1 Institute of Molecular Plant Science, School of Biology, University of
Edinburgh, Mayfield Road, Edinburgh EH9 3JH, UK
2 Department of Plant and Microbial Biology, University of California, Berkeley,
CA 94720, USA
Author for correspondence (e-mail:
justin.goodrich{at}ed.ac.uk)
Accepted 16 August 2004
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SUMMARY |
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Key words: Polycomb, Flowering, VEFS domain
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Introduction |
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In Drosophila and other animals, the epigenetic control of
developmental patterning is mediated by members of the Polycomb group (Pc-G)
and trithorax group (trx-G) of genes (for a review, see
Francis and Kingston, 2001). A
general feature of these genes is that they are required not for pattern
initiation, but rather to ensure that the transcriptional output of early
patterning events is stably inherited through somatic development. The two
groups act antagonistically, so that the Pc-G genes are required for
maintenance of transcriptional repression and the trx-G genes for maintenance
of transcriptional activation. Recently, biochemical characterisation of their
protein products has provided some mechanistic insight. The Pc-G products,
which are structurally disparate from one another, are found in at least two
distinct complexes, termed the Polycomb Repressive Complex 1 and 2 (PRC1 and
PRC2) (Cao et al., 2002
;
Czermin et al., 2002
;
Francis et al., 2001
;
Kuzmichev et al., 2002
;
Muller et al., 2002
;
Saurin et al., 2001
).
Consistent with the epigenetic function of Pc-G proteins, the PRC2 was
recently shown to modify chromatin. Thus, several groups have shown that the
PRC2 has a histone methyltransferase (HMTase) activity, methylating specific
residues (lysine 9 and lysine 27) on the N-tail of histone H3
(Cao et al., 2002
;
Czermin et al., 2002
;
Kuzmichev et al., 2002
;
Muller et al., 2002
). The
precise biochemical function of the different PRC2 members is not well
defined, with the exception of one member, the Enhancer of zeste [E(z)]
protein, which has been shown to confer HMTase activity via a conserved motif,
the SET domain. Unlike most other SET domain proteins, E(z) itself does not
show HMTase activity in vitro unless associated with other members of the PRC2
complex (Czermin et al.,
2002
). The mK9 H3 and mK27 H3 modifications catalysed by the PRC2
are associated with the repression of transcription, although how they are
interpreted and inherited through mitosis is not well understood. The Polycomb
protein appears to recognise and bind mK27 H3, and may recruit other members
of the PRC1 complex, which probably have roles in mediating transcriptional
silencing and its propagation through mitosis
(Cao et al., 2002
;
Czermin et al., 2002
;
Francis et al., 2001
).
The PRC2 components are also found in plants, and were identified
independently through genetic screens in Arabidopsis aimed at
dissecting various developmental pathways. Thus, the FERTILISATION
INDEPENDENT SEED (FIS) genes were mostly identified through
screens for mutants that showed some aspects of seed development in the
absence of fertilisation (Chaudhury et al.,
1997; Grossniklaus et al.,
1998
; Guitton et al.,
2004
; Ohad et al.,
1996
). Currently four FIS genes have been identified:
MEDEA (MEA), FERTILISATION-INDEPENDENT SEED2 (FIS2),
FERTILISATION-INDEPENDENT ENDOSPERM (FIE) and MULTICOPY
SUPPRESSOR OF IRA 1 (MSI1). These encode products with homology to the
Drosophila PRC2 proteins E(z), Suppressor of zeste 12 [Su(z)12],
Extra sex combs (Esc) and P55, respectively
(Grossniklaus et al., 1998
;
Kiyosue et al., 1999
;
Kohler et al., 2003a
;
Luo et al., 1999
;
Ohad et al., 1999
). The
FIS genes repress expression of the MADS box gene PHERES1
(PHE1) during early seed development, and presumably affect many
other as yet unidentified target genes
(Kohler et al., 2003b
). A
second group have been identified based on a common function in repressing
floral homeotic gene expression. Mutants in this class are early flowering and
exhibit mild homeotic transformations in flowers. The first two members
identified were CURLY LEAF (CLF) and EMBRYONIC
FLOWER2 (EMF2), which encode proteins with homology to E(z) and
Su(z)12, respectively (Goodrich et al.,
1997
; Yoshida et al.,
2001
). Recently, the FIS genes MSI1 and
FIE have also been implicated in repressing flowering homeotic genes
during vegetative development. Because mutant alleles of the FIS
genes all cause early embryo lethality when inherited maternally, this
obstructed the phenotypic analysis of fis homozygotes during later
developmental stages. However, studies of transgenic lines that confer a
partial loss of FIS gene activity have revealed roles for
FIE and MSI1 beyond seed development
(Hennig et al., 2003
;
Katz et al., 2004
;
Kinoshita et al., 2001
). A
third class of Arabidopsis Pc-G genes was identified on the basis of
the function of the genes in the epigenetic memory of vernalisation. In
Arabidopsis, as with many other plant species originating from
temperate latitudes, flowering is accelerated if plants are first vernalised
by growing for 3-6 weeks at low temperatures (4-10°C). The vernalisation
response has several epigenetic features, including stability during somatic
development and resetting from generation to generation. Recent studies
indicate that the underlying basis for the response involves transcriptional
repression of FLC, a gene that itself represses flowering
(Michaels and Amasino, 1999
;
Sheldon et al., 1999
). The
VERNALIZATION2 (VRN2) gene is required so that the
cold-induced repression of FLC is mitotically stable during later
periods of growth at warm temperatures
(Gendall et al., 2001
).
