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First published online April 30, 2007
doi: 10.1242/10.1242/dev.001891
1 National Research Laboratory of Plant Developmental Genetics, Department of
Biological Sciences, Seoul National University, Seoul, 151-742, Korea.
2 Global Research Laboratory for Flowering at SNU and UW, Seoul, 151-742,
Korea.
3 Environmental Biotechnology National Core Research Center, Gyeongsang National
University, Jinju 660-701, Korea.
4 Plant Metabolism Research Center, Kyung Hee University, Suwon 449-701,
Korea.
* Author for correspondence (e-mail: ilhalee{at}snu.ac.kr)
Accepted 13 March 2007
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Flowering, Chromatin remodeling, SWR1 complex, FLC, AtARP6
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). In Arabidopsis, molecular genetic analyses have
revealed many chromatin modifiers regulating the transition to flowering.
Flowering time is regulated in Arabidopsis by a complex genetic
network that consists of at least four interdependent genetic pathways, the
long day, vernalization, gibberellin (GA) and autonomous pathways (reviewed by
Putterill et al., 2004;
Henderson and Dean, 2004).
Among the genes involved in this complex genetic network, FLOWERING LOCUS
C (FLC) is a central flowering regulator integrating the
autonomous and vernalization pathways. FLC acts as a floral
repressor, downregulating the expression of common downstream target genes -
the so-called flowering pathway integrators FT, SUPPRESSOR OF
OVEREXPRESSION OF CO 1 (SOC1, AGL20) and LEAFY
(Simpson and Dean, 2002).
Ultimately, the expression level of these integrators determines the exact
flowering time (Hepworth et al.,
2002). Recently, the regulation of FLC through chromatin
modifications has been intensively studied (reviewed by
Lee, 2005).
Vernalization, a process accelerating flowering by sensing a long period of
winter cold, suppresses the expression of FLC through a series of
histone modifications, including deacetylation and the subsequent methylation
of histone H3 at lysine 9 (K9) and lysine 27 (K27)
(Sung and Amasino, 2004;
Bastow et al., 2004).
Deacetylation, and the subsequent K9 and K27 methylations, require
VERNALIZATION INSENSITIVE 3 (VIN3), which encodes a
PHD-domain protein, and the methylations (particularly K9) require
VERNALIZATION 1 (VRN1) and VRN2, which encode a
Myb-related DNA-binding protein and a Polycomb group protein homologous to
Drosophila Su[z]12, respectively
(Sung and Amasino, 2004;
Bastow et al., 2004). After
returning to warm temperature, the epigenetically repressed state of
FLC is maintained by LIKE HETEROCHROMATIN PROTEIN 1
(LHP1; also known as TFL2 - TAIR), through the maintenance
of the increased levels of H3K9 methylation at FLC chromatin
(Sung et al., 2006).
In summer annuals lacking the strong FLC activator
FRIGIDA (FRI), FLC expression is repressed by
autonomous pathway genes. If any of them is mutated, FLC expression
is increased resulting in late flowering (reviewed by
Henderson and Dean, 2004;
Lee, 2005). Among the
autonomous pathway genes, FVE (AtMSI4), FLOWERING LOCUS
D (FLD) and RELATIVE OF EARLY FLOWERING 6
(REF6) are involved in histone modification at FLC. Mutation
in any one of them causes hyperacetylation of H3 and H4 tails, resulting in
upregulation of FLC (He et al.,
2003; Ausin et al.,
2004; Noh et al.,
2004). FVE encodes a homolog of yeast MSI (multicopy
suppressor of IRA1) and mammalian retinoblastoma-associated proteins RbAp46
and RbAp48 (also known as Rbbp7 and Rbbp4, respectively), which have a
function in the histone deacetylase (HDAC) complex
(Ausin et al., 2004;
Kim et al., 2004).
FLD encodes an amine oxidase, homologous to the human LSD1
(lysine (K)-specific demethylase 1; also known as AOF2 - Human Gene
Nomenclature Database), whereas REF6 encodes one of the plant
jumonji-family proteins, the mammalian and yeast homologs of which function as
histone demethylases (He et al.,
2003; Shi et al.,
2004; Noh et al.,
2004; Tsukada et al.,
2006). Thus, it is likely that FLD and REF6 have
roles in the deacetylation of FLC chromatin through histone
demethylation.
