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E. DEPARTMENT OF
INTEGRATED GENETICS
E-b. Division of Agricultural Genetics - Keiichi
Shibahara Group
RESEARCH
ACTIVITIES
(1)
Mechanism of nucleosome assembly during DNA
replication
Yasunari Takami1, Tatsuya Ono, Tatsuo
Nakayama1 and Kei-ichi Shibahara
(1Department of Biochemistry, Miyazaki
Medical College, University of Miyazaki)
--A newly
replicated DNA is assembled into nucleosome soon
after the passage of replication fork. This rapid
formation of nucleosome is functionally linked to
DNA replication machineries and plays a critical
role for the maintenance of genome integrity and
epigenetic states of chromatin in proliferating
cells. Two histone binding proteins, CAF-1
(Chromatin Assembly Factor-1) and ASF1
(Anti-Silencing Function1), are involved in some
process of this nucleosome assembly reaction.
--In collaboration
with Takami and Nakayama in University of Miyazaki,
using CAF-1p150-or p60-deficint chicken DT40 cells,
we recently showed that without CAF-1 function,
S-phase progression was delayed and a rapid
nucleosome assembly during DNA replication was
disturbed in vivo. This is the first direct
evidence for the involvement of CAF-1 in a rapid
nucleosome assembly during DNA replication in
vivo. In addition, CAF-1 was suggested in
involving in Chk1-dependent checkpoint pathway
after the treatment with HU.
(2)
Physiological implications of CAF-1 and
CAF-1-dependent nucleosome assembly in higher
eukaryotes
Tatsuya Ono, Hidetaka Kaya1, Shin
Takeda2, Jerzty Paszkowski2,
Takashi Araki1 and Kei-ichi Shibahara
(1Department of Botany, Graduate School
of Science, Kyoto University,
2Department of Plant Biology, University
of Geneva)
--The genetic
approach of Arabidopsis is a powerful tool
to see physiological implications of CAF-1 and
CAF-1dependent nucleosome assembly in higher
eukaryotes. We analyzed loss-of-function mutants of
caf-1 (fasciata) in Arabidopsis and
showed that those caf-1 mutants displayed
severely disturbed cellular and functional
organization of both meristems (Cell 104,
131-142. 2001). We recently showed that
transcriptional gene silencing (TGS) of endogenous
CACTA transposons was released infrequently
in a stochastic manner in fas, without
decreasing DNA methylation. Other endogenous silent
genes at different chromosomal sites were also
transcriptionally activated non-concomitantly with
each other. Furthermore, TGS of the silent
transgene β-glucuronidase (GUS) was
also de-repressed randomly in fas mutant
plants, irrespectively of developmental
abnormalities, and the activated state of
GUS was maintained during growth to produce
clusters of cells expressing GUS. We suggest
that CAF-1 contributes to the stable inheritance of
epigenetic states through multiple rounds of cell
divisions and that defects in CAF-1 functions
explain the stochastic occurrence of pleiotropic
phenotypes in fas mutants.
--In collaboration
with J. Paszkowski in University of Geneva, we
analyzed bru1 mutants and found striking
phenotypic similarities to fas mutants.
Epistatic analysis of bru1 and fas
mutants suggests that BRU1 and CAF-1 have
overlapping functions. BRU1 encodes a novel nuclear
protein containing two protein-protein interaction
domains. However, the molecular function of BRU1
and the molecular link between BRU1 and CAF-1 is
still unknown. The future analysis of BRU1 would
provide yet another clue to understanding the
mechanisms of epigenetic maintenance.
(3)
Role of histon macroH2A in gene
repression
Yuya Ogawa, Tatsuya Ono and Kei-ichi
Shibahara
--The unusual
histone variant macroH2A (mH2A) is associated with
transcriptional repression as it appears to be
enriched in the inactive X chromosome by
immunostaining. In vitro analysis showed
that non-histone region of mH2A in carboxy-terminal
prevents transcription factor from interacting with
chromatin reconstituted with mH2A, but, the
molecular function of mH2A in modulating chromatin
structures remains unknown.