VRN2, like FIS2 and EMF2, encodes a protein with
homology to Drosophila Su(z)12
(Gendall et al., 2001
). The
completion of the Arabidopsis genome sequence revealed that these
comprised most of the Arabidopsis homologues of the core members of
the PRC2. One exception was a third E(z) homologue, GenBank accession
At4g02020, that had not been characterised genetically. In addition, there are
four genes with weak similarity to MSI1, MSI2-5, with poorly defined
functions (Ach et al., 1997
;
Hennig et al., 2003
;
Kenzior and Folk, 1998
). Thus,
unlike Drosophila, in which the PRC2 members are single copy genes,
in Arabidopsis the different members are mostly small gene families.
The duplicated members appear to have acquired distinct functions; thus
CLF and MEA function in repressing flowering and repressing
endosperm proliferation, respectively. It is not clear how this has occurred;
for example, whether it simply reflects different expression patterns of
MEA and CLF, or whether their protein products have also
diverged in function.
In addition to the conservation of PRC2 members in plants, there is also
evidence that their proteins may act together. Thus, several studies have
shown that the FIE protein can interact with the E(z) homologues MEA, CLF and
At4g02020 (Katz et al., 2004;
Kohler et al., 2003a
;
Luo et al., 2000
;
Spillane et al., 2000
;
Yadegari et al., 2000
). Also,
compelling evidence for interaction of FIE and MSI1 proteins was recently
presented (Kohler et al.,
2003a
). However, the role of the plant Su(z)12 homologues
has remained obscure. Despite the similarities in the phenotype of
fis2 and the other fis mutants, no interaction between FIS2
(or any other Su(z)12 homologue) with the other Pc-G proteins has been
found.
Here, we show that the plant E(z) and Su(z)12 homologues interact both genetically and physically through their protein products. We localise the interactions to motifs that are conserved between the plant and animal proteins. We show that the third Arabidopsis E(z) homologue functions largely redundantly with CLF, so that CLF has a more general role in control of plant development than was apparent from its single mutant phenotype. Characterisation of the misexpression phenotypes for the three E(z) homologues indicates that they have diverged not only in expression, but also at the protein level. We suggest that in plants an evolutionarily ancient complex (the PRC2) has been conserved, but gene duplication and divergence has given rise to several complexes with partially discrete functions.
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Materials and methods |
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Yeast two-hybrid assay
Constructs for yeast two-hybrid analysis were generated using the vectors
pGBT9 and pGAD424 (Clontech) that express protein fusions to the GAL4
DNA-binding domain or transcriptional-activation domain, respectively. cDNA
inserts encoding plant Pc-G proteins were introduced as
EcoRI/SalI fragments. The Quik Change site-directed
mutagenesis system (Stratagene) was used to introduce in-frame EcoRI
and SalI restriction sites within cDNA clones, with the exception of
EMF2 clones, which were generated by PCR amplification using
mutagenic primers. PCR-generated clones were validated by sequence analysis.
The methods for two-hybrid analysis were as described in the yeast protocols
handbook (Clontech). The analysis was performed in yeast strain Hf7c
(Feilotter et al., 1994),
which carries HIS3 and LacZ reporters for reconstituted GAL4
activity, or in strain AH109 (James et
al., 1996
), which carries HIS3 and ADE2
reporters.