It has also been reported that Arabidopsis homologs of components
of the yeast PAF1 (RNA Polymerase Associated Factor 1) complex are required
for trimethylation of H3 K4 at the promoter region of FLC, a hallmark
of the active chromatin state (Krogan et
al., 2003; Ng et al.,
2003). Thus, mutation in any of the homologs of the PAF1 complex
causes complete suppression of FLC, and consequently causes early
flowering in winter annuals and autonomous pathway mutants
(Oh et al., 2004;
He et al., 2004). A similar
suppression is observed in a mutation in EARLY FLOWERING IN SHORT
DAYS (EFS), a homolog of the SET domain methyltransferase
(Soppe et al., 1999;
Kim et al., 2005). The
efs mutation causes reduced trimethylation of H3 K4 or dimethylation
of H3 K36 in FLC chromatin (Kim
et al., 2005; Zhao et al.,
2005).
It has also been reported that putative homologs of components of the
ATP-dependent chromatin remodeling complexes are involved in the regulation of
flowering time (reviewed by Reyes,
2006). In particular, several homologs of components of the yeast
SWR1 complex (SWR1C), a member of the SWI2/SNF2 superfamily, have been
reported to regulate FLC expression in Arabidopsis. The
biochemical function of yeast SWR1C is to catalyze the replacement of H2A with
its variant H2AZ (Mizuguchi et al.,
2004; Kobor et al.,
2004). H2AZ deposition by SWR1C is required for transcriptional
regulation, heterochromatic barriers and genome stability in yeast (reviewed
by Kamakaka and Biggins,
2005). For transcriptional regulation, the SWR1C deposits H2AZ
into the transcriptional start site of a myriad of genes
(Raisner et al., 2005;
Zhang et al., 2005). Yeast
SWR1C consists of 13 subunits and homologs of most of them exist in
Arabidopsis, although SWC3 and SWC7 homologs have not been detected
(see Table S1 in the supplementary material)
(Meagher et al., 2005). The
substrate of SWR1C, H2AZ, is also conserved in Arabidopsis: three
homologs of H2AZ - HTA8 (At2g38810), HTA9 (At1g52740) and HTA11 (At3g54560) -
are present (Yi et al., 2006).
PHOTOPERIOD-INDEPENDENT EARLY FLOWERING 1 (PIE1) is the
Arabidopsis gene most homologous to SWR1, although it
contains a SANT domain, which is usually found in ISWI family members
(Noh and Amasino, 2003). Two
other reported homologs of components of SWR1C are ACTIN-RELATED PROTEIN
4 (AtARP4) and SUPPRESSOR OF FRIGIDA 3 (SUF3;
also known as AtARP6 - TAIR)
(Kandasamy et al., 2005;
Choi et al., 2005;
Deal et al., 2005;
Martin-Trillo et al., 2006).
The mutation in any of these homologs causes an early flowering phenotype
through the reduction of FLC expression. Furthermore, the
pie1 and suf3 mutants show similar developmental defects,
including early flowering, leaf serration, production of extra petals and weak
apical dominance.
To extend the study of SWR1C in Arabidopsis, we isolated a T-DNA insertion mutant in the homolog of SWC6 (AtSWC6), another component of yeast SWR1C, and produced RNAi-mediated H2AZ gene-knockdown transgenic plants. The mutation in AtSWC6 resulted in the same phenotypes as suf3. Chromatin immunoprecipitation assays showed that both AtSWC6 and SUF3 bind to the FLC promoter region. Consistently, when SUF3:GFP and AtSWC6:RFP constructs were co-transfected into protoplasts, complete colocalization of the two proteins was observed. In yeast two-hybrid and coimmunoprecipitation analyses, PIE1 interacted with SUF3, AtSWC6 and H2AZ, and AtSWC6 interacted with SUF3 and AtSWC2, another SWR1C component homolog. Finally, H2AZ gene-knockdown transgenic plants exhibited a similar phenotype to atswc6. Our results strongly suggest the presence of a homolog of SWR1C that regulates diverse aspects of plant development, including flowering.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Lee et al., 2000). All
flowering-time mutant alleles used in this study have been described
previously (Lee et al., 2000;
Moon et al., 2005;
Choi et al., 2005). Plants were
grown as previously described (Choi et al.,
2005). Flowering time was measured by counting the number of
rosette leaves from at least 20 plants.
