INVESTIGATION
Ectopic Gene Expression and Organogenesis
in Arabidopsis Mutants Missing BRU1 Required
for Genome Maintenance
Yusuke Ohno,* Jarunya Narangajavana,*,† Akiko Yamamoto,* Tsukaho Hattori,* Yasuaki Kagaya,‡
Jerzy Paszkowski,§ Wilhelm Gruissem,** Lars Hennig,**,†† and Shin Takeda*,§,1
*Bioscience and Biotechnology Center, Nagoya University, Chikusa, Nagoya 464-8601, Japan, †Department of Biotechnology,
Faculty of Science, Mahidol University, Bangkok 10400, Thailand, ‡Life Science Research Center, Mie University, Tsu, 514-8507,
Japan, §Department of Plant Biology, University of Geneva, Sciences III, CH-1211, Geneva 4, Switzerland, **Department of Biology
and Zurich-Basel Plant Science Center, ETH Zurich, CH-8092, Zurich, Switzerland, and ††Department of Plant Biology and Forest
Genetics, Faculty of Natural Resources and Agricultural Sciences, Swedish University of Agricultural Sciences, Uppsala, Sweden
ABSTRACT Chromatin reconstitution after DNA replication and repair is essential for the inheritance of epigenetic information, but
mechanisms underlying such a process are still poorly understood. Previously, we proposed that Arabidopsis BRU1 functions to ensure
the chromatin reconstitution. Loss-of-function mutants of BRU1 are hypersensitive to genotoxic stresses and cause release of transcriptional gene silencing of heterochromatic genes. In this study, we show that BRU1 also plays roles in gene regulation in euchromatic regions. bru1 mutations caused sporadic ectopic expression of genes, including those that encode master regulators of
developmental programs such as stem cell maintenance and embryogenesis. bru1 mutants exhibited adventitious organogenesis,
probably due to the misexpression of such developmental regulators. The key regulatory genes misregulated in bru1 alleles were often
targets of PcG SET-domain proteins, although the overlap between the bru1-misregulated and PcG SET-domain-regulated genes was
limited at a genome-wide level. Surprisingly, a considerable fraction of the genes activated in bru1 were located in several subchromosomal regions ranging from 174 to 944 kb in size. Our results suggest that BRU1 has a function related to the stability of
subchromosomal gene regulation in the euchromatic regions, in addition to the maintenance of chromatin states coupled with
heritable epigenetic marks.
E
UKARYOTIC DNA is packaged into highly ordered chromatin structures. Chromatin can develop diverse states
that are characterized by specific epigenetic marks, such as
DNA methylation and histone modifications, or by the presence of histone variants (Felsenfeld and Groudine 2003;
Berger 2007). Chromatin structure is dynamic, but also stable and inherited through mitotic and/or meiotic cell division. This stability provides the basis for epigenetic
regulation. Classically, chromatin has been divided into
euchromatin and heterochromatin (Grewal and Jia 2007;
Copyright © 2011 by the Genetics Society of America
doi: 10.1534/genetics.111.130062
Manuscript received April 28, 2011; accepted for publication June 8, 2011
Supporting information is available online at http://www.genetics.org/content/suppl/
2011/06/24/genetics.111.130062.DC1.
Microarray raw data from this article have been deposited with Array Express under
accession no. E-TABM-1006.
1
Corresponding author: Bioscience and Biotechnology Center, Nagoya University,
Furo-cho, Chikusa, Nagoya 464-8601, Japan. E-mail: [email protected]
Exner and Hennig 2008). Heterochromatin is typically transcriptionally inactive, has a low density of genes, and has
a high density of repetitive sequences and transposable elements. Euchromatin is usually transcriptionally competent,
has a high density of genes, and has a low density of repeats
and transposons. In heterochromatic regions, genes and
transposable elements are silenced and are associated with
methylation of DNA and histone H3 lysine 9 (H3K9). During
the S-phase of the cell cycle, heterochromatic regions are generally replicated later than euchromatic regions (Hiratani
et al. 2009). It is still unclear, however, how chromatin structure is reproduced after DNA replication (Margueron and
Reinberg 2010).
Replication timing and the transcriptional status of genes
correlate not only with chromatin states but also with the
topology of chromosomes and gene positioning within the
nucleus (Cremer and Cremer 2001; Kosak and Groudine
2004; Chakalova et al. 2005; Misteli 2007). In yeast,
Genetics, Vol. 189, 83–95 September 2011
83
transcriptionally inactive telomeres and centromeres are localized to the nuclear periphery, while active chromosomal
regions are more randomly localized to the internal space
(Gasser 2002). In mammals and most other multicellular
organisms, individual chromosomes occupy distinct territories within the nucleus (Croft et al. 1999; Felsenfeld and
Groudine 2003). Within a chromosome territory, transcriptionally active genes reside in spatial regions that allow easy
access for transcription factors (Cremer and Cremer 2001;
Felsenfeld and Groudine 2003). Moreover, changes in the
subnuclear position of genes, along with changes in the local
structure of chromosomes, are often coincident with gene
activation or silencing (Tumbar and Belmont 2001; Lanctot
et al. 2007). These observations suggest that epigenetic information might be linked to the spatial distribution of chromosomal regions.
In addition to transcription and replication, DNA repair is
affected by chromatin structure, because it controls the
accession of DNA repair machinery to damaged sites (Groth
et al. 2007; Huertas et al. 2009). Several proteins involved in
both epigenetic regulation and DNA repair have been identified. These include the SIR proteins, which mediate chromatin compaction in yeast (Gasser and Cockell 2001), and
Chromatin Assembly Factor-1 (CAF-1), which deposits
newly synthesized histone H3 and H4 during nucleosome
assembly after DNA replication and repair (Smith and
Stillman 1989; Probst et al. 2009). Yeast cac mutants and
Arabidopsis fasciata (fas) mutants, which both lack CAF-1
subunits, are hypersensitive to ultraviolet (UV) light and
to reagents that cause double-strand breaks (DSB) and are
unable to maintain epigenetic gene silencing (Kaufman et al.
