated C
DNA
DNAglycosylases
glycosylasesremove
remove5-methylcytosine
5-methylcytosineand
and
replace
replace it with an unmethylated cytosine throughaa
it with an unmethylated cytosine through
base
baseexcision
excisionrepair
repairmechanism
mechanism
Arabidopsis has a family

of four DNA demethylases
(glycolases):
•REPRESSOR OF
SILENCING 1 (ROS1)
•DEMETER (DME)
•DEMETER-LIKE 2 (DML2)
•DEMETER-LIKE 3 (DML3)
Reprinted from Gehring, M., Reik, W. and Henikoff, S. DNA demethylation by DNA repair. Trends Genet. 25: 82-90 with permission from Elsevier.

DNA demethylation is involved in
diverse processes
Gene expression is affected both by DNA methylation and demethylation
Switching off DML activity interferes

with ripening in tomato
A triple mutant (ros1 dml2 dml3)

shows enhanced susceptibility to
Fusarium oxysporum
Reprinted from Le, T.-N., et al. and Wang, M.-B. (2014). DNA demethylases target promoter transposable elements to positively regulate stress responsive genes in
Arabidopsis. Genome Biology. 15: 1-18. Liu, R., et al. (2015). A DEMETER-like DNA demethylase governs tomato fruit ripening. Proc. Natl. Acad. Sci. USA 112:
10804-10809.

Histone proteins can be modified to
affect chromatin structure
DNA
Histone octamer
The amino
terminal regions
of the histone
monomers
extend beyond
the nucleosome
and are
accessible for
modification.
NUCLEOSOME

The Histone Code
• Histones can be modified by
– Acetylation (Ac)
– Ubiquitination (Ub)
– Methylation (Me)
– Phosphorylation (P)
– Sumoylation (Su)
• Depending on their position, these can
contribute to transcriptional activation or
inactivation.

Example – H3 modifications
Me Me P Ac Me Ac Ac Me Me P
H3 A R T K Q T A R K S T G G K A P R K Q L A T K A A R K S
4 9 10 14 1718 23 262728
The amino terminus of H3 is often modified at one or more positions,
which can contribute to an activation or inhibition of transcription.

Example – H3 modifications
Me Me P Ac Me Ac Ac Me Me P
H3 A R T K Q T A R K S T G G K A P R K Q L A T K A A R K S
4 9 10 14 1718 23 262728
The amino terminus of H3 is often modified at one or more positions,
which can contribute to an activation or inhibition of transcription.
Lysine can be acetylated, or mono-, di-, or tri-methylated
+ Methylated lysine +
NH3 NH2
Mono (Kme1) CH3
Lysine (K) + CH3

Di (Kme2) NH
CH3
N CH3
O CH3
+
Acetylated lysine Tri (Kme3) N CH3

CH3
(KAc)

Histone modification affects
chromatin structure
Open configuration
Me P Ac
H3 K4 S10 K14
Closed configuration
Me Me P
H3 K9 K27 S28

Different histone modifications are
associated with genes and transposons
Gene
mRNA
H3K4me
H3K4me is associated with actively

transcribed genes and mRNA.
H3K9me
Me-C
Transposon
Red = high correlation

H3K9me is associated with methylated Green = low correlation
DNA (Me-C) and transposons.
Reprinted by permission from Macmillan Publishers, Ltd: NATURE. Lippman, Z., Gendrel, A.-V., Black, M., Vaughn, M.W., Dedhia, N., McCombie, W.R., Lavine,
K., Mittal, V., May, B., Kasschau, K.D., Carrington, J.C., Doerge, R.W., Colot, V., Martienssen, R. (2004) Role of transposable elements in heterochromatin and
epigenetic control. Nature 430: 471-476 Copyright 2004.

H3K27me3 is associated with genes
BLUE = Gene H3K27me3 in

density Arabidopsis is
RED = Repetitive
element density
present within the
gene-rich region,
not the repeat-rich
region.
GREEN = H3K27me3
PURPLE = methylcytosine
Zhang, X., Clarenz, O., Cokus, S., Bernatavichute, Y.V., Pellegrini, M., Goodrich, J., Jacobsen, S.E. (2007) Whole-
genome analysis of histone H3 lysine 27 trimethylation in Arabidopsis. PLoS Biol. 5: e129.

H3K27me3 methylation is conferred
by Polycomb Repressive Complex 2
Polycomb
Repressive
Complex 2
H3K27me3
Active genes Silenced genes Polycomb repressive

complex proteins are
also known as
Polycomb group
proteins (PcG)

PRC2 core proteins in Drosophila
Drosophila
PRC2 has a
conserved core
of four proteins E(Z) ESC SU(Z)12 NURF55
Enhancer of zeste (E(Z))

Extra sex comb (ESC)
Suppressor of zeste 12 (SU(Z)12)
NURF55

Arabidopsis has multiple forms of
three of the core PRC2 proteins
Drosophila PRC2
E(Z) ESC SU(Z)12 NURF55

