Laboratory of Molecular and Cellular Interactions, Division of Biological Sciences, Graduate School of Science, Hokkaido University, Sapporo 060-0810, Japan
Author for correspondence (e-mail:
leej{at}ans.kobe-u.ac.jp)
Accepted 18 March 2003
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Summary |
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Key words: Rec8 protein, Chromosome cohesion, Sister chromatid, Homologous chromosome, Mammalian meiosis
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Introduction |
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Meiosis is an essential step in sexual reproduction to produce gametes that
have a reduced number of chromosomes from diploidy to haploidy. The reduction
of chromosome number is conducted by two rounds of meiotic division following
a single round of DNA replication. During prophase in meiosis I, homologous
chromosomes pair, recombine, and crossover with their partners, yielding
junctional sites called chiasmata between non-sister chromatids in a bivalent
chromosome. As a result, homologous chromosomes are aligned at the metaphase
plate and separate at anaphase in meiosis I without separation of sister
chromatids. A pair of sister chromatids in a homologous chromosome remains
attached through meiosis I until they finally separate at the onset of
anaphase in meiosis II. Therefore, meiotically dividing cells must be equipped
with special molecules that ensure these specific behaviors of meiotic
chromosomes. It has been shown that a meiosis-specific cohesin subunit in
yeast, Rec8p, replaces a mitotic cohesin subunit, Scc1p/Rad21p, during meiosis
and that this replacement is needed for preventing sister chromatids from
separating precociously in meiosis I
(Klein et al., 1999;
Watanabe and Nurse, 1999
).
Homologous chromosome separation in meiosis I is promoted by the proteolytic
cleavage of Rec8p by separin (Buonomo et
al., 2000
). In mammals, putative homologs of the rec8
gene have been isolated, and high levels of mRNA expression of mouse
rec8 has been found in both male and female germ line cells
(Parisi et al., 1999
;
Lee et al., 2002
). In
addition, meiosis-specific cohesin proteins, STAG3 and SMC1ß, whose yeast
homologs have not been found in the database, have been found on mammalian
meiotic chromosomes (Prieto et al.,
2001
; Revenkova et al.,
2001
). However, there has been no report on mammalian Rec8 protein
so far.
In this study, we investigated the protein expression of mammalian Rec8
during meiosis in the male mouse. For this purpose, specific antibodies
against mouse Rec8 were raised in the mouse and rabbit and used for
immunoprecipitation, immunoblotting, and immunohistochemistry. We found that
mammalian Rec8, which associated with SMC3 and SMC1ß but not with
SMC1, was expressed from the pre-leptotene stage and was localized
along the axial/lateral element (AE/LE) of synaptonemal complexes from
leptotene to diplotene stages in prophase I. During diakinesis and metaphase
I, Rec8 was localized in both centromeres and arm regions of chromosomal
interstices. At the metaphase I-to-anaphase I transition, Rec8 dissociated
from the arms but remained associated with centromere regions until metaphase
II. In anaphase II, Rec8 was no longer detected on chromosomes. The
localization and the step-wise dissociation of Rec8 from meiotic chromosomes
suggests that Rec8 is the protein that is responsible for both cohesions
between homologs and sisters and that its dissociation from arms and
centromeres causes the separation of homologous chromosomes and that of sister
chromatids, respectively.
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Materials and Methods |
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Concurrently with the investigation of mammalian Rec8, we have been
investigating cohesin proteins in fish. We have cloned a medaka (Oryzias
latipes) homolog of mammalian SMC1 from a cDNA library constructed from
the testis, and we have produced a mouse polyclonal antibody against the
recombinant C-terminal 337-amino-acid sequence of medaka SMC1
(DDBJ/EMBL/GenBank accession number, AB097255). The antigenic C-terminal amino
acid sequence of medaka SMC1
shows 89% and 59% homology to the
corresponding regions of mouse SMC1
and SMC1ß, respectively.
Hence, the anti-medaka SMC1 antibody has crossreactivity to both mouse
SMC1
and SMC1ß in western blotting and immunoprecipitation
analyses, although it is less reactive to mouse SMC1ß than to SMC1
in immunoprecipitation analysis.
Goat polyclonal anti-SMC3 antibody and mouse monoclonal
anti--tubulin antibody (DM1A) were purchased from Santa Cruz
Biotechnology (Santa Cruz, CA) and Sigma (St Louis, MO), respectively.