1997; Takeda et al. 2004; Ono et al. 2006). Defects in CAF-1
activate the DNA damage checkpoint, suggesting that efficient chromatin reconstitution after DNA replication is required for cell-cycle progression (Tchenio et al. 2001; Hoek
and Stillman 2003; Ye et al. 2003). Acetylation of histone
H3 lysine 56 (H3K56Ac) has an important role in regulating
chromatin assembly following DNA synthesis and DNA damage response (Masumoto et al. 2005; Driscoll et al. 2007;
Han et al. 2007; Li et al. 2008) and chromatin disassembly
during transcriptional activation (Williams et al. 2008).
Arabidopsis BRUSHY1 (BRU1) was identified as a protein
whose absence resulted in hypersensitivity to DNA damage
and release of transcriptional gene silencing (TGS) (Takeda
et al. 2004). Because of phenotypic similarity to CAF-1 defect, BRU1 was proposed to function in the inheritance of
chromatin states during DNA replication (Takeda et al.
2004; Endo et al. 2006). bru1 is proficient in DSB repair,
but exhibits high frequencies of homologous recombination
and a constitutive DNA damage response. The extent of TGS
release in bru1 is modest like in fas, however, compared to
typical TGS mutants, such as met1, ddm1, or mom1 (Vongs
et al. 1993; Jeddeloh et al. 1998; Amedeo et al. 2000).
Reactivation of silent loci in bru1 and fas is rather stochastic,
as reflected by the instability of heterochromatin compaction (Takeda et al. 2004; Ono et al. 2006; Schönrock et al.
84
Y. Ohno et al.
2006; Kirik et al. 2006). Overlapping functions of BRU1 and
CAF-1 during chromatin reconstitution after DNA replication and/or DNA repair was suggested by analysis of bru1
fas double mutant (Takeda et al. 2004). BRU1 was also
reported as TONSOKU (TSK) and MGOUN3 (MGO3) on
the basis of different phenotypic mutant screens (Suzuki
et al. 2004; Guyomarc’h et al. 2004). Collectively, the mutant alleles bru1, tsk, and mgo3 exhibit pleiotropic developmental abnormalities similar to those observed in fas,
including fasciation (shoot meristem disorganization), disordered phyllotaxy (Leyser and Furner 1992; Kaya et al.
2001), short roots, organ fusion, and changes in the
floral organ number (Suzuki et al. 2004; Takeda et al. 2004;
Guyomarc’h et al. 2004).
The activation or inactivation of key transcription factors
in eukaryotic developmental processes is often maintained
by epigenetic control, such as that mediated by the
Polycomb group (PcG) and Trithorax proteins (Köhler and
Villar 2008; Simon and Kingston 2009). PcG proteins are
required for the maintenance of repressive states and provide a basis for cellular memory (Schuettengruber et al.
2007; Hennig and Derkacheva 2009). PcG protein function
involves trimethylation of histone H3K27 (H3K27me3) by
Polycomb repressive complex 2 (PRC2) in animals and
plants. In mammals and Drosophila, H3K27me3 assists in
the recruitment of another PcG protein complex, PRC1,
which is required for transcriptional silencing (Hennig and
Derkacheva 2009). Plants lack clear homologs for some essential PRC1 subunits. Instead, LIKE HETEROCHROMATIN
PROTEIN1 (LHP1) is often associated with PcG target loci
and has been proposed to substitute for the function of PRC1
(Turck et al. 2007; Zhang et al. 2007a; Exner et al. 2009).
In this study, we assessed BRU1 function in euchromatic
gene regulation. We show that bru1 causes sporadic adventitious organogenesis and ectopic expression of developmentally regulated genes, some of which are normally repressed
by PcG proteins. Genome-wide analyses showed limited
but significant overlap between BRU1- and PcG proteinmediated regulation, and preferential distribution of genes
upregulated in bru1 on several subchromosomal domains.
Our results suggest that BRU1 ensures epigenetic regulation
of a subset of euchromatic genes at at least two levels, one
associated with H3K27me3 and/or repression by PcG proteins and the other associated with the subchromosomal
region.
Materials and Methods
Plant material and growth conditions
The bru1-1 (accession Wassilewskija, Ws) and bru1-2 (accession Columbia, Col) alleles were previously described
(Takeda et al. 2004). The tsk3 (accession Col) allele (Suzuki
et al. 2004) was kindly provided by K. Nakamura (Nagoya
University, Nagoya, Japan). To construct a pFUS3::GUS reporter line, 1.9 kb of the FUS3 promoter and 59-UTR
sequences were fused to a GUS cDNA and introduced into
Col wild-type (WT) plants. A bru1-2 pFUS3::GUS line was
generated by crossing. The presence of the GUS transgene
was determined using PCR with forward (59-TGAAGATGCG
GACTTACGTG-39) and reverse (59-TGAGCGTCGCAGAAC
ATTAC-39) primers. To select lines carrying homozygous
pFUS3::GUS, the segregation pattern of the progeny was
analyzed using GUS staining of seeds. The genotype of
bru1-2 was determined using PCR with forward (59-TGT
GAAGCCATTCAGAGTGCT-39) and reverse (59-TCTCACCT
CCTCTTCATCGCAAATCTGagGCC-39) primers, which introduce a StuI site specific for the bru1-2 allele.
