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A. DEPARTMENT OF
MOLECULAR GENETICS
A-a. Division of Molecular Genetics - Tatsuo
Fukagawa Group
RESEARCH
ACTIVITIES
(1)
Identification of new centromere proteins in higher
vertebrate cells
Masahiro Okada, Tetsuya Hori, Mi-Sun Kwon,
Mayumi Takahashi and Tatsuo Fukagawa
--The centromere
plays a fundamental role in accurate chromosome
segregation during mitosis and meiosis in
eukaryotes. Its functions include sister chromatid
adhesion and separation, microtubule attachment,
chromosome movement and mitotic checkpoint control.
Although chromosome segregation errors cause
genetic diseases including some cancers, the
mechanism by which centromeres interact with
microtubules of the spindle apparatus during cell
division is not fully understood.
--To understand the
function of the centromere, we were led to develop
a genetic analysis method that utilizes the
hyper-recombinogenic chicken B lymphocyte cell line
DT40. The high level of homologous recombination in
DT40 cells permit efficient targeted disruption of
genes of interest. We have improved this system and
have created several cell lines with conditional
knockouts of several centromere proteins to
investigate the molecular mechanism of centromere
assembly and function. We have proposed a model for
kinetochore assembly in vertebrate cells (Fukagawa
et al., EMBO J., 2001; Nishihashi et al., Dev.
Cell, 2002). In this model we explained that many
unidentified proteins could be involved in
kinetochore assembly. Therefore, we tried to
identify new kinetogore proteins using the
proteomics approach. We prepared cell lines in
which expression of CENP-H was replaced by
expression of CENP-H-Flag or CENP-H-GFP. We also
prepared cell lines in which expression of CENP-I
was replaced by expression of CENP-I-Flag or
CENP-I-GFP. We extracted chromosome fraction from
these cell lines and performed immunoprecipitation
with anti-Flag or anti-GFP antibodies.
Immunoprecipitates were then separated by SDS-PAGE
and we identified several common bands from all
cell lines. We analyzed these bands by LC-MS/MS and
identified amino acids sequence of all bands by a
peptide-mass-fingerprinting method. We newly
identified five proteins and cloned cDNA of these
proteins. We then made expression constructs for
these cDNAs fused by GFP and investigated
localization of these proteins. These proteins
perfectly co-localized with CENP-H throughout the
cell cycle. We conclude that these five proteins
are constitutive centromere proteins (manuscript in
preparation). We are creating conditional knockout
cells for these proteins to understand mechanism of
kinetochore assembly in vertebrate cells.
(2)
Molecular analysis of the Nuf2-Hec1 complex that
transiently localizes to centromere during
mitosis
Tetsuya Hori, Yoshikazu Mikami, Kazuko Suzuki
and Tatsuo Fukagawa
--Nuf2 and Hec1 are
evolutionarily conserved centromere proteins. To
clarify the functions of these proteins in
vertebrate cells, we characterized them in chicken
DT40 cells (Hori et al., J. Cell Sci., 2003). We
generated GFP fusion constructs of Nuf2 and Hec1 to
examine in detail localization of these proteins
during the cell cycle. We found that Nuf2 is
associated with Hec1 throughout the cell cycle and
that this complex is localized to the centrosomes
during G1 and S phases and then moves through the
nuclear membrane to the centromere in G2 phase.
During mitosis, this complex is localized to the
centromere. We also created conditional
loss-of-function mutants of Nuf2 and Hec1. In both
mutants, the cell cycle arrested at prometaphase,
suggesting that the Nuf2-Hec1 complex is essential
for mitotic progression. The inner centromere
proteins CENP-A, -C, and -H and checkpoint protein
BubR1 were localized to chromosomes in the mutant
cells arrested at prometaphase, but Mad2
localization was abolished. Furthermore,
photobleaching experiments revealed that the
Nuf2-Hec1 complex is associated stably with the
centromere and that interaction of this complex
with the centrosome is dynamic.
--We also observed
that CENP-H, which is a constitutive centromere
component that localizes to the centromere
throughout the cell cycle, interacts with the Nuf2
complex by yeast two-hybrid analysis.
Co-immunoprecipitation experiments revealed that
CENP-H interacts with the Nuf2 complex during
mitosis in chicken DT40 cells. Photobleaching
experiments showed that both Hec1 and CENP-H form
stable associations with the centromeres during
mitosis, suggesting that Hec1 acts as a structural
component of centromeres during mitosis. On the
basis of these results and published data, we
propose that the Nuf2 complex functions as a
connector between the inner and outer
kinetochores.
--We started the
proteomics approach to identify other proteins that
interact with the Nuf2-Hec1 complex. We identified
chicken homolog of Spc24 and Spc25. We also
identified several components that localizes
centrosome and centromere and are now
characterizing these components.
(3)
Functional roles of the RNAi machinery in
verterbrate centromeres
Tomoko Motohashi, Masahiro Nogami, Atsushi
Fukushima and Tatsuo Fukagawa
--RNAi-mediated
silencing of gene expression occurs when
double-stranded RNAs (dsRNAs) are cleaved by Dicer
into 21- to 23-nt small interfering RNAs (siRNAs).
These siRNAs guide a multicomponent nuclease,
RNA-induced silencing complex (RISC), to degrade
specific mRNAs. Although the Dicer-mediated
gene-silencing is evolutionarily conserved system,
the biological functions of the RNAi machinery are
not fully understood. Genetic strategies have been
used to examine the biological functions of the
RNAi machinery in C. elegans, Arabidopsis,
Drosophila and fungi. There are reports that
the RNAi machinery is related to chromosome
segregation in fission yeast. However, it is
unclear whether the RNAi machinery is associated
with chromosome segregation in vertebrate cells. To
examine the biological function of the RNAi-related
pathway in vertebrate cells, we generated a
conditional loss-of-function mutant of Dicer in a
chicken-human hybrid DT40 cell line that contains
human chromosome 21. Loss of Dicer leads to cell
death with accumulation of abnormal mitotic cells
that show premature sister chromatid separation.