Preparation of extracts
Nuclear extracts from tissues were prepared as follows. Tissues from
6-8-week-old C57BL/6 mice were minced with a surgical blade and homogenized by
a Teflon homogenizer in nine times volume (w/v) of 0.25 M sucrose-containing
TKM solution (50 mM Tris-HCl, pH 7.5, 25 mM KCl, 5 mM MgCl2). The
homogenized solution was filtered through a 70 µm cell strainer (Becton
Dickinson Labware, Franklin Lakes, NJ) and centrifuged for 10 minutes at 600
g. The pellet was resuspended in 0.25 M sucrose-containing TKM
solution. Then 2x volume of 2.3 M sucrose-containing TKM solution was
added to the cell suspension. After adding 150 µl of 2.3 M
sucrose-containing TKM solution to each 1.5 ml tube, 600-800 µl of cell
suspension and 200 µl of 0.25 M sucrose-containing TKM solution were loaded
in that order to the top. Nuclei were purified by centrifugation for 30
minutes at 12,000 g. The nuclear pellet was resuspended in
0.25 M sucrose-containing TKM solution and then centrifuged for 5 minutes at
2000 g. The pellet was extracted in RIPA buffer [20 mM
Tris-HCl (pH 7.4), 150 mM NaCl, 2 mM EDTA, 1% Nonidet P-40, 1% Na
deoxycholate, 0.1% SDS, 50 mM NaF, 5 mM 2-mercaptoethanol] containing
Protease-Inhibitor-Cocktail (Roche, Mannheim, Germany). After sonication, the
extraction buffer was centrifuged at 18,000 g for 1 hour. Then
the supernatant was recovered as nuclear extract.
Whole extracts of testes from 0-, 1-, 2-, 3- and 5-week-old mice were also prepared by extracting them in RIPA buffer containing Protease-Inhibitor-Cocktail. Briefly, after testes had been decapsulated, they were homogenized, sonicated in the buffer, and centrifuged at 18,000 g for 1 hour at 4°C. Then the supernatant was recovered as whole testis extracts.
Western blotting and immunoprecipitation
In immunoprecipitations, testis nuclear extracts were incubated with either
of the antibodies (anti-Rec8, anti-SMC1 and anti-SMC3 antibodies) in the
presence of Protein G Sepharose (Amersham Biosciences, Piscataway, NJ) at
4°C overnight with rotor agitation. Then, the sephoroses were washed six
times in the RIPA buffer, and the immunoprecipitates were analyzed by SDS-PAGE
followed by western blotting. As a control, testis extracts were incubated
without the primary antibody or with control preimmune serum in the presence
of Protein G Sepharose, and the immunoprecipitates were analyzed as above. For
the treatment of Rec8 with protein phosphatases (PPases), immunoprecipitates
obtained with anti-Rec8 antibody were washed five times in RIPA buffer and
twice in alkaline PPase buffer (50 mM Tris-HCl, pH 8.0, 1 mM MgCl2)
or in 1x -PPase buffer equipped with the
-PPase, then
incubated with 0.25 U/µl calf intestine alkaline PPase (Takara, Tokyo,
Japan) or 20 U/µl
-PPase (New England BioLabs, Beverly, MA) for 30
minutes at 30°C. As controls, the immunoprecipitates were incubated
without PPases in the buffers.
The immunoprecipitates, nuclear extracts, and whole testis extracts were separated by SDS-PAGE with 7.5% or 12.5% gels. After blotting onto an Immobilon membrane, the proteins were probed with primary antibodies. The antigen-protein complex was detected with alkaline phosphatase (AP)-conjugated secondary antibodies [AP-goat anti-mouse IgG (American Qualex International, San Clemente, CA), AP-goat anti-rabbit IgG (Zymed Laboratories,San Francisco, CA), AP-rabbit anti-goat IgG (Chemicon International, Temecula, CA)] and visualized by incubation in a color-substrate solution (0.2 mM nitoroblue tetrazolium, 0.2 mM 5-bromo-chloro-3-indolyl phosphate, 100 mM Tris-HCl, pH 9.5, 5 mM MgCl2).