Seeds were sown on plates containing half-strength
Murashige and Skoog (MS) medium (Wako, no. 39200591, Tokyo, Japan) supplemented with 0.5 mg/liter
nicotic acid, 0.1 ml/liter thiamine HCl, 0.5 mg/liter pyridoxine HCl, 2 mg/liter glycine, 50 mg/liter myo-inositol,
10 g/liter sucrose, and 1.5% agar. After stratification at 4°
for 2–3 days, plates were placed vertically and incubated
for 7 days under continuous light (100 mmol/m2/sec) at
22°. Seedlings were transferred to pots filled with rock-fiber
supplemented with 3000 times diluted Hyponex (Hyponex
Japan, Osaka, Japan). For the expression analysis of seedspecific genes in seedlings, at least 15 seedlings were transferred into 10 ml of liquid half-strength MS medium and grown
for 4 days at 22° under continuous light (40 mmol/m2/sec)
with gentle rotation at 90 rpm. For microarray experiments,
plants were grown in soil in long-day photoperiods (16 hr
light/8 hr dark) at 21° during the day and at 16° during the
night.
RT–PCR
Total RNA was extracted from plant tissues with the
RNeasy mini kit (QIAGEN, Valencia, CA); 1 mg of total RNA
was treated with 10 units of DNase I (Roche, Mannheim,
Germany) at 37° for 30 min and was then extracted with
phenol-chloroform-isoamylalcohol (25:24:1), before reverse
transcription with an oligo(dT) primer and the Omniscript
Reverse Transcription kit (QIAGEN). For quantitative RT–
PCR, 1 mg of total RNA was used for reverse transcription
with the Quantitect Reverse Transcription kit (QIAGEN). Real-time PCR was performed with Power SYBR Green PCR
Master Mix (Applied Biosystems, Warrington, UK) and ABI
PRISM 7000 (Applied Biosystems). Aliquots of cDNA were
amplified using PCR with gene-specific primers as listed (supporting information, Table S5).
Histochemical GUS staining
Seeds or 3-week-old plants were soaked in GUS staining
solution [1 mM 5-bromo-4-chloro-3-indolyl-b-d-glucuronide
(X-Gluc), 10 mM EDTA, 0.5 mM potassium ferricyanide,
0.5 mM potassium ferrocyanide, 50 mM sodium phosphate
(pH 7.0)], gently degassed twice for 30 min, incubated at
37° for 3 days and cleared with 70% ethanol. Stained spots
or sectors were observed under a stereomicroscope (Leica
MS5, Wetzlar, Germany).
Transcriptome analysis and statistical analyses
RNA was extracted from rosette leaves of 24-day-old plants
grown under long-day photoperiods. Labeling and hybridization to Affymetrix ATH1 microarrays (Affymetrix, Santa
Clara, CA) was performed as described (Hennig et al. 2003).
Signal values were derived from .CEL files using GCRMA in
the statistic language R (http://www.r-project.org/). To
identify differentially expressed genes between WT and
bru1-2, the linear models for microarray data (limma) algorithm (Smyth 2004) was used with multiple testing correction according to (Benjamini and Hochberg 1995) [critical
P-value = 0.05, signal log ratio (SLR) .1 or ,21]. The
chromosomal localization of genes upregulated in bru1-2
was determined by using the chromosome map tool at TAIR
(http://www.arabidopsis.org/jsp/ChromosomeMap/tool.jsp).
When the probe set hybridized to transcripts from more than
one gene, a gene associated with H3K27me3, located in the
five subchromosomal domains, or with the lowest AGI code
number was displayed as a representative. Subchromosomal
domains were defined with nonrandom distribution of at
least five neighboring genes upregulated in bru1 (SLR .
1). The domains are selected on the basis of the number
of the upregulated genes per length of the chromosomal
region and further verified considering the degree of the
overrepresentation of the genes upregulated in bru1 among
the probed genes (.10-fold enrichment) and the statistical
significance (P-value , E-05) determined by hypergeometric test (the total number of successes: 148 [total upregulated genes in bru1], parent population: 24,122 [total genes
that were examined by ATH1 arrays]). For mapping of differentially expressed genes in fus3, we selected the genes
from the transcriptomic data set for the developing seeds
(12 DAF; Yamamoto et al. 2010). Analysis of tissue-specific
expression using Shannon entropy was performed as described (Schug et al. 2005; Zhang et al. 2006) using the
AtGenExpress developmental gene expression atlas (Schmid
et al. 2005).
Genes that were expressed in rosette leaves at levels
lower than those in other organs in WT were selected using
data from AtGenExpress (Schmid et al. 2005) [expression
profiles in root (sample ID ATGE_9), rosette leaf 4 (1 cm
long), 2, 4, 6, 8, 10, 12 (ATGE_10, 12, 13, 14, 15, 16, 17),
entire rosette leaves (ATGE_22), shoot apex (ATGE_29),
sepals, petals, stamen, carpels (stage 15; ATGE_41, 42,
43, 45), mature pollen (ATGE_73), seeds (stages 4-10;
ATGE_77, 78, 79, 81, 82, 83, 84)]. We defined an ideal
nonleaf gene expression pattern as 0 in rosette leaves of
any stage and 1 in at least one nonleaf sample. Genes with
expression excluded from leaves were selected on the basis
of the Pearson correlation coefficients for all 22,746 probe
sets with the defined reference pattern (correlation coefficient .0.6 and SLR $4 between the nonleaf organ with
highest expression and the rosette leaf with highest expression). Microarray raw data are available at ArrayExpress
(accession no. E-TABM-1006).
Euchromatic Gene Regulation by BRU1
85
Figure 1 BRU1 functions in the nucleus in Arabidopsis. (A) Complementation of bru1-1 by the
BRU1-GFP fusion gene. Sensitivity to MMS of
wild-type (WT) Ws, bru1-1, and bru1-1 transformants carrying pBRU1::BRU1:EGFP (lines 1–4) is
shown. (Left) Two representative seedlings after
treatment with the indicated concentrations of
MMS are shown. (Right) The survival rate of the
seedlings after treatment with MMS is plotted.
