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215

Genetic approaches to understanding the role of epigenetic


regulation of gene expression in plants and its mechanisms
have revealed several new components. Rapidly accumulating
information from other eukaryotes provides complementary
knowledge with important implications for plant research.
Comparison of epigenetic events across species is proving
critical for defining the mechanisms and functions of epigenetic
modification, including those specific to plants.
Addresses
*

Friedrich Miescher Institute, Novartis Research Foundation,


Maulbeerstrasse 66, 4058 Basel, Switzerland

National Institute of Genetics, Mishima, Shizuoka 411-8540, Japan;


e-mail: tkakutan@lab.nig.ac.jp
*e-mail: habu@fmi.ch

e-mail: paszkowski@fmi.ch
Current Opinion in Genetics & Development 2001, 11:215220
0959-437X/01/$ see front matter
2001 Elsevier Science Ltd. All rights reserved.
Abbreviations
AG AGAMOUS
ATRX X-linked alpha thalassaemia
CAF chromatin assembly factor
CHD chromodomain-helicase-DNA-binding domain
CLF CURLY LEAF
CMT chromomethylase
crc crabs claw
DDM decreased DNA methylation
Dnmt1 DNA methyltransferase 1
FI E FERTILIZATION-INDEPENDENT ENDOSPERM
FWA FLOWERING LOCUS WA
HDAC histone deacetylase
Lcyc Linaria vulgaris homologue of cycloidea
MET methyltransferase
MOM Morpheus molecule
PAI phosphoribosylanthranilate isomerase
PcG Polycomb group
SNF sucrose non-fermenting
SRP signal recognition particle
SUP SUPERMAN
SWI switching mating type
TGS transcriptional gene silencing
TSI transcriptionally silent information
Introduction
Gene function can be stably modified by epigenetic compo-
nents that can either silence or superactivate selected DNA
templates. Over the years, numerous functions and mecha-
nisms of epigenetic regulation in plants have been
proposed. The recent isolation of Arabidopsis mutants
impaired in epigenetic gene silencing and the cloning of the
affected genes has extended our understanding of the uni-
versal mechanisms of epigenetic control significantly. Both
the structures and suggested functions of these genes imply
novel links between chromatin components, DNA methyla-
tion and transcription. Here we review the molecular
characteristics of recently identified plant genes involved in
transcriptional gene silencing (TGS), DNA methylation and
other chromatin-associated activities for which in vivo func-
tions have been assayed. We also review their target
templates, which include genes controlling plant develop-
ment. Plant genomic imprinting, epigenetic components of
the vernalization process and nucleolar dominance have
been reviewed recently and are not discussed here [13].
The cloning and characterization of several plant DNA
methyltransferases have also been reviewed adequately
elsewhere [4

].
Proteins required for transcriptional gene
silencing
Although several mechanisms for TGS have been suggest-
ed [5,6], only a few protein components have been
identified to date.
Decreased DNA methylation 1
The Decreased DNA methylation 1 (DDM1) gene, which
encodes a member of the SWI2/SNF2 protein family
involved in chromatin remodeling [7

], was one of the first


genes shown to be required for maintenance of TGS [8,9].
Mutations in DDM1 cause a genome-wide hypomethyla-
tion and developmental abnormalities that accumulate
over generations in plants [10,11]. Surprisingly, in addition
to reduced genome-wide methylation in general, in partic-
ular in repetitive DNA, mutation of the DDM1 gene also
causes increased methylation and transcriptional inactiva-
tion of selected single-copy genes (see below). Thus, it
seems that DDM1 in addition to its role in maintenance of
TGS and proper levels of genome-wide methylation also
functions in the correct distribution of methylation within
chromosomal regions containing active genes. An interest-
ing similarity to DDM1 function was noticed during
characterization of mutations in the human ATRX gene,
which encodes an SWI2/SNF2-like protein. These muta-
tions also caused changes in patterns of DNA methylation
[12