Tissue staining
Testes from 6-8-week-old mice were frozen in liquid nitrogen, and
10-µm-thick cryostat sections were prepared. The sections were air-dried on
Vectabond (Vector Laboratories, Burlingame, CA)-coated slides and fixed in
cold 1% paraformaldehyde in PBS for 15 minutes. After washing in PBS twice,
sections were blocked in 10% goat serum (Sigma) in PBS for 1 hour and
incubated with the primary antibodies at appropriate dilutions in the blocking
buffer at 4°C overnight. After washing three times in PBS, the Rec8 and
SCP3 signals were detected with secondary antibodies [Alexa 488-conjugated
anti-rabbit IgG antibody, Alexa 488-conjugated anti-mouse IgG antibody, Alexa
546-conjugated anti-mouse IgG antibody (Molecular Probes, Eugene, OR)]. DNA
was counterstained with propidium iodide in the single-labeled sections.
Preparation of nuclear spreads and immunocytochemistry
Preparation of meiotic nuclear spreads was performed according to the
methods for surface spreading of meiotic chromosomes described previously
(Moens and Pearlman, 1991)
with some modifications described below. Testicular cell suspension was
prepared according to the method described by Heyting and Dietrich
(Heyting and Dietrich, 1991
),
and the cells were put on poly-L-lysin-coated coverslips. The cells on the
coverslips were placed in 85 mM NaCl for 3 minutes, transferred to 1%
paraformaldehyde solution (pH 8.2 with 0.01 M sodium borate) containing 0.03%
SDS for 3 minutes, and then to 1% paraformaldehyde solution (pH 8.2) without
SDS for 3 minutes. The coverslips were rinsed three times for 1 minute each in
4% (v/v) Photo-Flo (Kodak, Rochester, NY) in distilled water (pH 8.0) and
air-dried overnight. For preservation of chromatin loops in the nuclear
spreads, the cells on coverslips were incubated in 75 mM KCl solution for 3
minutes instead of 85 mM NaCl solution. Further, for the preservation of
meiotic chromosome shape, the operation to diffuse chromatin by SDS during
fixation was omitted. Instead, after the incubation in 85 mM NaCl for 3
minutes, the cells were fixed in 1% paraformaldehyde in PBS for 15
minutes.
According to the labeling methods described by Heyting and Dietrich
(Heyting and Dietrich, 1991),
the coverslips were placed for 10 minutes in PBS, for 20 minutes in 1 µg/ml
DNase I (Sigma) in PBS, for 10 minutes in detergent (5 mM EDTA, 0.25% gelatin,
and 0.05% Triton X-100 in PBS), for 10 minutes in PBS, and for 30 minutes in
blocking buffer (3% BSA, 10% goat serum, 0.05% Triton X-100 in PBS). The
coverslips were incubated with the first antibodies at appropriate dilutions
in the blocking buffer at 4°C overnight. After washing for 10 minutes in
PBS, for 10 minutes in detergent and for 10 minutes in PBS, the coverslips
were incubated with the secondary antibodies at appropriate dilutions in the
blocking buffer. After washing three times in PBS, DNA was counterstained with
propidium iodide in single-labeled samples. The samples were mounted with
Vectashield Mounting Medium (Vector Laboratories) and observed under a Bio-Rad
MicroRadiance confocal microscope.
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Results |
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Using the anti-Rec8 antibody, we first examined the expression of Rec8
protein in various mouse tissues. Nuclear extracts from kidney, liver and
testis were analysed by western blotting with the rabbit polyclonal anti-Rec8
antibody (Fig. 1C). We also
examined the expressions of other cohesin subunits (SMC1 and SMC3) and a
component of synaptonemal complexes (SCP3) to guarantee that cohesin proteins
and synaptonemal complex proteins were extracted properly in those extracts.
SMC3 was observed in all of the extracts as a 145-kDa band. SMC1 was
detected as a 160-kDa band in kidney, liver and testis extracts, whereas
SMC1ß protein was detected as a 155-kDa band only in testis extract as
has been previously reported (Revenkova et
al., 2001
). SCP3 protein was detected as 30/33-kDa bands only in
testis extract as has been previously reported
(Heyting et al., 1987
;
Lammers et al., 1994
). Thus,
proteins of mitotic and meiotic cohesin and synaptonemal complexes were
extracted properly in each extract. Of these extracts, anti-Rec8 antibody
detected three bands (82 to 95 kDa) only in testis nuclear extracts
(Fig. 1C). Essentially the same
result was obtained with mouse polyclonal anti-Rec8 antibody (data not
shown).