Yeast split-ubiquitin assay
Vectors were used as described in Kim et al.
(Kim et al., 2002). CLF-C5 was
cloned into pENTRY 3c (Invitrogen) and recombined into the bait vector using
the Gateway system (Invitrogen), resulting in a CLF-C5-Cub-URA3 gene fusion.
EMF2-VEFS was fused to a gene encoding the N-terminal part of ubiquitin in the
vector pCGK. The plasmids were transformed into the Saccharomyces
cerevisiae strain JD53 and interaction of the fusion proteins was
monitored as ability to grow on 5-fluoroorotic acid (5-FOA) plates, containing
yeast nitrogen base without amino acids (Difco) and glucose, supplemented with
lysine, leucine, uracil, and 1 mg/ml 5-FOA.
In-vitro pull down assay
A similar protocol to that described in Kohler et al.
(Kohler et al., 2003a) was
applied. The coding region for the CLF C5 domain (amino acids 257-331) was
cloned into the pGEX-4T expression vector (Amersham) as a GST-fusion, whereas
the EMF2 VEFS domain (amino acids 427-631) was cloned in the pET30a expression
vector (Novagen) as a HIS6-fusion. Escherichia coli strain
BL21 DE3 Codon-plus (Stratagene) was freshly transformed with the pGEX-CLF-C5,
pGEX or pET-EMF2-VEFS plasmids and grown in LB medium at 37°C overnight.
After diluting the cultures 1:100 in 250 ml LB, they were grown at 37°C
(pGEX-CLF-C5 and pGEX) or 18°C (pET-EMF2-VEFS) until OD600=0.7.
Production of recombinant protein was induced by adding
isopropyl-ß-D-thiogalactopyranoside (IPTG) to 0.2 mM and after growing
the cells for 3 hours at 18°C, they were harvested and resuspended in 4 ml
binding buffer [BB; 20 mM Tris pH 7.5, 150 mM NaCl, 0.1% Triton X100, 1 µM
ZnSO4, 1 mM Pefabloc (Roche)]. The cells were lysed by the addition of
Lysozyme to 2 mg/ml and incubated for 20 minutes on ice. The solution was
centrifuged (20,000 g) for 10 minutes, the pellets discarded,
centrifuged again and a 100 µl sample of supernatant was mixed with SDS
sample buffer and frozen in liquid nitrogen (input control sample). Equal
volumes of extract containing HIS6-EMF2-VEFS were mixed with
extracts containing GST-CLF-C5 or GST and 150 µl of pre-equilibrated
glutathione-sepharose 4B beads (Pharmacia) and incubated with shaking for 2
hours at 4°C. The beads were washed four times with BB and then mixed with
SDS sample buffer, analysed on protein blots and the HIS6-EMF2-VEFS
fusion detected with anti-HIS6-antibodies (New England
Biolabs).
Misexpression of CLF, SWN and MEA
Constructs for expression of CLF, SWN and MEA cDNAs under
control of the cauliflower mosaic virus 35S promoter were assembled using the
pART7 and pART27 vector systems (Gleave,
1992). A full-length SWN cDNA clone (pda05864) was obtained from
the Riken Bioresource centre, Japan (Seki
et al., 2002
), CLF and MEA cDNA clones were isolated previously
(Goodrich et al., 1997
;
Spillane et al., 2000
). The
Quik Change site-directed mutagenesis system (Stratagene) was used to engineer
restriction sites within the cDNA clones that facilitated subcloning the
coding sequences into pART7. The constructs were introduced into
Agrobacterium strain GV3101 pMP90
(Koncz and Schell, 1986
) and
used to transform clf-50/+ heterozygotes by floral dip transformation
(Clough and Bent, 1998
). At
least 23 primary transformants were identified for each construct. Selected
plants in the T1 and T2 generations were genotyped for presence of a transgene
and for clf-50 and CLF+ alleles by Southern blot
analysis.
In-situ hybridisation
The methods for in-situ hybridisation analysis using digoxigenin-labelled
mRNA probes were described previously
(Narita et al., 2004).
SWN probes were generated from a poorly conserved 700 bp region at
the 5' end of the SWN coding region. WUS probes were
generated using the clone pMH WUS 16, generously provided by R. Simon.