For each treatment, 23 seedlings each of WT and
bru1-1 and 12 T2 seedlings for each of the respective transgenic lines were tested. The copy number
of the transgene in the parental T1 plants was one
(line 1), two (line 2), three (line 3), and more than
five (line 4). (B) Localization of BRU1–GFP in the
nucleus. (Left) GFP signals (green) in the root-tip
of a 10-day-old seedling of bru1-1 carrying
pBRU1::BRU1:EGFP (line 4) observed using a confocal laser microscope. The inset shows the root tip
cells of a non-transformed bur1-1 seedling as
a negative control. (Right) A magnified view of
the root tip cells of bru1-1 carrying pBRU1::BRU1:
EGFP. Cells were visualized by counter staining
with PI (red). Scale bars, 50 mm.
To select upregulated genes in clf swn, we used microarray raw data of clf swn at Gene Expression Omnibus (GEO;
accession no. GSE20256). Signal values were derived from
.CEL files using GCRMA in the statistic language R (http://
www.r-project.org/). Upregulated genes were selected using
the rankproduct algorithm (Breitling et al. 2004) [critical
P-value = 0.05, SLR .1].
Complementation of bru1-1 by pBRU1::BRU1:GFP
To express a BRU1-GFP fusion protein under the control of
the BRU1 promoter, a KpnI–NheI genomic fragment, which
contained the BRU1 promoter, the 59 UTR, the first exon and
intron, and a part of the second exon, was fused to the
BRU1–EGFP fusion gene (Takeda et al. 2004) and inserted
into the binary vector pCAMBIA1301 (Figure S1). The
resulting transgene was introduced into bru1-1. The copy
number of transgenes in the transgenic lines was determined by DNA gel blot analysis. For complementation
assays, MMS sensitivity (Takeda et al. 2004) and root length
of the transgenic lines were examined.
Subcellular localization of BRU1-GFP
For detecting GFP signals, root cells of bru1-1 carrying
pBRU1::BRU1:GFP were observed using a confocal laser
scanning microscope (FV500, Olympus, Tokyo, Japan) after
counterstaining with propidium iodide (PI).
Results
BRU1 functions in the nucleus in Arabidopsis
BRU1/TSK is localized to the nucleus when fused to GFP
and overexpressed in tobacco protoplasts or cells (Suzuki
86
Y. Ohno et al.
et al. 2004; Takeda et al. 2004). Here, we examined the
functionality of nuclear-localized BRU1 in Arabidopsis using
transgenic complementation. We created transgenic bru1-1
plants that express a BRU1-EGFP fusion protein under the
control of the native BRU1 promoter (pBRU1::BRU1:EGFP)
(Figure S1). The bru1 fasciation phenotype was fully complemented in four independent transgenic lines. The T2
progenies of these lines also showed partial recovery
from the methyl methanesulfonate (MMS; causing DSBs)hypersensitive and root growth phenotypes (Figure 1A and
Figure S1) The BRU1–EGFP fusion protein was detected
in the nucleus of root tip cells and was partially excluded
from the nucleolus (Figure 1B). These results indicate that
BRU1 functions in the nucleus in Arabidopsis, consistent
with a role for BRU1 in the DNA damage response and in
epigenetic gene regulation (Takeda et al. 2004).
Adventitious organ initiation in bru1
In addition to the previously reported morphological
aberrancies of bru1 alleles, we found that bru1-2 plants
exhibited sporadic adventitious organ formation on leaves
that were often accompanied by alterations in leaf shape
(frequency of 2–10%) (Figure 2). These adventitious
organs developed from the mid-rib on the abaxial side (Figure 2, B and C) or from the surface on the adaxial side
(Figure 2D) of leaves. Because of their apparent radial
symmetry, they resembled leaf needles with trichomes.
This phenotype was observed for the bru1-2 and bru1tsk3 alleles in the Col background (Figure 2E) but not for
bru1-1 in WS.
It is known that ectopic expression of SHOOT MERISTEMLESS
(STM), a class I KNOX family gene, which regulates meristem
Figure 2 Ectopic organogenesis in bru1. (A) A representative rosette leaf of WT (Col). (B–E) Adventitious organ
formation on the rosette leaves of bru1-2 (B–D) and tsk3
(E). The ectopic organogenesis is indicated with red
arrows. (B and C) Needle leaf-like organs, carrying trichomes, generated on the mid-rib of the abaxial side of
the leaf. Note that the rosette leaf in B has serrated edges.
(D) An adventitious organ was generated on the adaxial
side of the heart-shaped leaf. (E) An adventitious organ
was generated on the abaxial side of the heart-shaped
leaf.
organization (Long et al. 1996), induces ectopic organogenesis (Gallois et al. 2002). We therefore examined a possibility that bru1 causes ectopic activation of genes, such as
STM, that may lead to a reprogramming of differentiated
cells. In WT plants, STM is mainly expressed in meristems
and much less in leaves. In bru1 alleles, sporadic accumulation of STM transcripts was observed in rosette leaves,
even though at much lower abundance than in WT inflorescences (Figure 3A and Figure S2). Since such sporadic
activation of STM was observed in both Ws and Col backgrounds, the ectopic organogenesis in bru1 cannot be
explained by misexpression of STM alone and probably
correlates with the accession.
bru1 perturbs developmentally regulated
gene expression
In bru1-mgo3, several flower-specific genes were ectopically
expressed in leaves (Guyomarc’h et al. 2006). To further
assess the effect of bru1 on developmentally regulated
genes, we analyzed the expression of two seed-specific
marker genes: the transcriptional activator gene FUSCA3
(FUS3), which is essential for seed maturation and dormancy (Giraudat et al. 1992; Luerssen et al. 1998), and
the FUS3 target gene LOB40 (Yamamoto et al. 2010). Ectopic expression of FUS3 and LOB40 were often observed in
seedlings and leaves of bru1 mutants (Figure 3B and Figure
S2). However, misregulation of LOB40 in bru1 cannot be
explained by FUS3 activation alone. Under the condition
in which expression of FUS3 was activated in bru1-2 seedlings, but only slightly in bru1-tsk3, expression of LOB40 was
activated in both alleles (Figure 3B). This suggests that the
ectopic expression of these seed-specific genes is not primarily due to changes in the developmental state of the cells
and is likely locus dependent.