Next, we examined when Rec8 protein is expressed in mouse testis. Testis
extracts were prepared from 0-, 1-, 2-, 3- and 5-week-old mice
(Fig. 1D). In male mice,
meiosis starts between 1 and 2 weeks of age. Tubulin protein, as a loading
control, was expressed in extracts from 0- to 5-week-old mice at a similar
level. SMC1 and SMC3 were also expressed in extracts from 0-5-week-old
mice. By contrast, protein expression of Rec8 began at 2 weeks of age. Other
meiosis-specific proteins, SMC1ß and SCP3, were also expressed in
extracts from 2-week-old mice. These results suggest that Rec8 is a
meiosis-specific protein that is expressed only when spermatogenesis starts in
the testis.
Association of Rec8 protein with other cohesin proteins
To examine the association of Rec8 protein with other cohesin proteins, we
immunoprecipitated testis nuclear extracts with the antibodies against cohesin
proteins and analyzed the immunoprecipitates by western blotting. Anti-Rec8
immunoprecipitates contained Rec8, SMC1ß, SMC3, and SCP3 but not
SMC1 (Fig. 2).
Immunoprecipitates with anti-SMC1 or anti-SMC3 antibodies contained Rec8,
SMC1, SMC3 and SCP3-reactive bands, although the intensities of Rec8 bands
were very faint in anti-SMC1 immunoprecipitates, probably due to weaker
crossreactivity to SMC1ß than to SMC1
in immunoprecipitation
analysis (compare the relative band intensity of SMC1ß to SMC1
in
testis nuclear extracts in Fig.
1C with that in anti-SMC1 immunoprecipitates in
Fig. 2). No band was detected
in immunoprecipitates without the primary antibodies or with control preimmune
serum (data not shown). These results demonstrate that Rec8 protein associates
with other cohesin proteins, SMC1ß and SMC3, and a synaptonemal complex
protein, SCP3 (30 kDa), but not with SMC1
. The results also demonstrate
that the phosphorylation state of Rec8 protein does not affect its association
with SMC1ß and SMC3, since both phosphorylated and dephosphorylated Rec8
proteins were coimmunoprecipitated with these proteins
(Fig. 2).
|
Localization of Rec8 on synaptonemal complex in meiotic prophase
To examine the expression pattern of Rec8 protein in mouse testis cells,
frozen sections of mouse testis were subjected to indirect immunofluorescence
labeling with anti-Rec8 antibody or anti-SCP3 antibody or both
(Fig. 3). SCP3 is one of the
major components of the axial/lateral element (AE/LE) of synaptonemal
complexes (Lammers et al.,
1994). In the sections, SCP3 was detected as several lines, which
represented the synaptonemal complexes in spermatocytes. Similarly, Rec8 was
mainly detected along the synaptonemal complexes, but the signal was dotty
lines in spermatocytes. In contrast to SCP3, Rec8 was also detected in round
and elongated spermatids although the signals were weak compared to those in
spermatocytes. No labeling was essentially observed in spermatogonia and other
somatic cells. Immunofluorescent double labeling with anti-Rec8 antibody and
anti-SCP3 antibody showed that most signals of Rec8 and SCP3 overlapped with
each other. However, some spermatocytes showed Rec8 labeling but hardly showed
SCP3 labeling (indicated by arrows in the lower panels in
Fig. 3), indicating that Rec8
protein expression starts at an earlier stage of meiosis than does the
expression of SCP3 in spermatocytes, probably from pre-leptotene stage. In
control sections incubated with anti-Rec8 antibody preabsorbed with antigenic
proteins or with secondary antibodies alone, no specific signals were observed
(data not shown).
|
To examine the localization of Rec8 on synaptonemal complexes in detail, we performed immunofluorescence double labeling of Rec8 and SCP3 on nuclear spreads of mouse testis cells. Anti-SCP3 immunocytochemistry showed the presence of short and fine AEs of synaptonemal complexes in leptotene spermatocytes (Fig. 4A). In this stage, Rec8 was detected as dotty signals along the AEs. In the zygotene stage, when AEs have become longer and have started to form synapsis (AEs are now called LEs), Rec8 signals were more concentrated and detected as dotty lines along the AE/LEs (Fig. 4B). The dotty lines of Rec8 signal were also detected along LEs of synapsed chromosomes and unsynapsed XY chromosomes (indicated by an arrow) in the pachytene stage (Fig. 4C) and along LEs of desynapsed chromosomes in the diplotene stage (Fig. 4D).