Scanning electron microscopy and cell size measurements
Scanning electron microscopy (SEM) was performed on a Hitachi 4700 with a
Gatan Alto cryo-stage. The methods for cryo-SEM were as described previously
(Jeffree and Read, 1991). For
measuring cell sizes, fully expanded rosette leaves were fractured in
transverse section and photographed using the cryo-SEM. The cell outlines were
traced onto transparencies, scanned, and quantified using image analysis
software (image tool, University of Texas, available at
http://ddsdx.uthscsa.edu/dig).
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Results |
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The similarities in phenotypes suggested that the moe leaf phenotype, like
that of clf mutants, could be caused by misexpression of floral
homeotic genes during vegetative and floral development. Previous studies have
shown that the AG and AP3 genes, whose expression is
normally confined to flowers, are misexpressed in leaves of clf
mutants (Finnegan et al.,
1996; Goodrich et al.,
1997
; Serrano-Cartagena et
al., 2000
). We therefore used RT PCR to compare AG and
AP3 expression in leaves of wild-type and emf2-10 plants.
This indicated that both genes were expressed in emf2-10 leaves (data
not shown). To confirm this, we introduced reporter constructs for AG
and AP3 expression into the emf2-10 background. The
pAG-I::GUS construct contains AG upstream promoter sequences
and intragenic sequences fused to the GUS reporter and has been shown to
contain the cis-acting sequences necessary for repression by
CLF (Sieburth and Meyerowitz,
1997
). This construct was strongly misexpressed in seedlings of
both emf2-10 and clf-2 mutants
(Fig. 1G,H). In addition, both
mutants showed misexpression in inflorescence stems
(Fig. 1J,K) and occasional
misexpression in the outer floral whorls. We also tested reporter constructs
containing the AG second intron upstream of a GUS reporter gene (KB9)
(Busch et al., 1999
). This
construct also confers the wild-type AG expression pattern in
flowers, presumably because the second intron contains many AG
regulatory elements (Busch et al.,
1999
). However, when the KB9 construct was introduced into
clf or emf2-10 mutant backgrounds, no expression was seen in
seedlings (data not shown). This suggested that the AG promoter
contains additional enhancers that are required for misexpression of
AG in clf and emf2-10 mutant backgrounds. An
AP3 reporter construct containing 3.7 kb of upstream regulatory
sequences (Jack et al., 1994
)
also showed weak expression in emf2-10 and clf seedlings but
not wild-type (Fig. 1L,M).
Genetic data have indicated that the phenotype of clf mutants is
chiefly caused by ectopic AG expression. Thus in clf ag
double mutant plants, in which AG activity is eliminated, leaf
morphology is restored to near wild-type
(Goodrich et al., 1997;
Serrano-Cartagena et al.,
2000
). To test whether the moe leaf phenotype was also a result of
ectopic AG activity, we made emf2-10 ag-2 double mutants.
Although the double mutants had larger, less curled leaves than
emf2-10 single mutants, there was less restitution of wild-type
morphology than in the case of clf ag double mutants. Thus
emf2-10 ag-2 plants were still much smaller than wild type, their
leaves retained some curling, and they flowered earlier
(Fig. 1N). This indicated that
although AG+ activity contributes to the moe leaf
phenotype, misexpression of other genes is also probably involved. Taken
together, these results indicated that EMF2 and CLF shared
common functions in repressing floral homeotic gene expression, with
EMF2 required to repress a broader range of targets than
CLF. These similarities suggested that EMF2 might act in a
common pathway with CLF.
The moe leaf phenotype is conferred by a weak emf2 allele
To determine the molecular basis for the moe leaf phenotype, we employed a
map-based cloning strategy and initially localised the mutation responsible to
a 10 cm interval between markers nga129 and ATTED2 on the lower arm of
chromosome 5. It was striking that the plant Pc-G member EMF2 had
also been located within this interval. All nine emf2 mutant alleles
previously described have much more severe phenotypes than moe leaf, producing
minute plants that appear to flower soon after germination without undergoing
a prior phase of vegetative development
(Sung et al., 1992;
Sung et al., 2003
;
Yang et al., 1995
). Instead, a
few flowers and sessile cauline leaves are produced on an inflorescence with a
severely shortened bolt (Fig.
1O, Fig. 3C).