]. Thus, it is likely that a subset of chromatin-remod-


eling factors controls both the level and the allocation of
DNA methylation in both plants and vertebrates.
Morpheus molecule 1
A novel, recently described protein required for TGS is
Morpheus molecule 1 (MOM1) [13

]. In contrast to ddm1,
the mom1 mutant maintains dense DNA methylation, char-
acteristic of silent loci, also after transcriptional activation.
It is, therefore, possible that MOM1 functions downstream
or independently of methylation signals. The downstream
function may be the recognition of methylation. The mom1
mutant has no morphological abnormalities even after
inbreeding for many generations, suggesting that MOM1
Epigenetic developmental mechanisms in plants: molecules and
targets of plant epigenetic regulation
Yoshiki Habu*, Tetsuji Kakutani

and Jerzy Paszkowski

epigenetically regulates only a narrow subset of genetic


information or that a mom1 mutation produces develop-
mental phenotypes over a much longer time scale than
ddm1. The MOM1 gene encodes a novel protein of
2001 amino acids whose middle section has significant sim-
ilarity only to the second half of an SWI2/SNF2-like
ATPase/helicase domain [13

]. This domain is present in


diverse proteins involved in chromatin remodeling and
DNA repair. This unusual half-helicase structure of the
MOM1 protein implies an interesting function, namely that
the protein exerts ATPase/helicase activity as a het-
erodimer with an as yet unknown protein which provides
the missing half of the ATPase/helicase domain. Such a
heterodimer of ATPases/helicases has not yet been report-
ed, however, and the function of the MOM1 protein
remains the subject of speculation.
Methyltransferase 1
The Arabidopsis Methyltransferase 1 (MET1) multigene fami-
ly comprises several members, with MET1 encoding a
predominant methyltransferase similar to mouse DNA
methyltransferase 1 (Dnmt1) (reviewed in [4

]). Expression
of MET1 antisense RNA results in release of TGS [14

].
Morphological and developmental changes in antisense
MET1 plants also indicate the involvement of MET1 in
global gene regulation, similar to that of DDM1 and histone
deacetylase 1 (HDAC1) (see below).
Histone deacetylase 1
Local levels of histone acetylation are inversely correlated
with the degree of chromatin compaction and with TGS.
Inhibition of HDAC1 gene expression by transgenic produc-
tion of its antisense RNA releases silencing in Arabidopsis
[15]. Interestingly, although TGS is released by the inhibi-
tion of HDAC1 without changes in DNA methylation,
transgenic plants show developmental abnormalities, some
of which resemble the ddm1-induced phenotypes [15].
Although in organisms other than plants, only a single type
of HDAC has been characterized to date, maize has at least
three different types of HDAC, each with a distinct sub-
strate specificity [16]. It will be interesting to determine the
involvement of each type of HDAC in well characterized
epigenetic processes, such as paramutation in maize, possi-
bly in conjunction with histone acetyltransferase, which was
also described recently in this plant [17].
Plant genes encoding chromatin components
In plants, several mutations initially identified through
developmental alterations have been revealed to be in
genes encoding chromatin components.
Chromodomain-helicase-DNA-binding domain 3/ Mi-2
In mammals, members of the Chromodomain-helicase-
DNA-binding domain 3 (CHD3)/Mi-2 protein family are
components of NuRD transcriptional repression complexes
[18]. The gene PICKLE/GYMNOS encodes an Arabidopsis
homologue of CHD3/Mi-2 [1921]. Various developmental
effects have been assigned to different mutations in this gene.
For example, mutants described as pkl show derepression of
a transcription factor implicated in embryo development. gym
mutants show enhancement of the effect of the crabs claw (crc)
mutation on the polarity of carpels, probably as a result of
incorrect temporal regulation of downstream targets.
Considering that the pkl/gym mutations modulate penetrance
of the primary mutations in genes influencing complex devel-
opmental decisions, it can be envisaged that plant
CHD3/Mi-2 orthologues are indeed in part similar to NuRD
protein complexes influencing plant chromatin structure.
Chromatin assembly factor 1
Chromatin assembly factor 1 (CAF-1) is a protein complex
required for inheritance of epigenetic chromosomal states
and for the assembly of chromatin during DNA replication.
Two components of CAF-1 in Arabidopsis have been identi-
fied in a genetic screening for the maintenance of shoot and
root apical meristems (T Araki, personal communication).
Distorted morphological phenotypes of the mutants suggest
that the plant CAF-1 plays a critical role in the establishment
of meristem organization, possibly by facilitating stable
inheritance of epigenetic states during DNA replication.
Chromodomain proteins
A chromodomain is a highly conserved amino acid
sequence motif which was originally identified as an
homologous region in a heterochromatin protein HP1 and
Polycomb protein of Drosophila. Although several plant pro-
teins carrying chromodomains have been characterized so
far, a subset of proteins carrying both chromodomain and
methyltransferase domains is unique to plants (reviewed in
[4