|
In the above observations, chromatin was mostly dispersed except for the proximal to synaptonemal complexes (Fig. 5A). Hence, if Rec8 protein existed on chromatin loops expanding from the axis of synaptonemal complexes, the signal would not be observable. By treatment of cells with KCl instead of NaCl prior to fixation in the presence of SDS, we found that chromatin diffusion was suppressed and chromatin loops could be seen (Fig. 5B,C). Using this method, we examined the localization of Rec8 on chromatin loops. Rec8 existed on the portion of chromatin loops that came in contact with synaptonemal axes, but it was hardly detected along chromatin loops extending from the synaptonemal axes, implying that Rec8 is involved in the conjunction of chromatin loops with the synaptonemal axes and that Rec8-mediated cohesion of sister chromatids is limited to the sites of chromatin adjacent to synaptonemal complexes.
|
Localization of Rec8 on meiotic chromosomes
To examine the localization of Rec8 protein on meiotic chromosomes,
chromosome spreads were prepared by the method described in Materials and
Methods to prevent dispersion of chromatin by SDS during fixation, thereby
preserving chromosome shape. In meiotic prophase I at the pachytene stage
(Fig. 6A), Rec8 was detected as
lines of dotty signals, representing synaptonemal complexes. At later stages,
however, the intensity of the Rec8 signal tended to decline as meiosis
proceeded. In diakinesis and metaphase I stages
(Fig. 6B,C,I), a considerable
amount of Rec8 signal was still detected along the chromosomal axes (indicated
by arrows), which covered both the regions of the centromere and the
chromosome arm proximal to chiasmata. In these stages, Rec8 was also observed
along chromosome arms distal to chiasmata (indicated by blue arrowheads in
Fig. 6B,C) or on the sites at
which the homologs were seen overlapped (indicated by an white arrowhead in
Fig. 6I). After metaphase
I-to-anaphase I transition (Fig.
6D,E,J), homologous chromosomes separated from each other, whereas
sister chromatids were still attached to each other at their centromere
regions. In these stages, the Rec8 signals along chromosome arms were no
longer detected, while the signals were detected at the conjunction sites of
sisters (centromere regions) (indicated by arrows in
Fig. 6J). The centromeric Rec8
signals often observed as doublets. The reason why centromeric Rec8 signals
were observed as doublets is not clear, but we speculate that it may reflect
the antibody's inability to access deep into the inner centromere regions. The
Rec8 signals at centromere regions were detected up to metaphase II
(Fig. 6F). In anaphase II,
however, the signals of centromeric Rec8 were diminished
(Fig. 6G). The Rec8 signal was
not observed on mitotic chromosomes in spermotogonia
(Fig. 6H). The localization and
selective loss of Rec8 from meiotic chromosomes suggest that mammalian Rec8
protein plays pivotal roles in chromosome cohesion and separation in both
meiosis I and II.
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Discussion |
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The possible components of mitotic and meiotic cohesin complexes in budding
yeast and mammals, based on accumulated knowledge from the previous and
present studies, are illustrated in Fig.
7. In budding yeast, mitotic cohesin complexes consist of at least
Scc1p, Scc3p, Smc1p and Smc3p, and only the Scc1p subunit is replaced by Rec8p
during meiosis. In fission yeast, two Scc3p homologs, Psc3p and Rec11p, have
been identified, and Rec11p is required for chromatid cohesion in meiosis
(Krawchuk et al., 1999;
Tomonaga et al., 2000
).
