Download Dr Nobuaki Kudo Chromosome Cohesion Group

Chromosome Cohesion Group
Dr Nobuaki Kudo
Institute of Reproductive and Developmental Biology (IRDB)
Hammersmith Hospital Campus
Imperial College London
Du Cane Road, London W12 0NN
United Kingdom
Tel +44 (0)20 7594 3803
Fax +44 (0)20 7594 2192
[email protected]
Research Background
In recent years it has become more
commonplace for women to consider
pregnancy at around the age of 35 or
older, even though it is at this time that
the risk of pregnancy loss, as well as
innate diseases such as Down syndrome,
sharply increases. Aneuploidy (i.e. an
abnormal number of chromosomes in the
cells) is one of the leading causes of such
incidents, and is also associated with
cancer in somatic cells. Most early
embryonic aneuploidy is generated by
the fertilization of aneuploid eggs or sperm
that have originated from chromosome
mis-segregation during a specialized type
of cell division, called meiosis. We wish
to understand, at the molecular level,
what causes aneuploidy and why it
increases in a maternal age-dependent
manner, thereby allowing us to devise
methods or treatments to avoid it.
Following a mitotic cell division, the
chromosome number is the same in both
the mother and daughter cells. In contrast,
meiotic cell division, which takes place
in the germ line, produces cells with half
the number of chromosomes (see Fig.
2). This is the result of two sequential
rounds of chromosome separation without
an intervening round of DNA duplication.
As in mitotic cells, meiotic cells also use
the tension from microtubules from
opposite poles of the cell to align and
segregate chromosomes. However, the
requirement of homologous chromosome
segregation (meiosis I) in addition to sister
chromatid segregation (meiosis II) adds
a few functional modifications: firstly, pairs
of homologous chromosomes must be
linked to establish tension between them.
Secondly, unlike in mitosis, sister
chromatids must go to the same spindle
pole at the first meiotic division. Thirdly,
sister chromatids have to remain
connected to facilitate their bi-orientation
Figure 1. A full set of chromosomes (blue)
from a mouse oocyte, at metaphase I
stage, spread on a glass slide and stained
with antibodies detecting cohesin Rec8
(magenta) and kinetochores (green).
Nineteen homologous paternal and
maternal chromosome pairs are
connected with crossovers and cohesin
distal to crossover sites, however one
pair has been precociously segregated
(circles). This situation is thought to be
the most common causes for embryonic
aneuploidy leading to infertility,
spontaneous abortion and Down
syndrome in humans.
Figure 2. Chromosome segregation patterns in meiosis and mitosis. See main text for details.
© Copyright 2009 N. Kudo Imperial College London
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in meiosis II. These changes are achieved
by three meiosis I-specific mechanisms:
(i) synapsis and crossing-over between
homologous chromosomes, (ii) monopolar attachment of sister kinetochores
to spindle microtubules, (iii) chromosome
arm-specific loss of sister chromatid
cohesion. Malfunction in any of the above
mechanisms can cause chromosome
mis-segregation and thus aneuploidy,
and therefore it is important to understand
their precise molecular mechanisms.
Cohesion between sister chromatids is
generated during DNA replication and
maintained until their segregation into
two daughter cells. It is mediated by an
evolutionally-conserved hetero-tetrameric
protein complex, called cohesin; cohesion
is essential for faithful chromosome
segregation in mitosis as well as meiosis.
In meiosis, DNA replication is followed
by recombination of sister chromatid
strands between homologous paternal
and maternal chromosomes, which
creates crossovers. Crossovers are
physical linkages between homologous
chromosomes, and are in fact supported
by sister chromatid cohesion distal to
crossover sites. Loss of cohesion between
chromosome arms allows segregation of
homologous chromosomes during
meiosis I while loss of cohesion between
sister centromeres allows segregation of
sister chromatids in meiosis II. Therefore,
accurate regulation of the cohesin
complex is one of the most important
aspects of faithful chromosome
segregation.