Plasmid construction
Primer pairs for amplification of cDNAs or genomic DNAs for plasmid
construction are detailed in Table
1. To construct AtSWC6 and SUF3 overexpressors,
full-length AtSWC6 and SUF3 cDNAs amplified by PCR were
cloned into a binary vector myc-pBA (Zhou
et al., 2005). For 35S-AtSWC6:GFP construction,
full-length AtSWC6 cDNA without the stop codon was cloned into the
modified pPZP211 (KH24) vector with the 35S promoter, GFP coding sequence, and
NOS terminator derived from p326-GFP (Choi
et al., 2005). For generation of AtSWC6 p-AtSWC6:GUS
transgenic plants, 329 bp of upstream sequence from the stop codon of
At5g37060 (the neighboring gene) to the translational start site of
AtSWC6, and the full-length genomic DNA of AtSWC6 except for
the stop codon, were amplified and cloned into the T-DNA binary vector pDW137
(Blázquez et al., 1997)
as a translational fusion with the ß-glucuronidase (GUS) gene.
To prepare the construct expressing AtSWC6:GFP or AtSWC6:RFP fusion proteins
for protoplast transient expression assays, the open reading frame of
AtSWC6 was cloned into p326-GFP and p326-RFP
(Choi et al., 2005). For yeast
two-hybrid assays, AtSWC6, SUF3, AtSWC2 (At2g36740),
HTA8, HTA9, HTA11, PIE1-N (1-1560 bp), PIE1-M (1560-3660 bp)
and PIE1-C (3660-6168 bp) cDNAs were amplified and cloned into the
BamHI site or BglII site (for AtSWC2) of pGBKT7 and
pGADT7 vectors, respectively. To produce the H2AZ RNAi construct, an
inverted-repeat construct including the HTA8, HTA9 and HTA11
cDNAs was made using the pKANNIBAL vector in which a spliceable intron
separates the two repeats (Helliwell and
Waterhouse, 2003). Then, the region including the 35S promoter,
two repeats, intron and OCS terminator was subcloned into the NotI
site of the binary vector pART27 for transformation of FRI.
Artificial miRNAs to specifically knockdown HTA8, HTA9, HTA11, and
both HTA9 and HTA11, were prepared by overlapping PCR using
the pGEM-T Easy vector containing MIR319a as template
(Schwab et al., 2006). PCR
products including the amiRNA sequences were inserted into the binary vector
pCGN18 (Choi et al., 2005). For
Agrobacterium-mediated transient expression, the full-length
AtSWC2, HTA11 and PIE1 cDNAs were cloned into myc-pBA or the
modified pPZP211 (KH24).
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). Polyclonal anti-myc (Santa Cruz Biotechnology, sc-789)
was used for immunoprecipitation. PCR detection of FLC regions and
TUBULIN ß-2 (TUB2) was performed with the
primers shown in Table 1.
Analysis of gene expression
Total RNA was extracted from Arabidopsis using TRIZOL (Sigma). The
digoxigenin (DIG) labeled-mRNA probes for FLC, SOC1, SUF3 and
AtSWC6 were prepared as previously described
(Choi et al., 2005). RNA
blotting, hybridization and washes were performed as described in the DIG
application manual (Roche). RT-PCR and primers for SOC1, FLC, FT and
TUB2 are as previously described
(Lee et al., 2000;
Moon et al., 2005). Primers
for AtSWC6, HTA8, HTA9 and HTA11 are: AtSWC6,
5'-ATGGAGGAAGAGATGTCGAAC-3' and
5'-CTATGCAACAAATTTCTGACA-3'; HTA8,
5'-ATGGCTGGTAAAGGTGGGAAAG-3' and
5'-TCAATCCTTGGTGACTTTGTTG-3'; HTA9,
5'-ATGTCGGGGAAAGGGCTAAAG-3' and
5'-CTATTCCTTGGCGGATTTGTTG-3'; HTA11,
5'-ATGGCAGGCAAAGGTGGAAAAG-3' and
5'-TCACTCCTTGGTGGTTTTGTTG-3'.