However, several features made EMF2 a promising candidate. Firstly,
it was known to repress floral homeotic gene expression during vegetative
development (Chen et al., 1997
;
Moon et al., 2003
). Secondly,
transgenic plants that expressed an antisense EMF2 construct had a
phenotype resembling moe leaf, which probably reflected a partial loss of
EMF2 function (Yoshida et al.,
2001
). To test whether the moe leaf phenotype could be caused by
an unusual, weak allele of EMF2, we performed genetic complementation
tests. Because emf2 mutants are sterile, and moe leaf plants have low
fertility, we crossed heterozygotes for the two mutations. The resulting F1
population of 254 plants contained 71 mutants, consistent with the two
mutations being allelic (1/4 mutants expected,
2=1.2
P>0.1). We designated the new mutation responsible for the moe
leaf phenotype as the emf2-10 allele. The phenotype of
emf2-10/emf2-3 heterozygotes was intermediate between that of the two
parental alleles, consistent with emf2-10 being a weaker allele than
emf2-3 (Fig. 1O). To
identify the lesion causing the emf2-10 mutation, we compared the
sequence of the EMF2 locus from emf2-10 and the wild-type
progenitor. This revealed that the emf2-10 allele carried a 17 bp
deletion extending from the 3' end of the second exon (9 bp) into the
5' end of intron 2 (8 bp) followed by a cytosine to guanine substitution
(see Fig. S1A,C in the supplementary material). Because this deletion was
predicted to affect splicing of the EMF2 pre-mRNA, we used RT-PCR to
amplify EMF2 cDNA from emf2-10 and wild-type seedlings.
Whereas a single message corresponding to the spliced EMF2 transcript
was detected in wild type cDNA, five novel transcripts were identified in
emf2-10 cDNA (see Fig. S1B in the supplementary material). Molecular
cloning and sequencing of these aberrant transcripts indicated that four
contained frameshift mutations likely to abolish EMF2 activity. However, one
transcript was predicted to produce a variant EMF2 protein that was truncated
by 17 amino acids at the N-terminus (see Fig. S1C in the supplementary
material). The region deleted does not correspond to a conserved region or to
a known functional domain, so the variant protein is likely to retain
EMF2+ activity. The weak emf2 phenotype may arise because
only a small fraction (about 20%) of the various emf2-10 transcripts
are likely to produce a functional protein. In addition, the resulting
truncation of the protein may also reduce its activity.
|
The severity of the clf emf2 double mutant phenotype suggested that it was unlikely to result simply from misexpression of AG. To confirm this, we constructed ag-1 clf-2 emf2-10 triple mutants. The triple mutants were minute plants, similar to severe emf2 mutants in size, and had 1-3 flowers with normal petals and ag phenotype (Fig. 3E). This indicated that AG misexpression was not responsible for the severe effects of clf emf mutants on overall plant size, but did account for the poor development of petals in clf emf mutant flowers (Fig. 3D).
Molecular interactions of CLF and EMF2
To test whether the genetic interaction of CLF and EMF2
might reflect a direct interaction between their protein products, we
performed yeast two-hybrid assays. We expressed full-length EMF2 protein, and
a series of EMF2 truncations, as `prey' fusions with the GAL4 transcriptional
activation (TA) domain. We tested these fusion proteins for interaction with a
`bait' comprising a fusion of a truncated CLF protein (lacking the C-terminal
SET domain) with the GAL4 DNA-binding domain. We did not observe an
interaction between full-length EMF2 proteins with CLF in yeast. However,
yeast strains expressing both CLF and a C-terminal portion of EMF2 expressed
both two-hybrid reporter genes, consistent with the two proteins interacting
(Fig. 4A). The C-terminal
portion of EMF2 contained the VEFS domain, a motif originally defined on the
basis of its conservation between plant and animal homologues of the Su(z)12
protein (Gendall et al.,
2001). It was not clear why the full-length EMF2 protein, which
includes the VEFS box, did not also interact with CLF.
|
The Arabidopsis FIS genes FIS2 and MEA encode
homologues of EMF2 and CLF, respectively. Although the FIS2 and
MEA genes share extremely similar mutant phenotypes, suggesting that
their products may interact, we were previously unable to demonstrate any
interaction between the full-length proteins using the two-hybrid assay
(Spillane et al., 2000).
However, the observation that the interaction of EMF2 with CLF was mediated by
the VEFS box domain suggested that FIS2 and MEA might also interact via the
VEFS box. We therefore specifically tested the VEFS box of FIS2 against MEA in
two-hybrid assay and in this case were able to demonstrate an interaction
(Fig. 5A).
|
To determine the SWN expression pattern, we localised its mRNA by
in-situ hybridisation to sections of seedlings and inflorescences.
SWN was expressed throughout the apical meristem and leaf primordia
of 8-day-old wild-type seedlings (Fig.