--To reveal the roles
of mH2A, we purified mH2A-containing nucleosome by
affinity purification with anti-epitope tag
antibodies, and eventually, we found
mono-ubiquitinated form of mH2A.
Mono-ubiquitination of H2A and H2B has been shown
to be important in regulating chromatin structure
and transcription. We are currently trying to
identify the ubiquitinated site of mH2A and reveal
its implications in modulation of chromatin
structures and transcriptional regulations.
(4)
Differential methylation of Xite and CTCF
sites in Tsix in mouse
X-inactivation
Rebecca Maxfield Boumil1, 2, 3, Yuya
Ogawa, Bryan K. Sun1, 2, 3 and Jeannie
T. Lee1, 2, 3 (1Massachusetts
General Hospital, 2Harvard Medical
School and 3Howard Hughes Medical
Institute)
--In X-chromosome
inactivation (XCI) in mammalian female cells, one
of two X-chromosomes is inactivated in the
imprinted or stochastic manner. Although some loci
influencing the choice decision have been
genetically identified, the primary marks for
imprinting and random choice remain undefined. We
examined the role of DNA methylation, a mechanism
known to regulate imprinting in autosomal loci, in
XCI. We found differentially methylated domains
(DMDs) in Tsix and Xite, two genes
previously shown to influence choice. The DMDs
occured in gametes and disappeared in ES cells with
two active X chromosomes. Because the DNA
methylation pattern reflects events of XCI, we
propose that DMDs in Tsix and Xite
are candidates for the primary marks of epigenetic
regulation in XCI.
PUBLICATIONS
Papers
1. Takeda, S., Tadele, Z., Hofmann, I.,
Probst, A.V., Angelis, K.J., Kaya, H., Araki, T.,
Mengiste, T., Mittelsten Scheid, O.,
Shibahara, K., Scheel, D. and
Paszkowski, J. (2004). BRU1, a novel link between
responses to DNA damage and epigenetic gene
silencing in Arabidopsis. Genes Dev
18, 782-93.
2. Chromatin Assembly Factor-1 Dependent Nucleosome
Assembly Coupled with DNA Replication, is Essential
for Cell Proliferation in Vertebrate Cells. Takami,
Y., Ono, T., Fukagawa, T., Shibahara,
K. and Nakayama. T. Under revision in
Mol Cell Biol.
3. Mutations in CAF1 of Arabidopsis release
transcriptional gene silencing in a stochastic
manner. Ono, T., Kaya, H., Takeda, S., Abe, M.,
Ogawa, Y., Kato, M., Kakutani, T., Scheid, O.M.,
Araki, T. and Shibahara, K.
Submitted
Reviews
4.
久保知大,柴原慶一（2004）DNA複製及びヌクレオチド除去修復に伴うヌクレオソーム形成,わかる実験医学「DNA複製とDNA修復」.
特許
特願2005-65442,遺伝子導入方法,ジーンターゲッティング方法及びトランスジェニック植物の製造方法,柴原慶一
ORAL
PRESENTATIONS
1.
柴原慶一:CAF-1依存的なヌクレオソームアセンブリー機構とその生物学的役割、ヒストンの修飾とその機能、大阪大学蛋白質研究所セミナー「ヒストンの修飾とその機能」、2004年6月23日
2. Werner, M. H., Ahujas, S., Mendelev, N., Ito,
Y., Huang, G., Shigesada, K., Osato, M., Liu, P.
P., Krude, T. and Shibahara, K-i. Chromatin
assembly and acute human leukemia. HFSP Awardees
Meeting in Japan, May 2004
POSTER
PRESENTATIONS
1. Kei-ichi Shibahara, Hideaki Kaya, Takashi
Araki, Bruce Stillman: CAF-1 maintain states of
gene expression through rounds of cellular
divisions in Arabidopsis. 69th
Cold Spring Harbor Symposium on quantitative
Biology: Epigenetics June 2nd-7th 2004
2.
高見恭成、柴原慶一、深川竜朗、中山建男:高等動物細胞におけるクロマチンアセブリーファクター-1（CAF-1）の機能解析.第27回日本分子生物学会年会.神戸ポートアイランド.2004年12月8日
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