In addition to cohesin’s canonical role in
sister chromatid cohesion, recent studies
provide evidence that cohesin regulates
gene expression. Human developmental
diseases such as Cornelia de Lange and
Roberts syndromes were shown to be
caused by mutations in genes encoding
cohesin or its regulators. Latest
investigations in mammalian mitotic cells
show that many cohesin binding sites on
chromosome arms are co-occupied with
an insulator element binding protein called
CTCF and they co-operate to regulate
expression of a subset of genes. One of
the genes regulated by these is the
human H19/Igf2 locus, whose expression
is controlled by parental origin-dependent
DNA methylation of the H19 imprinting
control region (ICR). Therefore, a certain
fraction of cohesin, together with CTCF,
is thought to be required for maintaining
expression and repression of imprinted
genes by defining chromosomal
boundaries, possibly by affecting higher
order chromatin structure, in somatic cells.
Figure 3. Chromosomal development during meiotic prophase (leptotene, zygotene,
pachytene and diplotene) and 2 rounds of meiotic cell division in mouse
spermatogenesis. Interkinesis is the secondary spermatocyte in interphase
between the first and second meiotic division. Chromosome spreads were prepared
from the testis of a transgenic mouse expressing Rec8-myc and stained with
antibodies against the myc epitope (green) and a synaptonemal complex protein
Sycp3 (magenta). Transgene-derived Rec8-myc is perfectly functional and is a
useful tool to study cohesin function during meiotic chromosomal development.
Studies in various model organisms have
revealed molecular mechanisms of
cohesin regulation and chromosome
segregation; however, much less is
understood in mammalian germ cells.
Studying mammalian meiotic
chromosome segregation with a special
focus on cohesin is particularly interesting
and clinically important, since cohesion
must be maintained for up to 40 years in
human oocytes - in cellular terms, an
exceedingly long period of time. The
significance of cohesin regulation for
mammalian fertility has been illustrated
by recent studies involving Smc1β and
Separase knockout mice. Furthermore,
the new discovery of a potential role for
cohesin in gene regulation has prompted
the hypothesis that deterioration of
cohesion in germ cells might cause an
aberrant gene expression pattern and
thus physiological aging of eggs.
Therefore, we are actively studying the
mechanisms of chromosome cohesion
and segregation in mammalian germ cell
development.
Ongoing Research
Where does cohesin bind on meiotic
chromosomes?
Recently numerous research groups
mapped multiple cohesin binding sites
on chromosome arms in mammalian
mitotic cells and found that cohesin
preferentially binds to DNase I
hypersensitive sites with co-localization
of the insulator protein CTCF. In mitotic
division, sister chromatid cohesion at
chromosome arms is essentially
dispensable for bi-orientation of sister
kinetochores and accurate sister
chromatid segregation. In fact, most
cohesin complexes are released from
arms before all chromatid pairs are
aligned on the cell equator plane. In
striking contrast, chromatid arm cohesion
is essential for bi-orientating homologous
chromosomes during meiosis I. Therefore
it is very interesting to explore the
distribution of cohesin during meiosis and
to determine whether or not mitotic and
meiotic cohesins share the same sites.
© Copyright 2009 N. Kudo Imperial College London
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We are performing chromatin
immunoprecipitation (ChIP) assays for
the meiotic cohesin subunit Rec8 in
spermatocytes. Our long-term aims
include genome-wide mapping of meiotic
cohesin sites, comparison between young
and aged oocytes and the relationship of
cohesin positions and reprogramming of
the sex-specific genome imprinting pattern
that is also a unique feature of germ cell
development.
How are sister kinetochores monoorientated during meiosis I?
One of the modifications that distinguishes
meiosis I chromosome segregation from
that in meiosis II and mitosis is
kinetochore mono-orientation. Molecular
mechanisms accomplishing this have
been studied in other eukaryotic model
organisms; however, it is assumed that
they are not applicable to mammalian
germ cells. One reason for this is that the
centromeric DNA structure, on which the
kinetochore is assembled, exhibits a large
degree of diversity between species;
another is that the molecules which have
been found to play a role in monoorientation, show very low conservation.
Therefore the mechanism that determines
kinetochore orientation in mammalian
meiosis I remains totally unclear. We are
taking a candidate approach, as well as
interaction-based screening, to identify
the relevant molecules. We are also
testing whether the bi-orientated sister
kinetochore configuration in mitotic cells
can be converted to a mono-orientated
arrangement.