Stochastic expression of FUS3-GUS induced by bru1
In a previous study, TGS was released in bru1 in a stochastic
manner (Takeda et al. 2004). We tested whether the ectopic
expression of a euchromatic gene was also induced stochastically in bru1 by using a FUS3–GUS reporter (pFUS3::GUS)
crossed into bru1-2. GUS reporter activity was detected as
scattered spots or sectors more frequently (.3.3-fold) in
bru1 than in the segregating WT plants (Figure 4). Large
spots or sectors in leaves or roots (Figure 4, D–F) were
observed in bru1-2 but seldom in WT. In one of the bru1-2
seedlings, pFUS3::GUS was remarkably activated in a whole
ectopic shoot emerging from the hypocotyl tissue adjacent to
the base of a cotyledon (Figure 4, G and H). The three leaflike structures on the ectopic shoot resembled cotyledons
rather than true leaves because they did not bear trichomes.
Ectopic expression of FUS3 was reported to cause conversion
of vegetative leaves to embryonic leaves (Gazzarrini et al.
2004). Considering that pFUS3::GUS was activated throughout this shoot, it is likely that upstream activators of FUS3
were activated in the ectopic tissue. Potential upstream activators of FUS3, such as AGL15, LEC1, LEC2, or BBM (Karami
et al. 2009), can induce somatic embryogenesis and result in
the development of organs with embryonic characters (see
Discussion for details). Together, these results indicate that
BRU1 is needed to prevent stochastic reactivation of genes
that are tightly regulated by specific developmental programs in WT.
A fraction of genes upregulated in bru1 are targeted
by PcG proteins
It was reported that expression of STM and FUS3 is repressed by PcG proteins in vegetative tissues (Makarevich
et al. 2006; Schubert et al. 2006). Another direct PcG protein
Euchromatic Gene Regulation by BRU1
87
genes among the upregulated genes in the clf swn double
mutant of the PcG SET domain proteins SWINGER (SWN)
and CURLY LEAF (CLF) (Chanvivattana et al. 2004). We
found a more significant enrichment of clf swn-induced
genes in the upregulated genes in bru1 (Figure S3), although the genes upregulated in the clf swn double mutant
do not necessarily overlap with genes that are associated
with H3K27me3 (Figure 5, A and B). These results indicate
that a portion of genes upregulated in bru1 is under the
control of PcG proteins.
Genes upregulated in bru1 are often localized in a few
subchromosomal regions
Figure 3 bru1 causes ectopic expression of genes. (A) Expression of STM
in the rosette leaves of 31-day-old plants of WT and bru1 alleles. Expression levels were analyzed using quantitative realtime RT–PCR. For each
analysis, reverse transcription was performed with mixture of RNA samples prepared from four to five individuals. Data are normalized to ACT2,
and expression levels of WT (Ws or Col-0) are set as 1. Mean 6SE, n ¼ 3
(biological replicates). (B) Expression of FUS3 and LOB40 in 13-day-old
seedlings in WT and bru1 alleles. Data are normalized to ACT2, and
expression levels of WT (Ws or Col-0) are set as 1. Mean 6SE, n ¼ 3
(for Ws and bru1-1) or n ¼ 2 (for Col-0, bru1-2 and tsk3).
target gene AG (Schubert et al. 2006) was also activated in
bru1-mgo3 (Guyomarc’h et al. 2006). These raise a question
whether genes misexpressed in bru1 are target genes of PcG
proteins. Genes repressed by PcG complexes were targeted
by H3K27me3 and LHP1 (Turck et al. 2007; Zhang et al.
2007a,b). On the basis of published data for genome-wide
distribution of these marks, most of the genes that were
misregulated in bru1 alleles were associated with
H3K27me3 and LHP1 in vegetative tissues (Zhang et al.
2007a,b) (Table S1). We further examined whether genes
carrying H3K27me3 were enriched among the genes misregulated in bru1 on a genome-wide scale. We first identified
genes that were misregulated in bru1-2 leaves using ATH1
microarrays. We found 148 genes whose expression levels
were increased more than twofold in bru1-2 (SLR . 1)
(Figure 5, A and B). Among them, 37 genes (25%) were
associated with H3K27me3, which is a 1.6-fold enrichment
over the fraction found for the whole genome (16%) (Zhang
et al. 2007b) (Figure S3). The significance of this enrichment was not as striking as that of H3K27me3-associated
88
Y. Ohno et al.
We examined the chromosomal positions of the 148 genes
upregulated in bru1. Surprisingly, 56 genes (38% of the
upregulated genes) were found to be localized to five subchromosomal segments, ranging from 174 to 994 kb in size
(regions I–V; Figures 5, B and C, and 6, Table 1, and Figure
S3). For example, 13 genes (9%) were found in a 174-kb
region of chromosome 4 (region I). The ATH1 microarray
contains probe sets for 38 genes in this region, indicating
that 34% of the genes in this region were upregulated in
bru1. Similarly, 10 upregulated genes were located in
a 452-kb region of chromosome 3 (region II). Such nonrandom positioning of the affected genes on chromosomes
was not obvious for genes upregulated in the clf swn, fus3
(Figure S4, Figure S5, and Table S2), nor for downregulated
genes in fus3 (Figure S6 and Table S2). Over the 195 kbregion containing region I, any changes in size were detected
in bru1 (at a resolution of 0.5 kb) in 24 PCR-amplified genomic fragments. Therefore, upregulation in the specific chromosomal regions was unlikely due to local rearrangements
or large insertions and/or deletions.