]). The recent finding that chromodomains bind to


RNA [22

] presents the interesting idea that plant chromo-


methylases (CMTs) are involved in RNA-directed DNA
methylation [23,24

,25]. A further Arabidopsis chromo-


domain protein, CHAOS, which is a component of a
chloroplast signal recognition particle (SRP) system, has
been identified recently [26]. Both prokaryotes and
eukaryotes possess a cytoplasmic SRP containing a 54 kDa
protein (SRP54) and an RNA molecule but the homologue
of SRP54 in chloroplasts was found to bind CHAOS rather
than RNA. Taken together with the RNA-binding activity
of chromodomains, this observation leads to the interesting
suggestion that CHAOS mediates interaction between
RNA and SRP54 through its chromodomain.
Polycomb group proteins
PcG proteins form a repressive chromatin structure mediating
gene silencing of homeotic genes in Drosophila. Several plant
proteins with homology to PcG proteins have been identified
by screening for developmental mutants [2731]. MEDEA
and CURLY LEAF (CLF) have the SET domain and FER-
TILIZATION-INDEPENDENT ENDOSPERM (FIE)
has the WD repeat, both characteristic of PcG proteins in
Drosophila. Recently, several members of the SU(VAR)39
family of mammalian and Drosophila proteins were shown to
have histone H3 methyltransferase activity [32

]. The SET
domain in conjunction with two cysteine-rich domains at both
216 Chromosomes and expression mechanisms
sides of the SET domain are required for this activity.
Although the Drosophila protein Enhancer of zeste has a similar
structure, it lacks the carboxy-terminal cysteine-rich region
and has no histone H3 methyltransferase activity [32

]. As
MEDEA and CLF have the same structure as Enhancer of
zeste [27,28], it is not likely that MEDEA and CLF are histone
H3 methyltransferases.
Endogenous targets of transcriptional gene
silencing
Using various methods for RNA analysis of mutants
impaired in gene silencing, several chromosomal templates
silenced in the wild type but reactivated in mutants impaired
in TGS have been characterized. In a directed screen for
activation of LTR (long terminal repeat) type endogenous
Arabidopsis retroelements in the ddm1 mutant, it was shown
that a particular class of the retroelement designated Tar17
was transcriptionally activated [33

]. In addition, another
class of endogenous Arabidopsis elements similar to
Robertsons Mutator of maize started transcription and was
able to transpose in the ddm1 background [34].
Applying an unbiased screen for transcripts present in the
mom1 mutant and absent in the wild type, a set of tem-
plates termed transcriptionally silent information (TSI)
which is transcribed only in the mutant background has
been identified. TSI RNAs are produced not only in mom1
but in all TGS mutants known at present [14