Protein extraction and protein gel blotting
Plant tissues were harvested and ground in liquid nitrogen and solubilized
in protein extraction buffer [10 mM HEPES pH 7.5, 100 mM NaCl, 1 mM EDTA, 0.2%
(v/v) Triton X-100, 1 mM PMSF, and 1xprotease inhibitor cocktail tablets
(Roche)]. The debris was cleared by centrifugation at 15,000 g
for 10 minutes at 4°C. Western blot analyses were performed to determine
the myc:AtSWC6 or AtSWC6:GFP protein levels in 35S-myc:AtSWC6 atswc6
or 35S-AtSWC6:GFP atswc6 using anti-myc or anti-GFP antibodies.
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).
Plant samples were incubated in 90% acetone at room temperature for 10
minutes. The samples were rinsed once with staining solution without X-Gluc
(0.2% Triton X-100, 50 mM sodium phosphate buffer pH 7.2, 2 mM potassium
ferrocyanide, 2 mM potassium ferricyanide). The staining solution was replaced
by staining solution containing 2 mM X-Gluc, and the plant samples were
incubated overnight at 37°C. Samples were then observed after incubating
in 50% ethanol at room temperature for 30 minutes.
Protoplast transient expression assay
Rosette leaves of Col plants grown for 4 weeks in long-day conditions were
sampled for the isolation and transformation of protoplasts as described
[Sheen, J. (2002). A transient expression assay using Arabidopsis
mesophyll protoplasts.
http://genetics.mgh.harvard.edu/sheenweb/].
Protoplasts were co-transformed with both GFP and RFP fusion constructs,
totalling about 10 µg of plasmid DNA (prepared using the Qiagen Plasmid
Maxi Kit) and cultured at 22°C in the dark. 12 hours after transformation,
protoplasts were observed as previously described
(Choi et al., 2005).
Yeast two-hybrid analysis and coimmunoprecipitation in tobacco transient expression system
The vectors and yeast strains (Matchmaker GAL4 Two-Hybrid System 3) were
obtained from Clontech. The yeast two-hybrid assay was performed as previously
described (Kim et al., 2006)
and according to the manufacturer's instructions. Binary constructs presented
in Table 1 were transiently
expressed in Nicotiana benthamiana leaves and the
coimmunoprecipitation assay performed as previously described
(Kim et al., 2006).
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Mizuguchi et al.,
2004). Because there is a single homolog of SWC6, At5g37055, in
Arabidopsis, we analyzed a T-DNA insertion mutant
(SAIL_1142/CS841940) (Fig. 1).
This mutant has a T-DNA insertion in the second intron of At5g37055,
which results in the complete loss of expression
(Fig. 1A-C). We named this gene
Arabidopsis thaliana SWC6 (AtSWC6). The AtSWC6 gene
encodes a HIT-type zinc-finger protein whose homologs, SWC6 and ZNHIT1, are
subunits of the yeast SWR1 and mammalian SRCAP (SWI2/SNF2-related CBP
activator protein) complexes, respectively
(Cai et al., 2005;
Mizuguchi et al., 2004). All
three proteins have seven cysteines and one histidine highly conserved in a
C-terminal region (see Fig. S1 in the supplementary material). The
atswc6 mutant showed an early flowering phenotype as well as leaf
serration, flowers with extra petals, small siliques and weak apical dominance
(Fig. 1D-H;
Table 2;
Table 3). When the
atswc6 mutation was introduced into FRI-containing Col
(Col:FRISF2, referred to as FRI below)
(Michaels and Amasino, 1999;
Lee et al., 2000), which has
very late flowering phenotype, the atswc6 suppressed
FRI-mediated late flowering (Fig.
1D,F; Table 3). The
atswc6 FRI showed much earlier flowering than FRI, but
similar flowering with Col. When FLC expression was checked, the
atswc6 FRI line showed an approximately 50% reduction as compared
with FRI, but fivefold higher expression than Col
(Fig. 1C). Such a partial
suppression of FLC was also observed in suf3 FRI
(Fig. 1C)
(Choi et al., 2005). Thus, the
phenotypes caused by atswc6 were almost identical to that of
suf3 reported previously (Choi et
al., 2005). Consistently, the double mutant of atswc6
suf3 was indistinguishable from any of the single mutants, irrespective
of the presence of FRI (Fig.