6A,B). Expression was also detected in the vasculature of
hypocotyls and cotyledons (Fig.
6B). In inflorescences, SWN was expressed throughout the
inflorescence meristem and young stage 1-3 floral meristems
(Fig. 6C). In older flowers,
expression was weak in the sepals and stronger in the inner whorls containing
developing petals, stamens and carpels
(Fig. 6D,E). In stage 12
flowers, strongest expression occurred in the ovules, particularly in the
funiculus and maternal tissues of the ovule
(Fig. 6F). Expression was also
seen in the female gametophyte, but the tissues were too poorly preserved to
distinguish the different cell types within the gametopyhte
(Fig. 6F). Little signal was
observed when seedlings and inflorescences were hybridised with a probe from
the sense strand of the SWN cDNA
(Fig. 6G,H), confirming that
the signal was specific for the SWN antisense probe. As a positive
control, we also hybridised seedlings with a probe for the WUSCHEL
(WUS) gene and detected expression confined to the centre of the
shoot meristem (Fig. 6I) as
previously described (Mayer et al.,
1998). The SWN expression pattern was therefore similar
to that of CLF (Goodrich et al.,
1997
), with both genes being generally expressed during vegetative
and reproductive development but with strongest expression in meristems and
other regions of dividing cells.
|
To identify the function of SWN, we exploited facilities for
reverse genetics in Arabidopsis to identify a series of mutant
alleles caused by T-DNA insertions within the locus. The swn-1 allele
contained an insertion 3 bp upstream of the predicted ATG start codon. This
allele is unlikely to be null, as RT-PCR analysis of swn-1 mRNA
revealed chimeric transcripts that initiated within the T-DNA insertion and
extended the full length of the SWN coding sequences (data not
shown). The swn-2 insertion is within an intron and swn-3
within an exon, but both are upstream of the catalytic SET domain and are
therefore likely to represent null alleles. All three alleles were viable as
homozygotes and had no obvious phenotype that we could discern from inspection
of gross plant morphology, embryo or endosperm development (data not shown).
However, all three alleles strongly enhanced the clf mutant phenotype
in clf swn double mutant combinations, confirming that the two genes
exhibit redundancy. The swn-1 allele gave a less severe enhancement
than did swn-2 or swn-3, consistent with its being a weaker
allele. The swn-1 clf-50 double mutant gave extremely small, early
flowering plants with few flowers that resembled emf2 mutants
(Fig. 3F,G). SEM analysis
indicated that the floral organs showed weak homeotic conversion to carpelloid
structures (Fig. 2K). In
addition, filamentous organs were observed in place of stipules, a phenotype
that has also been observed in plants that have a partial loss of
FIE+ activity (Katz et al.,
2004). Double mutants of the null clf-50 allele with
either swn-2 or swn-3 were more extreme, and viable plants
were recovered only when seedlings were grown in sterile tissue culture. The
seed germinated and produced seedlings with narrow, but relatively normal,
cotyledons, hypocotyl and roots. As the plants aged they became increasingly
abnormal. The cotyledons developed finger-like projections on their margins.
The shoot apex did not initiate leaves, but instead developed into a
disorganised mass of green tissue on which poorly differentiated organs formed
(Fig. 3H). In SEM analysis of
the plants, the epidermi of these organs lacked trichomes and comprised small,
isodiametric cells, which did not have the surface cuticular thickening or
elongated cell shape that is characteristic of epidermal surfaces of most of
the mature floral organs (Fig.
2M,N). In addition, colourless callus-like tissue formed and
eventually gave rise to somatic embryos and roots
(Fig. 3H,I). Unlike the single
mutants, which had normal roots, the primary root of the double mutants became
opaque, swollen and eventually produced green shoot-like tissue
(Fig. 3J). A similar phenotype
has been observed in seedlings of rescued fie homozygotes
(Kinoshita et al., 2001
).
Together, these observations suggested that weak clf-50 swn-1 double
mutants resembled emf2 mutants, whereas the null clf swn
doubles were more extreme and resembled plants lacking
FIE+ activity.