Genes in the five subchromosomal regions were not
preferentially associated with H3K27me3 or PcG regulation
(Figure 5, A and B). In the subchromosomal region I, upregulation of genes in bru1 did not correlate with well-known
repressive histone and DNA modifications (Figure 5C) and
thus were not primarily due to reactivation of repressed
genes. Moreover, none of these regions were close to heterochromatic regions marked with H3K9me2 and DNA
hypermethylation. This suggests that bru1 can affect gene
regulation in the subchromosomal regions independently of
H3K9me2, H3K27me3, and DNA methylation.
A subset of genes upregulated in bru1 outside of the
subchromosomal regions is developmentally regulated
We next tested whether bru1 preferentially affected developmentally regulated genes. As reported before, genes carrying H3K27me3 have lower Shannon entropy than the
genome-wide average, which indicates tissue-specific expression (Zhang et al. 2007b) (Figure 7). The same tendency was observed with the genes upregulated in clf swn,
consistent with the role of PcG in developmental regulation
of genes (Köhler and Villar 2008). Similarly, upregulated
genes in bru1 had reduced entropy values on average,
Figure 4 bru1 reactivates the FUS3 promoter in a stochastic manner. Three-week-old bru1-2 or WT plants that were
homozygous for a pFUS3::GUS transgene were examined
by histochemical GUS staining. The patterns of GUS expression were classified into the following four types in
terms of staining frequency and intensity: Type 1, 1 or 2
small stained spots of ,0.2 mm diameter, observed in
leaves; type 2, 3–10 small spots observed in leaves; type
3, at least 10 small spots, or larger sectors with .0.2 mm
diameter observed in leaves; and type 4, GUS activity
detected in other tissues than leaves. Tissues (A) without
or (B–H) with representative expression patterns are
shown. (A and B) Rosette leaves of the segregated WT
plants carrying the homozygous pFUS3::GUS. The GUS
staining is indicated by a red arrow. (C–E) Rosette leaves
of the segregated bru1-2 plants carrying the homozygous
pFUS3::GUS reporter. (F) A GUS signal (red arrow) in the
root of a segregated bru1-2 plant carrying the homozygous pFUS3::GUS reporter. (G) A segregated bru1-2 plant
with ectopic organogenesis, carrying the homozygous
pFUS3::GUS reporter. GUS activity was detected in an ectopic organ generated on the hypocotyl (red arrow) and in
a sector of a petiole (red arrowhead). (Inset) The adaxial
side of a leaf detached from the ectopic branch. co, cotyledon. hy, hypocotyl. (H) A magnified view of the intact
ectopic organ shown in G. (I) Frequency of GUS activities
detected in the segregated WT and bru1-2 plants. Percentages of plants with respective types of GUS expression
among total plants examined (WT, n ¼ 64; bru1-2, n ¼
38) are indicated. Bars, 1 mm.
suggesting that some of them are expressed in a tissuespecific manner (Figure 7). However, tissue specificity of
the upregulated genes located in the five subchromosomal
regions did not differ from the genome average. In contrast,
a subset of genes upregulated in bru1 outside of the subchromosomal regions is developmentally regulated. We further selected genes that were expressed at least 16 times
higher in nonleaf tissues than in leaves, on the basis of
published gene expression data (Schmid et al. 2005). The
respective tissue-specific gene sets were not significantly
enriched in genes upregulated in bru1 leaves, except for
seed-specific gene sets (Table S3).
Five genes that upregulated in bru1 are responsive to
genotoxic stress (Schönrock et al. 2006) (Figure 6). This
is consistent with previous observations that genotoxic
stress-responsive genes were upregulated in bru1 (Takeda
et al. 2004). These upregulated genotoxic stress-responsive
genes were neither associated with H3K27me3 nor located
in one of the subchromosomal regions described above.
Taken together, most euchromatic genes with altered expression in bru1 can be classified into the three following
groups: (1) genes associated with H3K27me3 or misregulated in PcG mutants; (2) genes located in specific subchromosomal regions; and (3) genes responsive to DNA
damage. Interestingly, the other genes often encoded transcriptional regulators or DNA binding proteins (10 of 46;
2.6-fold enrichment, P-value = 3.2E-3, hypergeometric
test), which often were related to stress responses (5 of
10) (Table S4).
Recently, UVB has been shown to induce the release of
TGS for certain transgenes (Lang-Mladek et al. 2010). This
indicates that environmental stressors, such as UVB can affect chromatin states, potentially perturbing the regulation
of a subset of endogenous genes. Indeed, genes responsive
to UVB stress (Kilian et al. 2007) were significantly enriched
in the upregulated genes in bru1 (Table S3). So far, we
found nothing striking about the genes downregulated in
bru1 (Figure S3).
Discussion
Chromatin reconstitution after DNA replication and repair is
essential for genome integrity and is the foundation for the
inheritance of epigenetic information. Histone chaperones
such as CAF-1 play a critical role in chromatin assembly
following DNA synthesis and deactivation of DNA damage
checkpoints (Kim and Haber 2009), where incorporation of
H3K56Ac is suggested to be a signal for completion of repair
(Chen et al. 2008). It has been suggested that BRU1 function
overlaps with that of CAF-1 and that bru1 causes instability
in chromatin replication that results in a constitutive activation of the DNA repair response and a perturbation of
epigenetic regulation (Takeda et al. 2004). In bru1, TGS
of heterochromatic genes, which are associated with
H3K9me2 and DNA hypermethylation, is released. In addition, WUSCHEL, that is specific for the shoot apical meristem
(SAM) (Mayer et al. 1998), is misregulated in bru1 as well
as in fas mutants (Takeda et al. 2004; Kaya et al. 2001).