1F). However, the expression analyses showed that the level of
SUF3 was not affected by atswc6 and that of AtSWC6
was not affected by suf3, indicating that the two genes do not
regulate each other at the transcriptional level
(Fig. 1B,C).
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Because the target of SUF3 is FLC
(Choi et al., 2005;
Deal et al., 2005), we used
chromatin immunoprecipitation (ChIP) to check whether AtSWC6 and SUF3 bind to
the chromatin of the FLC promoter region. For this, we used
35S-myc:AtSWC6 atswc6 and 35S-myc:SUF3 suf3 transgenic lines
showing complementation of the mutant phenotype
(Fig. 2E). Both transgenic
lines showed enrichment of AtSWC6 and SUF3 proteins at the proximal (FLC-B and
FLC-C) region of the FLC promoter, but not at the distal (FLC-A)
region of the promoter or first intron (FLC-V), suggesting that these proteins
bind to the promoter region of FLC chromatin.
Expression patterns of AtSWC6
The expression of AtSWC6 was examined by RT-PCR, northern blot
analysis and histochemical GUS reporter assay in AtSWC6p-AtSWC6:GUS
transgenic lines. AtSWC6 transcripts were detected in all tissues
tested at variable levels (Fig.
3A). AtSWC6 expression was strongly detected at early
stage, and increased slightly with developmental age
(Fig. 3B,E). The level of
AtSWC6 transcripts did not show any daily rhythm, although that of
SUF3 increased at night (Fig.
3C). Furthermore, AtSWC6 transcript levels were not
affected by photoperiod or vernalization
(Fig. 3D). In the transgenic
plants expressing the translational fusion of AtSWC6 genomic DNA with
GUS (AtSWC6p-AtSWC6:GUS), GUS expression was detected
strongly in actively dividing cells such as root and shoot apex, lateral root
primordia, inflorescence, flowers, axillary bud, developing siliques and
premature seeds (Fig. 3E).
However, GUS expression was rarely detected in mature seeds and siliques, old
leaves and stems (Fig. 3E).
To investigate whether AtSWC6 expression is regulated by other flowering time genes, we examined the transcript level of AtSWC6 in flowering-time mutants. The presence of FRI did not change the transcript level nor did an flc mutation (Fig. 1C; see Fig. S3 in the supplementary material). When we checked all the mutants involved in positive regulation of FLC (suf4, vip4, pie1, suf3 and abh1), the autonomous pathway (ld, fld, fve, fca, fy and fpa), the photoperiod pathway (gi and co) and flowering pathway integrators (ft and soc1), the AtSWC6 transcript level remained the same (see Fig. S3 in the supplementary material).
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AtSWC6 and SUF3 colocalize in the nucleus of Arabidopsis protoplasts
To investigate the cellular localization of AtSWC6, we examined the
florescence of GFP in the root of 35S-AtSWC6:GFP atswc6 transgenic
plants. The AtSWC6:GFP proteins were localized in the nuclei of root cells
(see Fig. S4 in the supplementary material). We also performed protoplast
transient assays using constructs expressing AtSWC6:GFP, AtSWC6:RFP and
SUF3:GFP fusion proteins under the control of the 35S promoter so as to
compare in detail the localization of AtSWC6 and SUF3. Consistent with the
localization of AtSWC6:GFP in roots, both AtSWC6:GFP and AtSWC6:RFP were
localized in the nuclei of leaf protoplasts. When they were transfected
simultaneously, the fluorescence overlapped entirely and the AtSWC6 proteins
formed nuclear speckles (Fig.
5, upper panels). By contrast, when AtSWC6:RFP proteins were
expressed with SUF3:GFP, most of the AtSWC6:RFP proteins moved to where
SUF3:GFP proteins were localized, such that the fluorescence overlapped
(Fig. 5, lower panels).
Previously, it was reported that SUF3 is localized at the nuclear periphery
(Choi et al., 2005). Our data
thus indicate that AtSWC6:RFP relocates to the nuclear periphery where
SUF3:GFP is localized, and strongly suggests that the two proteins
interact.
Interactions among homologs of SWR1C components
We performed yeast two-hybrid assays to determine whether the
Arabidopsis homologs of the yeast SWR1C components directly interact
with each other. For this assay, we included three homologs of H2AZ - HTA8,
HTA9 and HTA11 (Yi et al.,
2006) - and a homolog of SWC2 (At2g36740), another component of
SWR1C, which directly binds to H2AZ in yeast
(Wu et al., 2005). The results
showed that AtSWC6 interacts with SUF3 and AtSWC2, and revealed
homodimerization of AtSWC6 (Fig.
