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E. DEPARTMENT OF
INTEGRATED GENETICS
E-b. Division of Agricultural Genetics - Tetsuji
Kakutani Group
RESEARCH
ACTIVITIES
(1)
Developmental abnormalities induced by DNA
methylation mutation of Arabidopsis
Tetsuji Kakutani, Tetsu Kinoshita, Asuka Miura,
Yuki Kinoshita, Masaomi Kato and Hodetoshi Saze
--Arabidopsis
provides a genetically tractable system to learn
role of DNA methylation, since viable mutants with
reduced genomic DNA methylation are available.
Arabidopsis MET1 (METHYLTRANSFERASE 1, ortholog of
mammalian DNA methyltransferase Dnmt1) is necessary
for maintaining genomic cytosine methylation at
5'-CG-3' sites. Arabidopsis additionally methylates
non-CG sites using CHROMOMETHYLASE3 (CMT3). A third
Arabidopsis gene necessary for DNA methylation is
DDM1 (DECREASE IN DNA METHYLATION), which
encodes a chromatin remodeling factor (Jeddeloh et
al., 1999, Nat. Genet 22, 94-). The ddm1
mutation affects both CG and non-CG methylation. A
striking feature of ddm1 mutation is that it
induces a variety of developmental abnormalities by
causing heritable change in other loci (Kakutani et
al., 1996, PNAS 93, 12406-). The molecular basis
has been clarified in two of the loci directly
causing the developmental abnormalities (see
below). In addition, we are starting genetic
characterization of other types of developmental
abnormalities, such as bonsai (Kakutani et al.,
2005).
(2)
Epigenetic behavior of CACTA
transposon
Asuka Miura, Masaomi Kato, Kazuya Takashima,
Yuki Kinoshita and Tetsuji Kakutani
--Through genetic
characterization of one of the ddm1-induced
developmental abnormalities, we identified a novel
endogenous Arabidopsis transposon, named
CACTA1. This transposon transposes and
increases in the copy number in DNA hypomethylation
background in ddm1 mutant (Miura et al.,
2001, Nature 411, 212-). Loss of DNA methylation
seems to be sufficient for mobilization of
CACTA1, because it was mobilized in mutants
of DNA methyltransferase genes, MET1 and
CMT3. High frequency transposition of
CACTA elements was detected in
cmt3-met1 double mutants. Single mutants in
either met1 or cmt3 were much less
effective in mobilization. Thus CG and non-CG
methylation systems redundantly function for
immobilization of transposons (Kato et al., 2003
Curr Biol 13, 421-426). CMT3 gene and non-CG
methylation in plants may have evolved as an
additional epigenetic tag dedicated to transposon
control.
--CACTA1
activated by the ddm1 mutation remained
mobile in the presence of the wild type DDM1
gene, suggesting that de novo silencing is not
efficient for the defense of the genome against
CACTA movement. The defense depends on
maintenance of transposon silencing over
generations (Kato et al., 2004).
--Differentiation of
gene-rich and transposon-rich (gene-poor) regions
is a common feature in plant genomes. That may be
due to preferential integration of transposons to
gene-poor regions or may be due to purifying
selection against transposon insertion to gene-rich
regions in natural populations. To evaluate the
possible contribution of natural selection to the
formation of transposon distribution pattern, we
examined the distribution of the CACTA
transposons in genomes of 19 natural variants
(ecotypes) and compared that to integration induced
in laboratory in the ddm1 mutants. Sequences
similar to mobile CACTA1 copy distribute
among the ecotypes and show high polymorphism in
genomic localization. Despite the high
polymorphism, the copy number was low in all the
examined ecotypes and they localized preferentially
in pericentromeric and transposon-rich regions
(Miura et al, 2004). This contrasts to
transposition induced in laboratory, in which the
integration sites are less biased and the copy
number frequently increases. In addtion,
transposition induced in DDM1 wild type background
also showed unbiased integration sprcificity (Kato
et al., 2004). The differences in the integration
sites may be due to natural selection against
deleterious insertion into chromosomal arm
regions.
(3)
Inheritance of epigenetic developmental
abnormality
Yuki Kinoshita, Asuka Miura, Tetsu Kinoshita and
Tetsuji Kakutani
--Another
developmental abnormality, late flowering trait,
was induced by ectopic expression of FWA
gene associated with hypomethylation of tandem
repeat upstream of the coding region (Soppe et al.,
2000, Mol Cell 6, 791-). Interesting thing is that
change in nucleotide sequence was also not observed
in fwa-1 and fwa-2 alleles isolated
by conventional mutagenesis. In both cases,
over-expression associated with the hypomethylation
resulted in the phenotypes. Combining ddm1
mutation and linkage analysis is useful for
identifying epigenetically regulated genes
affecting plant development (Kakutani, 1997, Plant
J., 12, 1447-). The ddm1-induced late
flowering trait as well as the hypomethylation and
ectopic expression of the FWA gene were stably
inherited over generations even in the presence of
the wild type DDM1 copy (manuscript in
preparation).