(methylase)
Arabidopsis PRC2
FERTILIZATION- MULTICOPY
CURLY LEAF (CLF) FERTILIZATION INDEPENDENT SEED 2 SUPPRESSOR
INDEPENDENT (FIS2) OF IRA1
MEDEA (MEA) ENDOSPERM (MSI1,2,3,4,5)
(FIE) EMBRYONIC FLOWER 2
SWINGER (SWN) (EMF2)
VERNALIZATION 2 (VRN2)

Plants make multiple PRC2 complexes
with different targets
Seed development
MEA + FIS2 complex
Transition to flowering
CLF/SWN + VRN2 complex
Floral organogenesis
CLF/SWN + EMF2 complex

In animals, H3K27me3 silencing is
maintained by PRC1
Polycomb Polycomb
Repressive Repressive
Complex 2 Complex 1
H3K27me3
Active genes Silenced genes Stably silenced genes

In plants, PRC1-like functions
similarly
Polycomb PRC1-like
Repressive
Complex 2
LHP1
H3K27me3
Active genes Silenced genes Stably silenced genes
LHP1 binds
specifically to
H3K27me3

LHP1 co-localizes with H3K27me3
Note the close correlation of LHP1

and H3K27me3 distributions
Turck F, Roudier F, Farrona S, Martin-Magniette M-L, Guillaume E, et al. 2007 Arabidopsis TFL2/LHP1 specifically associates
with genes marked by trimethylation of histone H3 lysine 27. PLoS Genet 3(6): e86.

LHP1 co-localizes with H3K27me3
Here’s a transposon
marked with
H3K9me2!
Turck F, Roudier F, Farrona S, Martin-Magniette M-L, Guillaume E, et al. 2007 Arabidopsis TFL2/LHP1 specifically associates
with genes marked by trimethylation of histone H3 lysine 27. PLoS Genet 3(6): e86.

An H2 variant, H2A.Z is associated
with some active genesActive gene
Inactive gene
SWR1/
SRCAP
complex
H2A.Z H2A
The histone variant H2A.Z promotes transcription and is
swapped into the nucleosome by the SWR1/SRCAP complex.

There are three histone H3
variants: H3.1, H3.3, and CENH3
H3 H3.1 and H3.3
CENH3 differ in only four
amino acids, but
their distribution
is very different.
H3.1 is
associated with
heterochromatin,
and H3.3 with
Nucleosomes at the centromere transcribed
incorporate an H3 variant called genes
CENH3 that is necessary for
centromere maintenance
Redrawn from Jiang, J., Birchler, J.A., Parrott, W.A., and Dawe, R.K. (2003) A molecular view of plant centromeres. Trends Plant Sci. 8: 570-575 with permission from Elsevier. Stroud, H., Otero, S., Desvoyes, B.,
Ramírez-Parra, E., Jacobsen, S.E. and Gutierrez, C. (2012). Genome-wide analysis of histone H3.1 and H3.3 variants in Arabidopsis thaliana. Proc. Natl. Acad. Sci. USA 109: 5370-5375. See also Shi, L., Wang, J., Hong,
F., Spector, D.L. and Fang, Y. (2011). Four amino acids guide the assembly or disassembly of Arabidopsis histone H3.3-containing nucleosomes. Proc. Natl. Acad. Sci. USA 108: 10574-10578.

Chromatin remodelers use energy to
move or alter histone octamers
Reprinted by permission of Annual Reviews from Clapier, C.R. and Cairns, B.R. (2009). The Biology of Chromatin Remodeling Complexes. Annu. Rev. Biochem. 78: 273-304.

Example: Model for reversal of
polycomb repression
In this example, the chromatin remodelers SPLAYED (SYD) and
BRAHMA (BRM) facilitate switching of a gene from OFF to ON
Polycomb Repressive Complex (PRC) Loss of PRC

Repressive histone marks (Red stars) Activating histone marks (Green stars)
Wu, M.-F., Sang, Y., Bezhani, S., Yamaguchi, N., Han, S.-K., Li, Z., Su, Y., Slewinski, T.L. and Wagner, D. (2012). SWI2/SNF2 chromatin remodeling ATPases
overcome polycomb repression and control floral organ identity with the LEAFY and SEPALLATA3 transcription factors. Proc. Natl. Acad. Sci. USA 109: 3576-3581.

Summary – epigenetic marks

Epigenetic controls in whole-plant
processes
• Transposon silencing
• Control of flowering time
• Developmental switches and stress responses
• Control of imprinted genes
• Gene silencing in trans; paramutation
• Resetting the epigenome

Transposons
• Fragments of DNA that can insert
into new chromosomal locations
• Some copy themselves and

increase in number within the
genome
• Responsible for large scale

chromosome rearrangements and
single-gene mutagenic events
Reprinted by permission from Macmillan Publishers, Ltd: Nature Review Genetics. Surridge, C. (2001) Plant genetics: Turning off transposons. 2: 404. Copyright 2001.

Transposons
Transposable
elements were
discovered in Zea
mays by Barbara
McClintock.
For her discovery, she

was awarded the Nobel
Prize in Physiology or
Medicine in 1983.
Corn kernels showing

Barbara McClintock transposition
Photos courtesy of the Barbara McClintock Papers, American Philosophical Society.