6A). By contrast, SUF3 showed neither homodimerization nor
interaction with AtSWC2, suggesting that AtSWC6 links SUF3 and AtSWC2. For
PIE1 interaction analysis, we divided the PIE1 protein into three regions: an
N-terminal region (PIE1-N, 1-520 aa) containing the HSA domain; a middle
region (PIE1-M, 520-1220 aa) containing two ATPase domains; and a C-terminal
region (PIE1-C, 1220-2055 aa) containing the putative SANT domain
(Noh and Amasino, 2003). It
has been suggested that the SANT domain is required for interaction with both
HATs or HDACs and chromatin (Sterner et
al., 2002; Noh and Amasino,
2003). PIE1-C interacted with both SUF3 and AtSWC6 but not with
AtSWC2, and PIE1-N interacted with the three H2AZs (HTA8, HTA9, HTA11) but not
with the other proteins tested (Fig.
6D,E). PIE1-M did not show any interaction, despite the fact that
it contains ATPase domains. AtSWC2 also interacted with all three H2AZs
(Fig. 6B,C). This suggests that
AtSWC2 and PIE1-N together are involved in binding to H2AZ. To confirm the
yeast two-hybrid result, we performed coimmunoprecipitation analysis using
tobacco leaves transiently expressing myc:AtSWC6, AtSWC6:GFP, SUF3:Flag,
myc:AtSWC2, HTA11:GFP and PIE1:GFP (Fig.
6F,G,H; see Fig. S6 in the supplementary material). The
interactions of AtSWC6-SUF3, AtSWC6-AtSWC2, AtSWC2-HTA11, PIE1-SUF3, and
PIE1-AtSWC6 were confirmed by the in planta coimmunoprecipitation assay. As
expected, the negative control SUF4 failed to interact with AtSWC6
(Fig. 6G). Together, our
results show interactions among PIE1, SUF3, AtSWC6, AtSWC2 and H2AZ.
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).
Although the targeted H2AZ was specifically knocked down, none of the
amiR-HTA8 and HTA11 lines (48 and 64, respectively), and
none (48 lines) of the amiR-HTA9 lines except two, showed any obvious
flowering phenotype, and the FLC expression level was the same as
that of the FRI (Fig.
7F). However, two lines of amiR-HTA9 showed earlier
flowering (flowered with 38 and 40 leaves), and all amiR-HTA9 lines
showed some degree of leaf serration similar to that seen in suf3 and
atswc6. When we checked the tissue specificity of each H2AZ, they were expressed in almost every tissue and there was no difference in the expression pattern between them, although HTA9 showed higher expression than the other two (see Fig. S5 in the supplementary material). Thus, the absence of an obvious flowering phenotype in the amiR-HTA lines is likely to be due to functional redundancy among the three H2AZs. To confirm this, we made amiR-HTA9&11 transgenic lines that knocked down both HTA9 and HTA11 (Fig. 7G). These lines showed early flowering, reduced expression of FLC, leaf serration and flowers with extra petals (Fig. 7F). Among 32 transformants of the amiR-HTA9&11, 16 T1 plants flowered with 15-20 rosette leaves, which is similar to that of suf3 FRI or atswc6 FRI, and the rest also flowered before producing 30 rosettes. Taken together, our results strongly suggest that HTA8, HTA9 and HTA11 are functionally redundant and that FLC expression is regulated by the overall level of the three H2AZs.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Mizuguchi et al.,
2004; Cai et al.,
2005), but the presence of a homologous complex in plants has not
been established. In addition, how SWR1C homologs affect development in higher
eukaryotes remains largely unknown. Homologs of most SWR1C components are
present in Arabidopsis, and thus the function of the relevant complex
can be genetically dissected. In this work, we characterized AtSWC6 and
analyzed its interaction with PIE1, SUF3 and AtSWC2, the homologs of
components of SWR1C. We also analyzed the function of Arabidopsis
H2AZ, a substrate of SWR1C. Our results strongly support the presence of an
SWR1C-like complex in Arabidopsis that ensures proper development,
including floral repression through full activation of FLC.