]. They are
encoded by pericentromeric repeated elements that have
no protein-coding capacity. TSI shares sequence homology
with the 3 terminal part of a putative retrotransposon
Athila. Not all endogenous targets of TGS are transposon-
like or highly repeated pericentromeric DNA sequences.
The phosphoribosylanthranilate isomerase (PAI) gene
family, which encodes enzymes of the tryptophan biosyn-
thetic pathway, is methylated and silenced in ecotypes
having an inverted repeat of a PAI region [3537]. These
silent PAI genes are also transcriptionally reactivated by
the ddm1 mutation [8].
Epigenetic silencing of genes controlling
plant development
Both ddm1 and antisense MET1 exhibit a variety of devel-
opmental abnormalities, probably as a result of the
unscheduled expression or repression of genes regulating
developmental decisions [11,3840]. Heritable epigenetic
changes in expression of these genes may account for at
least some of the abnormalities found in ddm1 and anti-
sense MET1 lines. Destruction of genomic methylation
patterns and epigenetic silencing are often stably inherited
through many generations [41], enabling identification of
epigenetically controlled target genes by conventional
linkage analysis, followed by positional cloning. To date,
epigenetic silencing correlated with DNA methylation
changes have been documented for four genes involved in
plant development namely, FLOWERING LOCUS WA
(FWA), SUPERMAN (SUP), AGAMOUS (AG) and Linaria
vulgaris homologue of cycloidea (Lcyc).
FLOWERING LOCUS WA
Late-flowering traits are frequently observed in ddm1
inbred lines and in lines expressing MET1 antisense RNA
[11,40]. Such an acquired phenotype is later inherited as a
monogenic dominant trait mapped to a chromosomal region
containing FWA one of the flowering-time loci identified
by conventional mutagenesis and linkage analysis [39]. Of
the many Arabidopsis mutations affecting flowering time,
mutations in FWA are unique in that there are two different
mutant alleles induced by EMS or irradiation which are
semi-dominant [42]. Mutagens usually lead to recessive,
loss-of-function mutations. The semi-dominance suggests
that fwa are gain-of-function mutations.
FWA was isolated by a map-based approach [43

].
Surprisingly, the DNA sequences of the wild type and the
two mutant alleles are identical; however, FWA is expressed
in mutant tissues but silenced in wild-type plants. The
silencing in wild type is associated with extensive methyla-
tion of two direct repeat sequences in the 5 region of the
gene. The ectopic FWA expression and the hypomethyla-
tion of the FWA repeats were also observed in the
late-flowering ddm1 lines. It is still not clear how irradiation
or EMS mutagenesis contributed to the formation of epige-
netic alleles of FWA but the observations support the
conclusion that the ddm1-induced late-flowering pheno-
type is the result of heritable epigenetic alterations in FWA.
SUPERMAN and AGAMOUS
Heritable epigenetic alleles affecting development were
first found in the SUP gene in Arabidopsis [44]. A mutation
in SUP leads to the growth of extra stamens by affecting
floral whorl boundaries [45]. Seven heritable but semi-sta-
ble alleles of the sup mutation were found which had no
change in nucleotide sequence at the locus but which
turned out to be heavily methylated, silent forms of the
gene [44]. Moreover, counter-intuitively, formation of
hypermethylated and transcriptionally repressed epi-alleles
of SUP occur more frequently in antisense MET1, ddm1,
and ddm2 (encoding MET1) strains with overall genome-
wide hypomethylation [43

,46

]. Similarly, transcriptional
repression of AG and hypermethylation of its intron are
observed in antisense MET1 plants, explaining their ag
phenotype [46

]. The mechanism for the local hyperme-


thylation is unknown but specific DNA secondary
structures may play a role because the hypermethylated
regions of SUP and AG contain pyrimidine-rich sequences
[46

]. As in the case of FWA, it is not clear how the muta-


genesis treatment contributed to the frequent formation of
the SUP epi-alleles. It could be that this reflects temporal
release of tight epigenetic control as a result of genotoxic
stress imposed on the genome during mutagen treatment.
Linaria vulgaris homologue of cycloidea
Developmental variants based on epigenetic modifications
may be more common in natural plant populations than
was previously anticipated. Cubas et al. [47