Transposons can cause inactive or
unstable alleles
Gene required Gene
for pigment interrupted by
biosynthesis transposon
Excision of the
transposon causes
unstable alleles
Wild-type allele Mutant allele

Pigmented kernel Unpigmented kernel Unstable allele
Partially pigmented kernel

Naturally occurring transposons are
a source of genetic variation
“Variation is the raw material

of evolutionary change”
- Stephen Jay Gould
(1941 – 2002)
21˚ 15˚
An Antirrhinum transposon that is only
active at low temperatures.
Hashida, S.-N. Uchiyama, T., Martin, C., Kishima, Y., Sano, Y., and Mikami, T., (2006) The temperature-dependent change in methylation
of the Antirrhinum transposon Tam3 is controlled by the activity of its transposase. Plant Cell 18:104-118.

Transposons are abundant
% of genome derived from
Organism transposons
Yeast - S. cerevisiae 3%
Nematode - C. elegans 6%
Arabidopsis thaliana 14%
Fruitfly - D. melanogaster 15%
Rice - Oryza sativa 14%
Homo sapiens 44%
Corn - Zea mays 60%
Source: Kidwell, M.G. (2002) Genetica 115: 49-63.

Human transposons are almost
completely silent
Number of
active
transposon
families
through
evolutionary
time
Pace , J.K., and Feschotte, C. (2007) The evolutionary history of human DNA transposons:
Evidence for intense activity in the primate lineage. Genome Res. 17: 422-432

Transposon silencing
• By contrast, maize has many

active transposons
• Epigenetic marks are thought to

have evolved to silence foreign
DNA (transposons, viruses)
• Mutants that interfere with

epigenetic silencing release
transposons from silencing, and
allow mutagenic transposon
activity
Photo by Damon Lisch, in Gross, L. (2006) Transposon silencing keeps jumping genes in their place. PLoS Biology 4(10): e353.

Transposon location affects mode of
silencing and biology impact
Reprinted from Sigman, M.J. and Slotkin, R.K. (2016). The first rule of plant transposable element silencing: Location, location, location. Plant Cell. (in press
doi:10.1105/tpc.15.00869)

DNA methylation is necessary to
silence transposons
BLUE = Gene
density
RED = Repetitive
element density
GREEN = Methylated
Loss-of-function met1 or ddm1 DNA
(decrease in DNA
BROWN =
methylation1) mutants have Methylated DNA in
hypomethylated DNA a met1 mutant
Reprinted from: McCue, A.D. et al. (2014) ARGONAUTE 6 bridges transposable element mRNA-derived siRNAs to the establishment of DNA methylation. EMBO J 34 : 20-35;
Zhang, X., Yazaki, J., Sundaresan, A., Cokus, S., Chan, S.W.-L., Chen, H., Henderson, I.R., Shinn, P., Pellegrini, M., Jacobsen, S.E., and Ecker., J.R. (2006) Genome-wide high-
resolution mapping and functional analysis of DNA methylation in Arabidopsis. Cell 126: 1189–1201. With permission from Elsevier.

Transposons are activated in ddm
mutants
Six generations after
DNA methylation was CAC3
reduced by DDM CAC1
inactivation, newly CAC4
inserted transposons CAC2
were distributed
throughout the
genome.
Yellow is site of original

insertion, blue and red are
new sites of insertion.
Reprinted by permission from Macmillan Publishers, Ltd: NATURE. Miura, A., Yonebayashi, S., Watanabe, K., Toyama, T., Shimada, H., and Kakutani, T. (2001)
Mobilization of transposons by a mutation abolishing full DNA methylation in Arabidopsis. Nature 411: 212-214. Copyright 2001.

Activated transposons induce
mutations
After one After three After five
Wild-type generation generations generations
After DDM inactivation, plants become more and more

abnormal as they accumulate transposon-induced mutations.
Kakutani, T., Jeddeloh, J.A., Flowers, S.K., Munakata, K., and Richards, E.J. (1996) Developmental abnormalities and epimutations
associated with DNA hypomethylation mutations. PNAS 93: 12406-12411. Copyright (1996) National Academy of Sciences, U.S.A.

Epigenetic silencing of transposons by
DNA methylation is necessary to
maintain genomic integrity.
Kakutani, T., Jeddeloh, J.A., Flowers, S.K., Munakata, K., and Richards, E.J. (1996) Developmental abnormalities and epimutations
associated with DNA hypomethylation mutations. PNAS 93: 12406-12411. Copyright (1996) National Academy of Sciences, U.S.A.

Initiating and maintaining silencing
at repetitive DNA and transposons
How does the genome specifically

recognize and silence repetitive elements
and transposons?
In other words, how does it recognize “self” (genes) from
“non-self”? What is the basis for this “genomic immune
recognition system”?