How is cohesin regulated during
meiotic prophase?
Though cohesin is required for crossover
formation in all model organisms so far
tested, its precise role in the process is
not understood. Even when crossovers
are successfully created, sister chromatid
cohesion distal to the crossover sites
remains essential for the maintenance of
such crossovers. This is particularly
important in human oogenesis, because
female germ cells undergo homologous
recombination during fetal development
before birth and homologous
chromosome segregation after puberty,
indicating that crossovers must be
maintained for approximately 10-40 years.
Interestingly, cohesin is not replenished
after DNA replication in an unchallenged
yeast cell cycle. Is this also true for
mammalian oogenesis? In addition, how
oocytes maintain the cell cycle arrest for
such a long time is also an interesting
question. We are studying genetically
modified mice to address these questions.
Our Team; Xiangwei, Kasia, Nobu and James (from left)
Human meiotic problems and germ
line stem cells
In close collaboration with Dr. Carol
Readhead and Dr. Sheba Jarvis in the
IRDB and the IVF unit at Hammersmith
Hospital, we are studying meiotic
chromosomal abnormalities in nonobstructive azoospermic patients. We are
also characterizing mammalian
spermatogonial stem cells with the aim
of identifying future application in
regenerative medicine.
Selected Reviews
Petronczki M, Siomos MF, Nasmyth K.
(2003) Un ménage à quatre: the
molecular biology of chromosome
segregation in meiosis. Cell. 112: 42340.
Hauf S, Watanabe Y. (2004) Kinetochore
orientation in mitosis and meiosis. Cell.
119: 317-27.
McNairn AJ, Gerton JL. (2008) The
chromosome glue gets a little stickier.
Trends Genet. 24: 382-9.
Hassold T, Hunt P. (2001) To err
(meiotically) is human: the genesis of
human aneuploidy. Nat Rev Genet. 2:
280-91.
of APC/C activity in oocytes by a Bub1dependent spindle assembly
checkpoint. Curr Biol. 19: 369-80.
Kudo NR, Wassmann K, Anger M, Schuh
M, Wirth KG, Xu H, Helmhart W, Kudo
H, McKay M, Maro B, Ellenberg J, de
Boer P, Nasmyth K. (2006) Resolution
of chiasmata in oocytes requires
separase-mediated proteolysis. Cell.
126: 135-46.
Wirth KG, Wutz G, Kudo NR, Desdouets
C, Zetterberg A, Taghybeeglu S,
Seznec J, Ducos GM, Ricci R, Firnberg
N, Peters JM, Nasmyth K. (2006)
Separase: a universal trigger for sister
chromatid disjunction but not
chromosome cycle progression. J. Cell
Biol. 172: 847-60.
McGuinness BE, Hirota T, Kudo NR,
Peters JM, Nasmyth K. (2005)
Shugoshin prevents dissociation of
cohesin from centromeres during
mitosis in vertebrate cells. PLoS Biol.
3: 433-49.
Funding
The Royal Society
Medical Research Council (MRC)
New Investigator Award
Institute of Obstetrics & Gynaecology
Trust (IOGT)
Selected Publications
Team Members
Kudo NR, Anger M, Peters AH,
Stemmann O, Theussl HC, Helmhart
W, Kudo H, Heyting C, Nasmyth K.
(2009) Role of cleavage by separase
of the Rec8 kleisin subunit of cohesin
during mammalian meiosis I. J. Cell
Sci. 122: 2686-98.
McGuinness BE, Anger M, Kouznetosva
A, Gil-Bernabe AM, Helmhart W, Kudo
NR, Wuensche A, Taylor S, Hoog C,
Novak B, Nasmyth K. (2009) Regulation
Nobuaki Kudo, PhD (Head of Group,
Non-clinical Lecturer)
Xiangwei Fu, PhD (Post-doc)
Kasia Kuleszewicz, MSc (PhD student)
James Crichton, BSc (MSc student)
Alumni
Joao Pedro Sousa Martins, MSc
Giulia Grimaldi, BSc
Emily Waiyaiya, BSc
(Dec 2009)
© Copyright 2009 N. Kudo Imperial College London
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