Euchromatic Gene Regulation by BRU1
89
Figure 5 Transcriptomic analysis of the upregulated genes in bru1. (A) Venn diagram illustrating the overlap of genes associated with
H3K27me3, genes upregulated in clf swn, and
genes upregulated in bru1-2 (SLR . 1) among
the total (22,746) genes probed by ATH1
arrays. The number of genes that belong to
each group is indicated in parentheses. (B)
Venn diagram of the classified genes upregulated in bru1-2. The 148 upregulated genes in
bru1-2 identified in the microarray experiment
were classified into the following three groups:
genes associated with H3K27me3, genes upregulated in clf swn, and genes localized within
the five subchromosomal domains (I–V, shown
in Figures 5C and 6 and Table 1). The number
of genes that belong to each group is indicated
in parentheses. (C, left) Heat map view of expression of genes on the upper part of the chromosome 4 (short arm) in bru1. Expression levels
in bru1-2 relative to those in WT (Col) (three
biological replicates) are shown with SLR. Note
that genes upregulated in bru1 were enriched
in a subchromosomal region (region I; for chromosomal location, see Figure 6). (Right) The
gene loci on the subchromosomal region I are
indicated with the levels of upregulation in bru1
and with repressive histone and DNA modifications in seedlings that were reported by Roudier
et al. (2011). H3K9me2, dimethylation at K9 of
histone H3. H3K27me3, tri-methylation at K27
of histone H3. mC, 5-methyl cytosine (DNA
methylation). The loci of genes that were upregulated in bru1 (SLR . 1) are shaded. Bars in
the graph show the expression levels in bru1-2
relative to those in WT (Col) (SLR, mean values).
Blue bars, three SLR scores were .1. Gray bars,
one of three SLR scores was ,1.
However, it is not clear whether the misexpression of
WUSCHEL is directly caused by local chromatin defects or
whether it is an indirect consequence of a disorganized meristem that results from uncoordinated cell-cycle progression
(Suzuki et al. 2004; Suzuki et al. 2005a,b).
In this study, we have demonstrated that SAM- and seedspecific genes are ectopically activated in leaves of bru1. It is
plausible that loss of BRU1 affects gene repression mediated
by heritable epigenetic marks. Indeed, a fraction, but by no
means a majority, of genes upregulated in bru1 were associated with H3K27me3 or PcG-mediated regulation. Adventitious organ induction in bru1 may reflect stochastic
misregulation of genes encoding key regulators of developmental processes. This is supported by the observed ectopic
expression of pFUS3::GUS, which was strongest at sites of
ectopic shoot formation on the hypocotyls tissue. Moreover,
the ectopic leaf-like organs lack trichomes, suggesting a reversion from vegetative to embryogenic identity. We speculate that the leaf-like organs arose from a somatic embryo.
90
Y. Ohno et al.
Ectopic expression of some key regulators of embryogenesis,
such as AGL15, LEC1, LEC2, or BBM, are known to induce
formation of somatic embryos (Harding et al. 2003;
Lotan et al. 1998; Stone et al. 2001; Boutilier et al. 2002;
Braybrook et al. 2006; Thakare et al. 2008). Because FUS3
is a direct target of AGL15 (Zheng et al. 2009), it is possible
that FUS3 functions in the formation of somatic embryos.
Interestingly, somatic embryogenesis is also induced in the
clf swn double mutant (Chanvivattana et al. 2004) and in
loss-of-function mutants of certain genes predicted to encode subunits of chromatin remodeling complexes. These
proteins include PICKLE (Ogas et al. 1997) and VP1/
ABI3-LIKE (VAL) (also known as HSI2) (Suzuki et al. 2007;
Tsukagoshi et al. 2007). Thus, formation of somatic embryos
is repressed by chromatin-based mechanisms. The reversion
from vegetative-to-embryogenic identity in bru1 may be
complete and involve formation of somatic embryos, but it
may also be incomplete, leading to the formation of leaves
with partial embryonic characters directly from vegetative
Figure 6 Chromosomal localization of the upregulated genes in bru1. The upregulated genes in bru1-2 (SLR . 1) identified in the microarray
experiment were plotted on a chromosomal map. Green stars represent genes associated with H3K27me3 (Zhang et al. 2007b). Solid blue circles
represent genes responsive to DNA damages. Overrepresentation of upregulated genes is observed in the five subchromosomal regions (I–V; see
Table 1) indicated by light blue bars.
tissue. In either case, BRU1 is required to maintain repression
of genes that are specific for seed development and/or embryogenesis, most likely by reestablishing repressive local epigenetic chromatin states after DNA replication.
Surprisingly, many genes that were upregulated in bru1-2
were located in the five subchromosomal regions. Such nonrandom positioning of the affected genes implies a function
of BRU1 in a yet-unknown machinery of gene regulation.
BRU1 might function to compact and repress both constitutive heterochromatin (Takeda et al. 2004) and sequences in
a euchromatic environment. However, it is unlikely that
these regions adapt a facultative heterochromatic state in
leaves, because genes from these regions are not necessarily
less expressed in WT leaves than in other organs (Schmid
et al. 2005). Recently, 12 epigenetic marks were collectively
mapped on chromosome 4 of Arabidopsis (Roudier et al.
2011). In the subchromosomal region I located on this chromosome, however, we could not find any striking overlap
between the altered gene expression in bru1 and the pat-
terns of these epigenetic marks identified by Roudier et al.
(2011). Their report also suggested that the distribution
pattern of these marks defines four main chromatin states,
translating into relatively shorter chromatin domains. These
chromatin domains were typically ,20 kb (except for the
long heterochromatic knob or centromeric regions) and
much smaller than the subchromosomal domains found in
our study, which were .174 kb. It is therefore reasonable
that bru1 mutants could have defects in a different level of
chromatin organization, such as subchromosomal structures
and/or their positioning in specific subnuclear spaces. In
animals, yeast, and plants, chromosomes are not randomly
positioned in the nucleus but are regularly arranged to form
chromosome domains, whose configuration could affect
transcriptional states and replication timing (Cremer and
Cremer 2001; Kosak and Groudine 2004; Chakalova et al.
2005; Misteli 2007; Lieberman-Aiden et al. 2009; Schubert
and Shaw 2011). The defect of BRU1 may perturb the configuration of such chromosome domains, which would be
Euchromatic Gene Regulation by BRU1
91
Table 1 Enrichment of genes upregulated in bru1 in the subchromosomal regions
Region no.