] find that vari-


ation in flower pattern present in natural populations of
Epigenetic developmental mechanisms in plants Habu, Kakutani and Paszkowski 217
Linaria vulgaris is caused by epigenetic inactivation of Lcyc,
which controls the flower bilateral pattern. Naturally
occurring flower variants with radial symmetry were found
to have a methylated and transcriptionally silent Lcyc gene.
The degree of phenotypic penetrance observed in occa-
sional revertants could be correlated with the degree of
Lcyc methylation and the level of its transcription. This
suggests that variation introduced by multiple epigenetic
alleles plays a significant role in the creation of natural vari-
ation required for evolution of plant populations in nature.
Multiple roles of transcriptional gene silencing
TGS involvement in defense against genomic parasites such
as transposons has been discussed repeatedly, but this may
not be the only role of TGS. Genetic interference with gene
silencing induces a great variety of developmental changes in
plants. Some of these abnormalities are most likely caused by
the disturbed epigenetic regulation of genes responsible for
developmental decisions, such as SUP, AG or FWA. Others
could be caused by the reactivation of transposons, resulting
in genetic changes based on transposon-induced genome
rearrangements (T Kakutani, unpublished data). It will be
fascinating to see how epigenetic mechanisms controlling
plant development overlap with other processes controlling
genome stability, such as transposon movement or regulation
of recombination levels between chromosomal repeats [48].
As the latter mechanisms appear to be more widespread, it is
possible that epigenetic control of chromosomal gene
expression evolved via control over repeated elements. In
fact, mammalian imprinted genes often have direct-repeat
clusters [49] and, similarly, targets of methylation and silenc-
ing are associated with unusual repeats in the case of both
FWA and SUP.
In any case, it is remarkable that non-allelic mutations
interfering with TGS were isolated in rather limited
mutant screenings [9,10,13

]. This suggests that many


silencing components are encoded by non-redundant
genes. Furthermore, in contrast to mammals, even loss-of-
function mutations in such genes, leading for example to
drastic reduction in chromosomal methylation, are found
in viable and fertile plants (at least in early generations).
This provides the possibility for their propagation and the
propagation of newly created epi-alleles in the mutant
backgrounds. Although individual plants may suffer from
progressive accumulation of epimutations in terms of their
development and fitness, the release of epigenetic control
in plant populations provides an attractive opportunity for
the rapid generation of multiple combinations of heritable
epi-alleles that can be subjected to selection. Thus, the
down-regulation or mutation of genes involved in epige-
netic control may be a powerful natural tool for the
efficient creation of novel epigenetic variants.
Conclusions
Recent major breakthroughs in understanding the molecular
mechanisms of TGS in plants were clearly linked to the
identification of novel molecules involved in the process. In
addition, an increasing number of plant chromatin proteins are
being annotated in databases (refer to the following web site:
The Plant Chromatin Database, http://ag.arizona.edu/chro-
matin/chromatin.html). Although, it appears that a similar set
of chromatin components is involved in epigenetic regulation
in both animals and plants, they contribute differently to
animal and plant physiology.
Compared with mammals, the forward genetic system in
plants is clearly more advanced but the biochemical analy-
sis of silencing mechanisms in mammals has progressed
much further than in plants, where it is only now really
beginning. The application of biochemistry with genomics
and other new technologies (e.g. bio-imaging) to studies of
plant epigenetics may provide crucial paradigms stimulat-
ing further research. Recently identified plant chromatin
components involved in TGS should now provide a focal
point for these types of studies. It will be important to
decipher epigenetic pathways and their functions by link-
ing the activities of common or diverse chromatin
elements with plant- or animal-specific functions in epige-
netic control. It is hoped that this will provide a clearer
picture of the mechanisms of TGS and its physiological
role. The functions of TGS may turn out to be broad,
reaching not only developmental control but also the gen-
eration of epigenetic variability and the transcriptional
control of repeats, including transposable elements.
Update
The work referred to as T Araki, personal communication,
has been published [50].
Acknowledgements
Y Habu and J Paszkowski are supported by Novartis Research Foundation.
T Kakutani is supported by CREST, Japan Science and Technology
Corporation. The authors thank J Chen, T Araki and R Martienssen for
providing unpublished data. We also thank O Mittelsten Scheid and P King
for critical reading of the manuscript.
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220 Chromosomes and expression mechanisms