Although the above data suggest that the CLF and SWN genes have very similar functions, the fact that clf mutants have a clear phenotype indicates that SWN is not identical in function to CLF, at least with respect to repression of AG. This might be due to subtle differences in level of expression between CLF and SWN, and/or changes in protein function. To clarify whether differences are solely due to changes in expression, we expressed full-length cDNA clones for each gene under control of a common promoter (the cauliflower mosaic virus 35S promoter) and introduced the two transgenes into the null clf-50 mutant background. Whereas the 35S::CLF construct fully complemented the clf-50 mutation, the 35S:SWN construct did not (Fig. 7). There are therefore subtle differences in function between the CLF and SWN proteins, as might be expected given the persistence of the CLF/SWN duplication within angiosperms. Expression of 35S::MEA failed to complement the clf-50 mutation, indicating that the MEA protein has also diverged from CLF (Fig. 7).
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Discussion |
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Conservation of the PRC2 complex between plants and animals
In animals, the core members of the PRC2 complex comprise four proteins
first identified in Drosophila as the Esc, P55, E(z) and Su(z)12
proteins (Cao et al., 2002;
Czermin et al., 2002
;
Kuzmichev et al., 2002
;
Muller et al., 2002
). There is
now strong evidence that structurally and functionally equivalent complexes
occur in Arabidopsis. Thus several previous studies have shown
genetic and physical interactions of FIE with MEA and CLF, and also of FIE
with MSI1 (for a review, see Reyes and
Grossniklaus, 2003
) (Fig.
8). In particular, Kohler et al.
(Kohler et al., 2003a
)
partially purified an FIS complex and showed that it contained FIE, MEA, MSI1
and, based on molecular weights, probably several other unidentified
components. However, the role of the FIS2, VRN2 and EMF2 proteins has remained
enigmatic, although the strong similarity between the fis mutant
phenotypes suggested that FIS2 might interact with one or more of the other
FIS proteins. We have now shown that EMF2 interacts physically and genetically
with CLF. We extend this to show a general potential for the Su(z)12
homologues FIS2 and VRN2 to interact with the E(z) homologues MEA, CLF, and
SWN, at least in yeast two-hybrid assays. Taken together, these observations
strongly suggest that there are Arabidopsis complex(es) that are
structurally equivalent to at least the core members of the animal PRC2
members. It is also likely that they have an equivalent biochemical function
in mK27 H3 histone methylation. Thus, the Arabidopsis VRN2 protein
was recently shown to be required for vernalisation-induced mK27 H3
methylation at the FLC gene
(Bastow et al., 2004
;
Sung and Amasino, 2004
).
However, biochemical purification of the plant PRC2 complexes will be
necessary to confirm that they have a direct HMTase activity.
|
It is unclear how these complexes might acquire specificity for different
target genes, as PRC2 members appear to lack intrinsic DNA-binding specificity
and the recruitment of Pc-G members to specific targets is not yet well
understood either in animals or plants
(Birve et al., 2001;
Carrington and Jones, 1996
).
One possibility is that PRC2 members are recruited to targets by interaction
with sequence-specific DNA-binding proteins
(Wang et al., 2004
). A recent
alternative model is that Pc-G members could achieve sequence specificity
through interactions with small RNAs
(Steimer et al., 2004
).
Because the FIE gene is a single copy, and its protein product is
probably common to all complexes, it is unlikely that FIE could distinguish
the activity of different complexes. However, small differences between the
EMF2/VRN2/FIS2 and/or MEA/CLF/SWN proteins could change their affinities for
protein partners that target the complex. It is striking that the
FIS2/VRN2/EMF2 class of protein is the only one of the PRC2 members that is
not also conserved in C. elegans. This implies that this protein is
not absolutely required for the biochemical activity of the complex, and might
therefore play a role in specifying its targets. In addition to differences
between complexes in their target gene specificities, there must also be
differences between the CLF family members and the EMF2 family members in
their affinity for one another. For example, if CLF has equal affinity for
FIS2, EMF2 and VRN2 and the FIS2 family members have equal affinity for CLF,
MEA and SWN, then proteins such as MEA and CLF should be able to
cross-complement one another when misexpressed. We did not observe such
differences in yeast two-hybrid assays; for example, CLF and SWN showed a
similar potential to interact with each of the EMF2, VRN2 and FIS2 proteins.
However, the interactions in yeast may not accurately reflect subtle
differences in affinity in plants. It will be interesting to test whether
swapping the C5 domains between CLF and MEA proteins, or other regions, can
alter their specificity in vivo.