Homologs of SWR1C components in Arabidopsis
Mutations in three homologs of SWR1C components - pie1, suf3 and
atswc6 - cause similar developmental defects
(Noh and Amasino, 2003;
Choi et al., 2005) (see also
this study). All three mutants show leaf serration, weak apical dominance,
flowers with extra petals, short siliques and early flowering. Genetically,
all three mutants cause suppression of late flowering in autonomous pathway
mutants as well as in an FRI-containing line. They also show earlier
flowering than an flc-null and an approximately 50% reduction in
FLC expression in an FRI-containing line. The FLC
level in suf3 FRI or atswc6 FRI is fivefold higher than that
in Col although the flowering time is similar, suggesting that SUF3
and AtSWC6 regulate additional floral repressor(s). In addition, the
expression patterns of SUF3, AtSWC6 and PIE1 are similar
(Fig. 3)
(Noh and Amasino, 2003;
Choi et al., 2005). Although
all three genes are involved in the regulation of flowering time, their
expression is affected neither by photoperiod nor vernalization, nor by any of
the flowering-time mutants. It is notable, however, that pie1 has a
somewhat stronger effect than suf3 or atswc6
(Noh and Amasino, 2003).
pie1 also has the additional phenotypes of reduced fertility and
short flowering stem in the Col background, phenotypes that were not obvious
in suf3 or atswc6 in the same genetic background
(Noh and Amasino, 2003;
Choi et al., 2005;
Deal et al., 2005). Thus,
PIE1 might have a function that is at least partially independent
from that of SUF3 and AtSWC6.
In yeast, ARP6 and SWC6 are the most-tightly coupled components in SWR1C
(Wu et al., 2005). They are
necessary for the association with SWC2 and for nucleosome binding. Consistent
with this, SUF3 and AtSWC6 have the most similar
developmental function in Arabidopsis. The phenotypes of
suf3 and atswc6 mutants are indistinguishable, and the
suf3 atswc6 double mutant has the same phenotype as each single
mutant. The only difference we found between SUF3 and AtSWC6
was in the daily rhythm of expression; SUF3, but not AtSWC6,
showed a daily rhythm, the significance of which is not clear because the
suf3 mutant does not show any conspicuous rhythmic defect. Together
with the fact that overexpression of SUF3 or AtSWC6 does not
cause any phenotype, our results strongly suggest that SUF3, AtSWC6 and PIE1
act together as a protein complex. The protein interaction analyses confirmed
that the three proteins physically interact
(Fig. 6).
Although we have not purified the SWR1C homolog, three lines of
experimental evidence strongly support its presence in Arabidopsis.
Firstly, protoplast transfection assays clearly showed that AtSWC6 and SUF3
are colocalized. AtSWC6 alone disperses throughout the nucleoplasm as speckles
(Fig. 5). But when AtSWC6 and
SUF3 are expressed together, AtSWC6 moves to the nuclear periphery where SUF3
is localized, as previously reported (Choi
et al., 2005). Such a relocation of AtSWC6 strongly supports their
interaction. In addition, the localization analyses may indicate that the
nuclear periphery is the place where chromatin remodeling occurs, as previous
reports suggested that gene activation or gene silencing occurs at the nuclear
periphery (Misteli, 2004;
Casolari et al., 2004).
Consistent with this, both AtSWC6 and SUF3 bind to the proximal region of the
FLC promoter in a ChIP assay. More direct evidence was provided by
protein interaction analyses (Fig.
6), which showed that all of the putative components of the
Arabidopsis SWR1C homolog are interconnected. Finally, when the
expression of H2AZ was reduced to below a certain threshold level by RNAi or
amiRNA, the plants showed similar developmental defects to those shown by
suf3, atswc6 and pie1, as expected
(Fig. 7). Thus, although we
have not directly measured the biochemical H2AZ replacement activity, our
results strongly suggest that the complex including PIE1, SUF3 and AtSWC6
interacts with H2AZs biochemically and genetically.