Small interfering RNAs also
contribute to transposon silencing
siRNA-mediated silencing
ARGONAUTE (AGO)
RNA protein. Guides
siRNA to target sites
DNA
AGO
RNA Pol
AGO
RNA-dependent
RdRP
RNA polymerase
DCL
(RdRP)
RNA Pol
Double-
stranded RNA Dicer-like protein
(dsRNA) (DCL). Cuts siRNA targeting to
dsRNA into nascent RNA transcript
smaller pieces
including small leads to DNA and
interfering RNA histone methylation
(siRNA) and silencing

Small interfering RNAs (siRNAs) are
preferentially derived from
pericentromeric regions
Small RNAs
The density of small RNA-homologous loci is highest in the

centromeric and pericentromeric regions which contain a high
density of repeat sequence classes, such as transposons.
Kasschau, K.D., Fahlgren, N., Chapman, E.J., Sullivan, C.M., Cumbie, J.S., et al. 2007 Genome-wide profiling and analysis of Arabidopsis siRNAs. PLoS Biol 5(3):
e57.

Transposon silencing can be
initiated through siRNA
Reprinted from Fultz D., Choudury S.G., and Slotkin, R.K. (2015). Silencing of active transposable elements in plants. Curr. Opin. Plant Biol. 27: 67-76 with permission from Elsevier.

Transposon silencing can be
maintained by MET1 and CMT2&3
Fultz D., Choudury S.G., and Slotkin, R.K. (2015). Silencing of active transposable elements in plants. Curr. Opin. Plant Biol. 27: 67-76.

Epigenetic programming controls
developmental transitions
Embryonic to Vegetative to
vegetative reproductive
transition transition
Embryonic Vegetative Reproductive

development development development

Epigenetic control of flowering time
Prolonged cold
treatment
Vegetative Reproductive
Development Development
Autumn Winter Spring
Some plants require a prolonged cold

period (vernalization) - as experienced
during winter, before they will flower.

FLOWERING LOCUS C (FLC)
mutants flower early
flc mutant
FLC is an inhibitor of flowering;

removing FLC removes the
vernalization requirement
FLC inhibits FT, an activator of
flowering
Wild-type
plant
FLC FT gene
Transcription of FT gene
repressed by FLC binding
flc mutant
plant
FT gene
FT

FLC is silenced by vernalization
After 40 days at 4ºC,

FLC is not expressed.
Ten days after return
to 22ºC, FLC
expression is still off.
FLC gene transcribed FLC gene silenced
Reprinted by permission from Macmillan Publishers, Ltd: NATURE Sung, S., and Amasino, R.M. (2004)
Vernalization in Arabidopsis thaliana is mediated by the PHD finger protein VIN3. Nature 427: 159-164. Copyright
2004.

FLC is regulated by epigenetic
modifications
H2A.Z incorporation
H3K9me2, H3K27me2
H3K4me, H3K36me
H3K9Ac, H3K14Ac
cold
FLC gene transcribed FLC gene silenced

The FLC gene is epigenetically
modified during vernalization
The FLC gene promoter (P),
intron (V) and 3’UTR (U) were
examined for histone
P V U modifications. Before
+ - - vernalization, P and V showed
activating modifications
(H3Ac), and after vernalization
they showed inhibitory
modifications (H3K27me,
H3K9me).
NV = no vernalization
VT0 = 40 days at 4°
followed by 7 days at 22°
2004.

The FLC gene is epigenetically
modified during vernalization
P V U
+ - -
NV = no vernalization
VT7 = 40 days at 4° followed by 7 days at 22°
2004.

VIN3 is induced by vernalization
VIN3 gene silent VIN3 gene transcribed
2004.

The VIN3 gene is necessary for FLC
modification during vernalization
Wild-type plants vin3 mutant plants
+ - - + - -
H3Ac H3K27me H3K9me
In the vin3 mutant, the FLC gene remains

epigenetially “ON”, even when vernalized.
2004.

VIN3 and the PRC2 complex
epigenetically silence FLC
VIN3
PRC2
FLC gene transcribed (including FLC gene silenced
VIN3)

LHP1, a component of PRC1-like, is
required to maintain FLC silencing
Days after removal from cold
NV 40DV 1 5 10
Wild-type FLC mRNA
Before After
vernalization vernalization
In wild-type plants, FLC levels

remain low after returning plants to
warm temperatures.
Reprinted by permission from Macmillan Publishers, Ltd: NATURE GENETICS. Sung, S., He, Y., Eshoo, T.W., Tamada, Y.,
Johnson, L., Nakahigashi, K., Goto, K., Jacobsen, S.E., and Amasino. R.M. (2006) Epigenetic maintenance of the vernalized
state in Arabidopsis thaliana requires LIKE HETEROCHROMATIN PROTEIN 1. Nature Genetics 38: 706 – 710. Copyright
2006.

LHP1 is required to maintain FLC
silencing
Days after removal from cold
NV 40DV 1 5 10
Wild-type FLC mRNA
lhp1 mutant FLC mRNA
Before After
vernalization vernalization
In lhp1 mutants, FLC levels

increase after returning plants to
warm temperatures indicating that
the silencing is not stable.
2006.