I
II
III
IV
V
a
Chr.
From (base)
To (base)
Distance (kb)
The number
of gene probes
The number of gene probes
upregulated in bru1-2
Degree of
enrichmenta
4
3
3
1
1
261655
10526444
3636789
23158125
19337026
435193
10978591
4051567
24152197
19836381
174
452
415
994
499
38
68
114
232
104
13
10
9
17
7
55.8
24.0
12.9
11.9
11.0
P-value
a
,2.20E-16
1.16E-11
3.40E-08
7.44E-14
3.41E-06
Statistical significance of enrichment of genes upregulated in bru1-2 in the respective regions is indicated with p-values (hypergeometric test).
linked to yet-unknown epigenetic information and heritable
over DNA replication, resulting in the alteration of the transcription states of genes in the particular chromosomal
regions.
In animals, patterns of distribution of chromatin domains
are often similar among differentiated cells of the same
lineage but vary depending on cell types, possibly reflecting
tissue-specific patterns of gene expression (Francastel et al.
2000; Leitch 2000). In contrast, however, the genes that
were upregulated in bru1 and in the subchromosomal
domains were not linked to tissue-specific expression.
Therefore, features of subchromosomal domains in plants
might be different from those known in animals. It was
reported in Arabidopsis that chromosomal arrangement is
relatively random and that no significant difference was
found in the subnuclear position of the FWA gene between
the active and inactive state of the gene (Pecinka et al.
2004). These findings imply that transcriptional regulation
linked to chromosomal domains is not necessarily a universal
system. Future research is needed to determine whether
Figure 7 Tissue specificity of genes with increased expression in bru1.
Values of Shannon entropy of genes in the indicated categories are
shown in a logarithmic scale. Lower entropy values mean higher tissue
specificity. Box plots indicate the interquartile range, and whiskers represent the range of maximum and minimum values. Median values are
represented by magenta lines. The difference in entropy values between
category 1 (total) and other categories, but not category 5 (subchromosomal domain I–V), was statistically significant (P-value , 0.05, multiple
comparison Steel–Dwass test).
92
Y. Ohno et al.
BRU1 is involved in the formation and organization of chromosomal domains.
Several lines of evidence suggest that in Arabidopsis, adjacent genes are coexpressed with higher frequencies than
expected by chance. For example, several genes that are
up- or downregulated in a mutant lacking ABI5, which is
normally involved in the gene regulation during late
embryogenesis, are locally clustered (Nakabayashi et al. 2005).
Zhan et al. (2006) reported that more than 10% of genes
fall into groups of two to nine neighboring genes that are
significantly coexpressed in Arabidopsis. In both cases, the
sizes of the gene clusters are much smaller than the size of
the subchromosomal regions found in this study. Therefore,
subchromosomal domains affected in bru1 may have a role
that differs from the short-range effects described before
(Nakabayashi et al. 2005; Zhan et al. 2006).
Importantly, BRU1 and CAF1 have been demonstrated to
have distinct but overlapping roles in epigenetic regulation
(Takeda et al. 2004). There are two nonexclusive models for
how histone chaperones are involved in heterochromatin
silencing (Huang et al. 2007). The first model is that histone
chaperones function to ensure the maintenance of heterochromatin. Defects in histone chaperones leads to a reduction in nucleosome density during chromatin reassembly
after DNA synthesis and results in heterochromatin instability. In this model, other repressive chromatin domains would
also be unstable after replication. The second model shows
that histone chaperones mediate heterochromatin formation
through direct interactions with silencing proteins or other
chromatin constituents, such as HP1 or the Sir proteins. In
yeast, the Sir proteins are mislocalized in cells lacking CAF-1
and Rtt106p, both of which interact with the Sir proteins
(Huang et al. 2007). In mouse cells, the interaction of the
p150 subunit of CAF-1 with HP1, a heterochromatin constituent, is critical for replication of heterochromatin (Quivy
et al. 2008). In plants, however, direct binding of CAF-1 to
proteins specific for heterochromatin has not been demonstrated. Our results suggest that, in addition to heterochromatin silencing, gene regulation in euchromatic domains is
also perturbed in bru1. Together with the observation that
bru1 causes instability in heterochromatin compaction
(Takeda et al. 2004), our results may imply that bru1 affects
a broad spectrum of epigenetic regulation. Further studies of
BRU1, especially the identification of components of a potential BRU1 protein complex, will provide a better understanding of how and to what extent transcription and chromatin
states are regulated by a gene’s position in particular subchromosomal domains.
Acknowledgments
The authors thank Daisuke Ogawa, Yosuke Toda, and
Tokunori Hobo for help with the statistical analysis of
microarray data; Chiharu Ueguchi, Hanin Moez, Ken-ichi
Kurotani, and Taisuke Nishimura for helpful comments; and
Miyuki Hattori for technical assistance. This work was
supported in part by Grants-in-Aid for Scientific Research
on Priority Areas (18075007) and for Young Scientists
(Start-up) (18870012), Ministry of Education, Culture,
Sports, Science and Technology, Japan. The authors have
declared that no competing interests exist.
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Communicating editor: C. S. Pikaard
Euchromatic Gene Regulation by BRU1
95
GENETICS
Supporting Information
http://www.genetics.org/content/suppl/2011/06/24/genetics.111.130062.DC1
Ectopic Gene Expression and Organogenesis
in Arabidopsis Mutants Missing BRU1 Required
for Genome Maintenance
Yusuke Ohno, Jarunya Narangajavana, Akiko Yamamoto, Tsukaho Hattori, Yasuaki Kagaya,
Jerzy Paszkowski, Wilhelm Gruissem, Lars Hennig, and Shin Takeda
Copyright © 2011 by the Genetics Society of America
DOI: 10.1534/genetics.111.130062
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