The homolog of SWR1C is necessary for full activation of FLC
Genome-wide analyses in yeast showed that H2AZ is preferentially enriched
at repressed promoters (Raisner et al.,
2005; Zhang et al.,
2005). However, H2AZ is more susceptible to loss from yeast
chromatin upon increasing ionic strength, and the acetylated form of H2AZ is
preferentially detected in active promoters
(Zhang et al., 2005;
Millar et al., 2006). Thus, it
was proposed that H2AZ is deposited at repressed promoters and facilitates
rapid activation, probably through acetylation of H2AZ by the NuA4 complex
(Zhang et al., 2005;
Millar et al., 2006).
Consistent with this, H2AZ promotes full activation of target genes but does
not impact upon repression. For example, the yeast deletion mutant of H2AZ
shows approximately twofold attenuation in YDC1 activation when
induced by heat sock (Zhang et al.,
2005). The deletion mutant also causes twofold reduction in
PHO5 and GAL1 expression when induced by phosphate
starvation and galactose treatment, respectively, but it always shows higher
expression than the wild type before induction
(Santisteban et al., 2000). We
observed a similar result in the mutants of Arabidopsis homologs of
SWR1C components. In the presence of FRI, FLC is strongly activated
(Michaels and Amasino, 1999).
However, if any of the SWR1C homologs are mutated, the expression of
FLC is reduced twofold but is still higher than that in the plants
without FRI (Fig. 1)
(Noh and Amasino, 2003;
Choi et al., 2005).
Consistently, the knockdown lines of H2AZ showed a similar reduction
(Fig. 7). Therefore, our
results suggest that the SWR1C homolog in Arabidopsis promotes full
activation of a target gene, FLC, as is proposed for the function of
SWR1C in yeast.
Divergence of the SWR1C homolog in Arabidopsis
Histone variant H2AZ is highly conserved in a wide variety of species
including yeast, Tetrahymena, Drosophila, chicken, Xenopus,
mouse and human (reviewed by Kamakaka and
Biggins, 2005). Arabidopsis also has three highly
homologous H2AZs (Yi et al.,
2006). H2AZ is essential for survival in Tetrahymena,
Drosophila and mouse, but is not essential for yeast
(Santisteban et al., 2000;
Faast et al., 2001;
Kamakaka and Biggins, 2005).
Whether H2AZ is essential for survival of Arabidopsis is currently
unknown owing to genetic redundancy. We also failed to knockdown all three
H2AZ genes by RNAi, and thus it remains an open question. However,
suf3 and atswc6 mutants are not embryo lethal, although
SUF3 and AtSWC6 are single-copy genes in
Arabidopsis. Because the homologs of SUF3 and AtSWC6 are found only
in SWR1C and are not shared with other chromatin modification complexes, such
as yeast INO80 or NuA4 or mammalian TRRAP/TIP60, this suggests that the SWR1C
homolog in Arabidopsis is not essential for survival. It also
indicates that the SWR1C homolog does not regulate the whole genome, but
rather a certain group of genes that includes floral repressors.
The Arabidopsis homolog seems to be divergent from yeast SWR1C.
Firstly, PIE1 is divergent from SWR1 in that it contains a SANT domain at the
C-terminus, which is usually found in ISWI-family proteins
(Noh and Amasino, 2003).
Interestingly, AtSWC6 and SUF3 interact with the C-terminal region of PIE1
that contains the SANT domain (Fig.
6). By contrast, SWR1 interaction with SWC6 and ARP6 is dependent
on the region between two ATPase domains
(Wu et al., 2005), which
corresponds to the middle region of PIE1 in our analysis. Secondly, SWC2 could
not pull down SWC6 and ARP6 in a yeast swr1 deletion mutant
(Wu et al., 2005), but AtSWC2
directly interacts with AtSWC6, a SWC6 homolog, indicating that AtSWC2 is
connected with AtSWC6 and SUF3 in Arabidopsis. Thirdly, the binding
of H2AZ to the N-terminal region of SWR1 is not suggested in yeast, but in
Arabidopsis H2AZ interacts with the N-terminal region of PIE1 and
with AtSWC2. This therefore indicates the divergence of Arabidopsis
and yeast SWR1C homologs. Further biochemical purification of the SWR1C
homolog and functional studies using microarray and chromatin
immunoprecipitation-coupled chip hybridization will help us to understand the
crucial role of H2AZ and the SWR1C homolog in leaf and flower development and
in flowering time control in Arabidopsis.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/10/1931/DC1
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ACKNOWLEDGMENTS |
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-GFP) or anti-Flag (