(4)
Epigenetic control of FWA gene expression in
endosperm
Tetsu Kinoshita, Asuka Miura, Yuki Kinoshita and
Tetsuji Kakutani
--Although
FWA is ectopically expressed in the
epigenetic alleles stated above, the role of FWA
gene product in normal development remained
unknown. To understand why the FWA gene is
epigenetically controlled, we further examined
expression of this gene during normal development
in wild type. Results of GFP reporter system and
direct detection of the transcript both suggest
that FWA is expressed specifically in the
endosperm. Endosperm is a plant tissue analogous to
mammalian placenta; it serves as nutritional
support to the embryo. Furthermore, the FWA
gene was expressed in parent-of-origin-specific
manner; only maternal gene is expressed. The
FWA imprint depends on the maintenance DNA
methyltransferase MET1, as is the case in mammals.
Unlike mammals, however, the FWA imprint is
not established by allele-specific de novo
methylation. It is established by maternal
gametophyte-specific gene activation, which depends
on a DNA glycosylase gene, DEMETER. Since
endosperm does not contribute to the next
generation, the activated FWA gene need not
be silenced again. Double fertilization enables
plants to use such ‘one-way' control of imprinting
and DNA methylation in endosperm (Kinoshita et al,
2004).
PUBLICATIONS
Papers
1. Kinoshita, T., Miura, A., Choi, Y.,
Kinoshita, Y., Cao, X., Jacobsen, SE., Fischer, RL.
and Kakutani, T. (2004). One-way control of
FWA imprinting in Arabidopsis
endosperm by DNA methylation. Science 303,
521-523.
2. Miura, A., Kato, M., Watanabe, K., Kawabe, A.,
Kotani, H. and Kakutani, T. (2004). Genomic
localization of endogenous mobile CACTA family
transposons in natural variants of Arabidopsis
thaliana. MGG 270, 524-532.
3. Kato, M., Takashima, K. and Kakutani, T. (2004).
Epigenetic control of CACTA transposon mobility in
Arabidopsis thaliana. Genetics 168,
961-969.
4. Kakutani, T., Kato, M., Kinoshita, T. and Miura,
A. (2005). Control of Development and Transposon
Movement by DNA Methylation in Arabidopsis
thaliana. Cold Spring Harbor Symposia on
Quantitative Biology. 69 in press.
5. Bachmair, A., Garber, K., Takeda, S., Sugimoto,
K., Kakutani, T. and Hirochika, H. (2004).
Biochemical analysis of long terminal repeat
retrotransposons. Methods in Molecular Biology
260, 73-82.
ORAL
PRESENTATIONS
1. Kakutani T, Epigenetic Inheritance and
control of transposons in Arabidopsis. CDB
symposium; Developmental Remodeling (March 2004,
Kobe)
2. Kakutani T, Epigenetic inheritance and
transposon movement in Arabidopsis. The
69th Cold Spring Harbor Symposia on
Quantitative Biology (June 2004, Cold Spring
Harbor, NY, USA)
3. Kinoshita T, Miura A, Choi Y, Kinoshita Y, Cao
X, Jacobsen S, Fischer R, Kakutani T. Genomic
imprinting of the FWA gene in Arabidopsis
endosperm. 15th International Conference on
Arabidopsis Research, (July 2004, Berlin,
Germany)
4.
角谷徹仁「植物の個体発生とゲノム構造のエピジェネティックな制御」第6回細胞性粘菌研究会（2004年3月東京）
5.
木下哲、三浦明日香、木下由紀、角谷徹仁「一方向性のDNAメチル化修飾によるシロイヌナズナFWA遺伝子のゲノムインプリンティング」第45回日本植物生理学会年会（2004年3月、東京）
6.
角谷徹仁「シロイヌナズナを用いたエピジェネティクス研究」弘前大学遺伝子実験施設第11回シンポジウム（2004年11月、弘前）
7.
角谷徹仁「シロイヌナズナを用いたエピジェネティクス研究」名古屋大学遺伝子実験施設第4回公開セミナー「核ゲノムのダイナミズムと遺伝子発現」（2004年12月、名古屋）
8.
三浦明日香、加藤正臣、高嶋和哉、角谷徹仁「トランスポゾンの挙動のエピジェネティックな制御」組換えワークショップ（2004年12月、兵庫県）
EDUCATION
1. 角谷徹仁
東京大学医科学研究所で講義,2004年5月
2. 角谷徹仁 京都大学農学部で集中講義,2004年7月
3. 角谷徹仁 東京大学農学部で集中講義,2004年11月
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