Aberrant accumulation of transcripts from
α-satellite sequences, which consist of human
centromeric repeat DNAs, was detected in
Dicer-deficient cells. Immunocytochemical analysis
revealed abnormalities in localization of
heterochromatin proteins, Rad21 cohesin protein,
and BubR1 checkpoint protein, but core kinetochore
proteins such as CENP-A and -C were normal. We
conclude that Dicer-related RNAi machinery is
involved in formation of the heterochromatin
structure in higher vertebrate cells. We also
examined expression profile of non-coding region
using high-density DNA-microarray, when expression
of Dicer was lost. We could identify several
non-coding RNAs from this method. We are
characterizing biological function these RNAs. We
also started to create conditional knockout cells
for other components involved in RNAi machinery
such as Ago-family proteins. We would like to
comprehensive understand relationship of RNAi
machinery with centromere function.
PUBLICATIONS
Papers
1. Fukagawa, T., Nogami, M., Yoshikawa, M.,
Ikeno, M., Okazaki, T., Takami, Y., Nakayama, T.
and Oshimura M. (2004). Dicer is essential for
formetion of the heterochromatin structure in
vertebrate cells. Nature Cell Biol. 6,
784-791.
2. Fukagawa, T. (2004). Centromere DNA, proteins
and kinetochore assembly in vertebrate cells.
Chromosome Res. 12, 557-567.
3. Fukagawa, T. (2004). Assembly of kinetochores in
vertebrate cells. Exp. Cell Res. 296,
21-27.
ORAL
PRESENTATIONS
1. Fukagawa, T. Formation of kinetochores and
heterochromatin structures in vertebrate cells. The
21st Radiation Biology Center International
Symposium, Kyoto, October, 2004.
2. Fukagawa, T. Kinetochore assembly and formation
of heterochromatin structures in vertebrate cells.
CSHL meeting on Dynamic organization of nuclear
function, Cold Spring Harbor, New York, October,
2004.
3. Fukagawa, T. Formation of kinetochores and
heterochromatin structures in vertebrate cells. The
15th International Chromosome Conference, London,
September, 2004.
4.
深川竜郎、堀哲也、野上正弘、三上剛和、岡田聖裕「ゲノム安定性を保障するセントロメアの機能構築」日本分子生物学会、神戸、2004年12月
5.
深川竜郎、野上正弘、池野正史、岡崎恒子、押村光雄「高等動物セントロメアにおけるRNAi装置の役割」染色体ワークショップ、湯河原、2004年1月
POSTER
PRESENTATIONS
1. Spence, J., Alonso-Gonzalez, L., Mills, W.,
Carpenter, A., Earnshaw, W., Fukagawa, T., Porter,
A., and Farr, C. Topoisomerase II and Vertebrate
centromere. The 15th International Chromosome
Conference, London, September, 2004.
2. Takami, Y., Fukagawa, T. and Nakayama, T.
Chromatin assembly factor1-mediated nucleosome
assembly, coupled with DNA replication, is
essential for cell proliferation in vertebrate
cells. The 77th annual meeting of the Japanese
Biochemical Society, Yokohama, October, 2004.
3. Mikami, Y., Hori, T., Kimura, H. and Fukagawa,
T. Functional region of CENP-H interacts with the
Nuf2 complex, which functions as a connector
between the inner and outer kinetochores. The 21st
Radiation Biology Center International Symposium,
Kyoto, October., 2004.
4.
岡田聖裕、深川竜郎、「脊椎動物セントロメアタンパク複合体の精製」日本分子生物学会、神戸、2004年12月
5.
堀哲也、小布施力史、原口徳子、平岡泰、木村宏、深川竜郎「高等動物Nuf2-Hec1複合体と相互作用する新規構成因子の解析」染色体ワークショップ、湯河原、2004年1月
6.
堀哲也、原口徳子、平岡泰、小布施力史、深川竜郎「高等動物M期セントロメアに局在する必須因子km23の機能解析」日本分子生物学会、神戸、2004年12月
7.
高見恭成、柴原慶一、深川竜郎、中山建男「高等動物細胞におけるクロマチンアセンブリーファクター1（CAF-1）の機能解析」日本分子生物学会、神戸、2004年12月
8. 三上剛和、堀哲也、木村宏、深川竜郎「Functional
region of CENP-H interacts with the Nuf2 complex,
which functions as a connector between the inner
and outer
kinetochores.」日本分子生物学会、神戸、2004年12月
EDUCATION
1. Dr. Fukagawa gave several lectures at The
Graduate University for Advanced Studies, Hayama
(in Japanese).
2. Dr. Fukagawa was invited a seminar on
“Kinetochore Assembly" at Kyoto University, Kyoto,
August, 2004 (in Japanese).
3. Dr. Fukagawa gave a public lecture of National
Institute of Genetics at National Museum of
Emerging Science and Innovation (MIRAIKAN), Tokyo,
October, 2004.
4. Dr. Fukagawa gave a special lecture at Tokyo
Institute of Technology, Yokohama, December, 2004
(in Japanese).
5. Dr. Fukagawa was invited a seminar on
“Assembly" at Tokyo Institute of Technology,
Yokohama, December, 2004 (in Japanese).
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