LHP1 is required to maintain FLC
silencing
Wild-type FLC mRNA
lhp1 mutant FLC mRNA
PRC2 PRC1-like
FLC gene transcribed FLC gene silenced Silencing maintained
2006.

The activity of FLC is also regulated
by trithorax group (trxG) proteins
In the regulation of FLC and
other genes, trithorax group
proteins are generally
antagonistic to polycomb
group proteins including the
PRC2 complex
The trxG SET DOMAIN

GROUP 26 (SDG26)
induces gene expression of
the flowering activator SOC1
by inducing H3 trimethylation
at SOC1 promoter
Pien, S., Fleury, D., Mylne, J.S., Crevillen, P., Inzé, D., Avramova, Z., Dean, C. and Grossniklaus, U. (2008). ARABIDOPSIS TRITHORAX1 dynamically regulates FLOWERING LOCUS C activation
via histone 3 lysine 4 trimethylation. Plant Cell. 20: 580-588. Holec, S. and Berger, F. (2012). Polycomb group complexes mediate developmental transitions in plants. Plant Physiol. 158: 35-43.

Long noncoding RNAs (lncRNAs)
also contribute to FLC regulation
In other systems,
lncRNAs have been
shown to contribute to
the recruitment of
silencing machinery.
COOLAIR and COLDAIR

are FLC lncRNAs that
are cold-induced
From Heo, J.B., and Sung, S. (2011). Vernalization-mediated epigenetic silencing by a long intronic noncoding RNA. Science. 331: 76-79. Reprinted with permission
from AAAS. See also Song, J., Angel, A., Howard, M. and Dean, C. (2012). Vernalization – a cold-induced epigenetic switch. J. Cell Sci. 125: 3723-3731.

FLC expression is also regulated by
chromatin remodeling proteins
BAF60 is a SWI/SNF
subunit. By
modulation of histone
density, composition,
and posttranslational
modification, BAF60
activity creates a
repressive chromatin
configuration at the
FLC locus.
Reprinted from Jégu, T., Latrasse, D., Delarue, M., Hirt, H., Domenichini, S., Ariel, F., Crespi, M., Bergounioux, C., Raynaud, C. and Benhamed, M. (2014). The BAF60 subunit of
the SWI/SNF chromatin-remodeling complex directly controls the formation of a gene loop at FLOWERING LOCUS C in Arabidopsis. Plant Cell. 26: 538-551.

Summary: Mechanisms underlying
FLC activation and repression
Reprinted from Hepworth, J. and Dean, C. (2015). Flowering Locus C’s lessons: Conserved chromatin switches underpinning developmental timing and adaptation. Plant Physiol. 168: 1237-1245.

Natural variation of flowering time
can be correlated to FLC responses
Reprinted from Hepworth, J. and Dean, C. (2015). Flowering Locus C’s lessons: Conserved chromatin switches underpinning developmental timing and adaptation. Plant Physiol. 168: 1237-1245.

Epigenetic control of flowering time
• During vernalization, silencing of FLC allows expression
of FT and other flowering promoters
• VIN3 and PRC2 complex proteins epigenetically modify
FLC to silence it
• Maintaining the silenced state requires PRC1-like
complex which includes LHP1
• Trithorax protein SDG26 induces expression of flowering
activator SOC1 by methylating H3 Lys residues at SOC1
promoter
• Long non-coding RNAs contribute to epigenetic control of
gene expression

Many developmental genes and stress-
responsive are epigenetically regulated
Holec, S. and Berger, F. (2012). Polycomb group complexes mediate developmental transitions in plants. Plant Physiol. 158: 35-43.

Many developmental genes and switches are
epigenetically regulated
Normal fruit,
Abnormal fruit, epiallele
normal allele
Toadflax (Linaria vulgaris) flowers.

The peloric phenotype (right) is due to
gene silencing via DNA methylation of
CYCLOIDEA gene, leading to radial
flower symmetry. The peloric Abnormal development of oil palm fruit,
phenotype is heritable but occasionally leading to less oil production, occurs
reverts to wild type due to DNA when a transposon (Karma) within a
demethylation of CYC gene. floral homeotic gene is hypomethylated
Reprinted by permission from Macmillan Publishers Ltd from Cubas, P., Vincent, C., and Coen, E. (1999). An epigenetic mutation responsible for natural variation in floral
symmetry. Nature 401:157-158. Ong-Abdullah et al. (2015) Loss of Karma transposon methylation underlies the mantled somaclonal variant of oil palm. Nature 525: 533-537

Biotic or Abiotic Stress
Stress signalling
Hormones may or may not
involve hormones
Many stress-
responsive
genes are
epigenetically
regulated
The stress-responsive
phenotype is usually not
transmitted to progeny,
but chromatin changes
can be heritable
Reprinted from Gutzat, R. and Mittelsten Scheid, O. (2012). Epigenetic responses to stress: triple defense? Curr. Opin. Plant Biol. 15: 568-573, with permission from
Elsevier.