Partial redundancy between CLF and SWN
The CLF and SWN genes show similar expression patterns
and encode closely related proteins that display identical interactions in
several yeast two-hybrid assays. We tested three independent swn
mutant alleles and all three strongly enhance the clf single mutant
phenotype, although they are without gross morphological phenotype by
themselves. This suggests that there is substantial functional redundancy
between the two genes, so that the roles of CLF are largely masked by
SWN activity in clf single mutants. For example, a role for
CLF and SWN in primary root development is not apparent from
either single mutant phenotypes but is revealed in null clf swn
doubles. The partial redundancy of CLF and SWN probably
explains why null clf mutants have much less extreme phenotypes than
null emf2 mutants, although the CLF and EMF2 proteins act together.
Consistent with this, weak swn-1 clf-50 double mutants resembled
emf2 mutants. However, null swn clf double mutants were more
extreme than emf2 mutants and resembled plants lacking
FIE+ activity. It is likely that the full function of EMF2
is also masked by partial redundancy; for example, with the related
VRN2 gene with which it shares overlapping expression.
Despite overlapping functions, CLF and SWN are not completely redundant with respect to one another: firstly, clf mutants have a phenotype, largely caused by ectopic AG expression, that is not complemented by SWN+ activity; secondly, 35S::SWN, unlike 35S::CLF, does not complement clf mutants; thirdly, phylogenetic comparisons indicate that CLF and SWN orthologues can be distinguished clearly in other plants, including monocotyledenous species such as rice and maize. This means that the CLF/SWN duplication is an ancient one within the angiosperm lineage. It is unlikely that the SWN gene would show such wide conservation if it did not have at least partially distinct functions from CLF. Although we did not identify gross morphological effects of null swn mutations, several of the phenotypes associated with other plant PRC2 members (for example, autonomous endosperm development or vernalisation response) are apparent only in specific phenotypic screens or genetic backgrounds. It is therefore likely that swn mutants do have a phenotype, but this was not manifest in our growth conditions or assays.
The potential for CLF and SWN to act in vernalisation response
Recently, it was shown that VRN2 is required, after vernalisation
treatments, for mK27 H3 methylation in chromatin of its target gene
FLC (Bastow et al.,
2004; Sung and Amasino,
2004
). In animals, mK27 H3 methylation by the PRC2 complex
requires the E(z) protein, which contains a SET domain known to have HMTase
activity (Cao et al., 2002
;
Czermin et al., 2002
;
Kuzmichev et al., 2002
;
Muller et al., 2002
). Together
these observations suggest that an E(z) homologue will be required
for the vernalisation response. Consistent with this, we show that the VRN2
protein has potential to interact, through its VEFS domain, with the C5 domain
of the E(z) homologues CLF and SWN. In preliminary experiments (data not
shown), we did not observe gross effects of null clf or swn
mutations on the vernalisation response comparable with those of vrn2
or other vernalisation response mutants. It is possible that CLF and
SWN act redundantly with respect to the vernalisation response, so
that defects will be manifest only in double mutants. Unfortunately, the
pleiotropic phenotype of clf swn double mutants makes it difficult to
characterise their vernalisation response, at least by straightforward
comparison of flowering times. One possibility will be to use chromatin
immunoprecipitation (ChIP) to test whether FLC chromatin becomes enriched for
CLF and/or SWN proteins following vernalisation treatments.
In summary, it is likely that the PRC2 complex is conserved between plant and animals, both structurally and also functionally in terms of its histone methylation activity. However, in plants there has been duplication of most components of the PRC2, and the duplicated members have diverged in protein function as well as in expression. This has given rise to several PRC2-like complexes in plants, with at least partially discrete functions in terms of target gene specificity. Expression of chimeric proteins that swap domains between duplicated components, such as CLF/SWN/MEA, may help identify how the changes in specificity are mediated. Despite the conservation of the PRC2 in plants, it is striking that there are no homologues of the animal Pc-G members that comprise the PRC1 complex. It is therefore possible that the mechanisms to interpret, maintain, and re-set epigenetic information conveyed by the PRC2 have evolved independently in plants. Alternatively, plants may employ similar protein motifs to those found in the animal PRC1 members, but in novel combinations.
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ACKNOWLEDGMENTS |
---|
![]() |
Footnotes |
---|
Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/131/21/5263/DC1
* Present address: National Centre for Genetic Engineering and Biotechnology
Thailand Science Park 113, Phahonyothin Rd., Klong 1, Klong Luang, Pathum
Thani 12120 Thailand
These authors contributed equally to this work
Present address: Department of Molecular Biology, Pusan National
University, Keumjung-ku, Busan 609-735, Korea
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