Stress can cause diverse epigenetic
changes including…
Unstressed
Transposon
activation
Stressed Gene activation

or silencing
Heterochromatin
decondensation
Reprinted from Probst, A.V. and Mittelsten Scheid, O. (2015). Stress-induced structural changes in plant chromatin. Curr. Opin. Plant Biol. 27: 8-
16.

Epigenetic responses can be
transient, primed or persistent
In some cases, stress-

induced chromatin
In some cases, such marks are retained after
as vernalization, stress, allowing a faster
chromatin marks and response to subsequent
gene expression stress. This is referred to
levels are both as priming
persistently altered
Reprinted from Eichten, S.R., Schmitz, R.J., and Springer, N.M. (2014). Epigenetics: Beyond chromatin modifications and complex genetic regulation. Plant Physiol. 165: 933-947.

Genomic imprinting
•The zygote receives two copies of

each gene, one from the mother’s
genome and one from the father’s.
•At most loci, both copies are active.

Genomic imprinting
•The zygote receives two copies of

each gene, one from the mother’s
genome and one from the father’s.
•At most loci, both copies are active.
•Some loci, imprinted loci, show a

“parent of origin effect”.
•Expression of these loci is

controlled by epigenetic factors.

Imprinting – nuclear transplant
experiment
Placing a sperm and an

egg nucleus into an
enucleated fertilized cell
leads to a normal embryo.

experiment
Zygotes that receive only

maternal or only paternal
nuclei do not survive.

experiment
The two parental Zygotes that receive only

genomes are not maternal or only paternal
equivalent nuclei do not survive.

Imprinting in angiosperms
Angiosperms show genomic imprinting

too, but their reproduction is a bit more
complicated than that of animals.....

Angiosperm reproduction
Meiosis Microspore
n Mitosis
Multicellular
haploid
n
gametophytes
Megaspore
Differentiation of
egg and sperm
Mitosis 2n
Fertilization
Single-celled
Sporophyte diploid zygote

Development of male and female
gametophyte
Vegetative nucleus
meiosis mitosis
2n n
Generative cell
Microspore Microspore 2-celled male
mother cell gametophyte
(aka pollen grain)
meiosis mitosis Central cell

2n n
Megaspore Megaspore
mother cell Egg cell
8-celled female
gametophyte
(aka embryo sac)

Double Fertilization
Mitotic division
of generative
cell to two
sperm cells
3n endosperm
Pollen tube
One sperm nucleus growth
fertilizes the egg cell to
produce diploid embryo.
The second sperm

nucleus fertilizes the
polar nuclei to produce a
triploid endosperm.
2n zygote
The MEDEA (MEA) gene is
imprinted
MEA/mea x MEA/MEA
All seeds viable
MEA/MEA x MEA/mea
50% of seeds abort
From: Grossniklaus, U., Vielle-Calzada, J.-P., Hoeppner, M.A., Gagliano, W.B. (1998) Maternal control of embryogenesis by
MEDEA, a Polycomb Group gene in Arabidopsis. Science 280: 446-450. Reprinted with permission from AAAS.

imprinted
MEA/mea x MEA/MEA
All seeds viable
MEA/MEA x MEA/mea
50% of seeds abort
In the second cross, 50% of the seeds receive the mutant mea allele
from their mother.
These seed abort, even though they also have a wild-type MEA allele
inherited from their father.
From: Grossniklaus, U., Vielle-Calzada, J.-P., Hoeppner, M.A., Gagliano, W.B. (1998) Maternal control of embryogenesis by
MEDEA, a Polycomb Group gene in Arabidopsis. Science 280: 446-450. Reprinted with permission from AAAS.

imprinted
MEA/mea x MEA/MEA MEA MEA

x
All seeds viable mea MEA
MEA/MEA x MEA/mea MEA MEA
50% of seeds abort MEA mea
The paternal allele is silent.

The phenotype of the
progeny is based on the
maternal genotype only.

MEA is silenced in most cells
MET1
The MEA allele inherited
from the father is silent.
MEA
MEA is
methylated by
MET1 in
vegetative tissues
and the male
gametophyte
MET1
MEA
MEA

MEA is silenced in most cells
MET1
MEA
MEA is MEA is demethylated and expressed

methylated by in the female gametophyte.
MET1 in
vegetative tissues
and the male
MET1
gametophyte
MET1 DME1 MEA

MEA
MEA
MEA

MEA regulates its own imprinting
•MEA encodes a component of PRC2.
•In the triploid endosperm, continued
silencing of the parental allele requires
histone modification by PRC2.
MEA PRC2
MEA
MEA
MEDEA

Summary of parental imprinting (1)
In females, the central cell DME removes DNA methylation from maternally
expressed genes, MEA and FIS2, and from the paternally expressed gene
PHE1. DNA methylation at these loci is maintained in the male gametophyte.
Reprinted from Iwasaki, M., and Paszkowski, J. (2014). Epigenetic memory in plants. EMBO J. 33: 1987-1998.

Summary of parental imprinting (2)
In endosperm, maternal alleles of MEA and FIS2 are expressed. The PRC2 complex mediates
silencing of the maternal allele of PHE. In addition to the PRC2 complex, maternal removal of
DNA methylation downstream of PHE gene is required for silencing of its maternal allele.
Reprinted from Iwasaki, M., and Paszkowski, J. (2014). Epigenetic memory in plants. EMBO J. 33: 1987-1998.

Silencing in trans: silencing of FWA
OFF ON
FWA
FWA is another imprinted gene. It is expressed

in the female gametophyte, like MEA.

FWA
fwa-1
The fwa-1 epiallele is hypomethylated and

expressed in vegetative tissues.

FWA
Using Agrobacterium tumefaciens-

mediated transformation, an
unmethylated FWA gene is inserted into
the plant chromosome.

Wild-type plant
FWA allele In plants with the
wild-type FWA
allele, the trans
trans allele allele is silenced.
fwa plant
fwa-1 allele In plants with the
fwa-1 allele, the
trans allele is
trans allele active.

Wild-type plant
In plants with the
FWA allele wild-type FWA
allele, methylation
trans allele is maintained by
DRM2 siRNA and DRM2,
which is capable of
also methylating the
fwa plant
FWA gene
fwa-1 allele introduced in trans.
trans allele

A FWA gene
with one repeat
deleted does not
promote DNA
methylation.
Chan, S.W.-L., Zhang, X., Bernatavichute, Y.V., and Jacobsen, S.E.(2006) Two-step recruitment of RNA-directed DNA
methylation to tandem repeats. PLoS Biol. 4: e363.

DRM2
The tandem repeat

upstream of the A FWA gene
FWA gene with one repeat
promotes siRNA deleted does not
production and is promote DNA
necessary for methylation.
methylation and
silencing.

DRM2
The tandem repeat Tandem repeats are common

upstream of the features of transposons and repetitive
FWA gene elements. The presence of the
promotes siRNA tandem repeats triggers the
production and is epigenetic mechanism that plants use
necessary for to silence potentially “foreign” DNA.
methylation and
silencing.

Silencing in trans: paramutation
Plants with an active

B-I allele B-1 allele have a lot
(highly active) of purple anthocyanin
pigmentation.
Paramutation: The interaction of two alleles of the same

locus, resulting in a heritable change of one allele that is
induced by the other allele. The maize b1 locus has a
paramutagenic allele.

B-I allele
(highly active)
The B' allele is

B' allele weakly active, and
(weakly active) plants are pale.

B-I allele
(highly active)
The B' allele is

heavily methylated.
B' allele
(weakly active)

X
B-I / B-1 B' / B'
What is the expected

phenotype of the
progeny of a cross
between these two
homozygous parents?

X
B-I / B-1 B' / B'
The F1 should be purple,

because the B-1 allele is
dominant.

X
B-I / B-1 B' / B'
Instead, the B' allele

paramutates B-1 to B', and
the plant is pale.

This system provides a

straightforward genetic
screen for mutations that
Paramutation interfere with paramutation
in maize. Such studies
have shown that siRNA
acting in trans is the basis
for paramutation.

An RNA-dependent DNA methylation
working model for paramutation
A transcript produced by
RNA Pol IV is copied by
RNA-dependent RNA
polymerase (RDR2)
A dicer-like protein produces

small RNAs, which, in
conjunction with AGO,
associate with Pol V nascent
RNAs, recruit a
methyltransferase (DRM2)
and lead to DNA methylation
Reprinted from Giacopelli, B.J. and Hollick, J.B. (2015). Trans-homolog interactions facilitating paramutation in maize. Plant Physiol. 168: 1226-1236.

Resetting the epigenome (or not…)
FLC ON FLC OFF

When does FLC

switch on again
between
generations?

In animals, imprinted genes are
reset during gametogenesis
Paternal allele silent Maternal allele active
Zygote and somatic cells
Both alleles
Both alleles
silenced during
activated during
spermatogenesis
oogenesis

Epigenetic reprogramming in plants
Large-scale epigenetic changes have been

observed in the endosperm and in the
vegetative nucleus of the male gametophyte.
Vegetative nucleus
Generative cell
3n endosperm

The heterochromatin fraction is
reduced in endosperm
EMBRYO NUCLEI ENDOSPERM NUCLEI
H3K9me
H3K27me2
DNA Histone Merged DNA Histone Merged

mark mark
In endosperm nuclei, heterochromatin marks such as H3K9me and K3K27me2

disperse into euchromatin, suggesting genome-wide epigenetic changes.
Baroux, C., Pecinka, A., Fuchs, J., Schubert, I., and Grossniklaus, U. (2007) The triploid endosperm genome of
Arabidopsis adopts a peculiar, parental-dosage-dependent chromatin organization. Plant Cell 19: 1782-1794.

DNA in endosperm is demethylated as
compared to embryo
Embryo
GENES Demethylation
Endosperm
Embryo
TRANSPOSONS
Endosperm
Demethylation
From Hsieh, T.-F., Christian A. Ibarra, C.A., Silva, P., Zemach, A., Eshed-Williams, L., Fischer, R.L., and Zilberman, D. (2009)
Genome-wide demethylation of Arabidopsis endosperm. Science 324: 1451-1454. Reprinted with permission from AAAS.

Transposons in pollen vegetative
cells are hypomethylated
Purified sperm cells
Filled boxes
indicate cytosine
methylation
Total pollen
Hypomethylation
Reprinted from Slotkin, R.K., Vaughn, M., Borges, F., Tanurdžić, M., Becker, J.D., Feijó, J.A., and Martienssen, R.A. (2009) Epigenetic
reprogramming and small RNA silencing of transposable elements in pollen. Cell 136: 461-472. Copyright 2009, with permission from Elsevier.

Transposons are specifically
activated in pollen
POLLEN RNA
Rows indicate
transcript levels for
different transposons.
Columns are different

tissue types.
Green indicates high

level of expression.
Reprinted from Slotkin, R.K., Vaughn, M., Borges, F., Tanurdžić, M., Becker, J.D., Feijó, J.A., and Martienssen, R.A. (2009) Epigenetic
reprogramming and small RNA silencing of transposable elements in pollen. Cell 136: 461-472. Copyright 2009, with permission from Elsevier.

Resetting of epigenetic marks
during development
• These results suggest
that resetting epigenetic
marks occurs during
angiosperm reproduction
• Activation of silenced
genes can activate RNA-
mediated DNA
methylation pathways

Resetting of epigenetic marks in
non-germ cells?
siRNAs generated by this

Vegetative
nucleus process could move into the
generative tissues to ensure
proper epigenetic
Sperm cells
reprogramming, without the
mutagenic potential of
activating transposons in the
germ cells themselves.
Redrawn from Slotkin, R.K., Vaughn, M., Borges, F., Tanurdžić, M., Becker, J.D., Feijó, J.A., and Martienssen, R.A.
(2009) Epigenetic reprogramming and small RNA silencing of transposable elements in pollen. Cell 136: 461-472.

Summary
• Expression of DNA is controlled by epigenetic marks
including DNA methylation and histone modifications.
• siRNAs contribute to epigenetic programming
• Epigenetic programming silences transposons and
controls the timing of many genes that control plant
development and stress responses.

Ongoing research
How can an
understanding of
epigenetics lead to
plants primed to better
withstand stress?
How can we use

epigenetic insights to
affect plant responses to
the environment, and
their ability to adapt to
How can an understanding of climate change?
epigenetics contribute to
increasing plant productivity, for How does clonal
example by fixing the vigor found propagation affect
in hybrids? chromatin marks
and plant
phenotypes?
Reprinted from Greaves, I.K., Gonzalez-Bayon, R., Wang, L., Zhu, A., Liu, P.-C., Groszmann, M., Peacock, W.J. and Dennis, E.S. (2015). Epigenetic Changes in
Hybrids. Plant Physiology. 168: 1197-1205; Eichten, S.R., Schmitz, R.J., and Springer, N.M. (2014). Epigenetics: Beyond chromatin modifications and complex genetic
regulation. Plant Physiol. 165: 933-947. Ong-Abdullah et al. (2015) Loss of Karma transposon methylation underlies the mantled somaclonal variant of oil palm. Nature
525: 533-537

Epigenetics: Any Potentially Stable and Heritable Change in Gene Expression That Occurs Without A Change in DNA Sequence

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Epigenetics: Any Potentially Stable and Heritable Change in Gene Expression That Occurs Without A Change in DNA Sequence

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Epigenetics

Any potentially stable and

© 2016 American Society of Plant Biologists

Usually this means

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© 2016 American Society of Plant Biologists

In female mammals, one copy of

Fur color in cats is determined in part by

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Embryonic Vegetative Reproductive

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© 2016 American Society of Plant Biologists

© 2016 American Society of Plant Biologists

Approximately 147 base

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DNA around the Less densely

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Euchromatin DAPI (DNA stain) Merged

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© 2016 American Society of Plant Biologists

© 2016 American Society of Plant Biologists

© 2016 American Society of Plant Biologists

© 2016 American Society of Plant Biologists

© 2016 American Society of Plant Biologists

© 2016 American Society of Plant Biologists

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© 2016 American Society of Plant Biologists

GREEN = DNA methylation is

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CMT2 and DRM2

are required for

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In plants, the DNA of introns and TEs

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Arabidopsis has a family

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Switching off DML activity interferes

A triple mutant (ros1 dml2 dml3)

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© 2016 American Society of Plant Biologists

© 2016 American Society of Plant Biologists

© 2016 American Society of Plant Biologists

Lysine can be acetylated, or mono-, di-, or tri-methylated

Lysine (K) + CH3

Acetylated lysine Tri (Kme3) N CH3

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H3K4me is associated with actively

Red = high correlation

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BLUE = Gene H3K27me3 in

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Active genes Silenced genes Polycomb repressive

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Enhancer of zeste (E(Z))

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E(Z) ESC SU(Z)12 NURF55

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MEA + FIS2 complex

CLF/SWN + VRN2 complex

CLF/SWN + EMF2 complex