Therefore, Rad21p and Psc3p are probably replaced by Rec8p and Rec11p during
meiosis, respectively. Vertebrate cohesin complexes consist of, at least,
SMC1, SMC3, SCC1/Rad21 and either one of SA1 and SA2 in mitosis
(Losada et al., 2000
;
Sumara et al., 2000
). In
addition, mammalian meiotic cells express two other cohesin proteins,
SMC1ß and STAG3 (SA3), of which homologs have not been found in yeasts
(Prieto et al., 2001
;
Revenkova et al., 2001
). Our
immunoprecipitation study revealed that mammalian Rec8 associates with
SMC1ß and SMC3 but not with SMC1
, suggesting that mammalian
meiotic cohesin consists at least of Rec8 (Rad21 isoform), SMC1ß (SMC1
isoform) and SMC3. However, close investigations into localizations of
SMC1ß and Rec8 in the previous and the present studies shed light on the
difference that dissociation of SMC1ß from chromosome arms occurs earlier
than that of Rec8 in meiosis I. SMC1ß dissociates from the chromosome
arms in late prophase and diakinesis stages, and very little SMC1ß, if
any, is localized on the arms at metaphase I, whereas a considerable amount of
Rec8 is still localized along chromosome arms at metaphase I. Therefore, Rec8
may associate with other as-yet-unidentified meiotic isoforms of SMC1 in
addition to SMC1ß. It is also possible that the difference between
dissociation time of Rec8 and that of SMC1ß from chromosome arms was
caused by the difference in fixations utilized in the previous and present
studies. STAG3 is known to be a meiotic isoform of SA1 and SA2 and to
associate with SMC1 and SMC3, although it is not clear whether the
STAG3-associated SMC1 is SMC1
or SMC1ß or both
(Prieto et al., 2001
). In
contrast to Rec8 and SMC1ß, STAG3 is localized only in the arm regions of
inter-chromatids in metaphase I. Furthermore, in late anaphase I and
thereafter, STAG3 is not detected (Prieto
et al., 2001
). Therefore, even if Rec8 and SMC1ß associate
with STAG3, the association should be limited spatially and temporally to the
arm regions of chromosomes until metaphase I. As has been previously proposed
(Prieto et al., 2001
), it is
very likely that two or more mammalian cohesin complexes participate in
meiosis. Furthermore, it has been shown recently that the mitotic cohesins,
STAG2 and Rad21, are localized on axial elements during diplotene stage, but
do not exist on the chromosome axes thereafter in meiosis
(Prieto et al., 2002
). Mitotic
cohesins may also participate in meiotic chromosome behavior at some
restricted time. In all cases, Rec8 is probably a common and essential
component of meiotic cohesin complexes from yeast to human.
|
Rec8 is a component of AE/LEs of synaptonemal complexes in meiotic
prophase I
The present study showed that Rec8 protein is expressed prior to the
expression of SCP3 in spermatocytes, suggesting that Rec8 expression starts
earlier than leptotene stage. Although it is not clear exactly when mammalian
Rec8 synthesis starts, yeast Rec8p starts to be expressed from pre-meiotic DNA
synthesis (Klein et al.,
1999). After pre-meiotic DNA synthesis, chromosomes are arranged
along proteinaceous axes called AEs, to which chromatin loops are attached
during early meiotic prophase. As meiotic prophase proceeds, the AEs become
aligned in parallel and incorporated in zipper-like structures (synaptonemal
complexes), which are now called LEs. The mature synaptonemal complexes are a
tripartite structure, two LEs and a central element (CE). Results of genetic
and cytological studies suggest that synaptonemal complexes play roles in
maintenance of homolog adhesion during meiotic prophase and possibly in
facilitation of meiotic exchange (Walker
and Hawley, 2000
). In mammals, three proteins have been identified
as the components of synaptonemal complexes; SCP1 (Syn1) is a component of CE,
and SCP2 and SCP3 (Cor1) are components of LEs
(Dobson et al., 1994
;
Lammers et al., 1994
;
Offenberg et al., 1998
). SCP3
is a main determinant of AEs of synaptonemal complexes since
SCP3-deficient spermatocytes fail to form AEs and thus synaptonemal
complexes (Yuan et al., 2000
).
Recently, cohesin proteins, Rec8p and Smc3p, have been found along the AEs in
yeast, and these proteins have been proved to be essential for the formation
of synaptonemal complexes, since AEs are not formed in rec8-deleted
or smc3-point mutants in budding yeast
(Klein et al., 1999
). In
mammals, SMC1
, SMC1ß, SMC3 and STAG3 are localized along AEs
throughout prophase I from leptotene to diplotene stages
(Eijpe et al., 2000
;
Prieto et al., 2001
;
Revenkova et al., 2001
). In
the present study, we found that mammalian Rec8 was also localized along the
AE/LEs throughout meiotic prophase I. Therefore, all cohesin proteins so far
examined are parts of components of AE/LEs of synaptonemal complexes. The role
of cohesin complexes in AE/LEs is not clear, but it has been proposed that the
cohesin core recruits recombination proteins and promotes synapsis between
homologous chromosomes, since cohesin-containing chromosomal cores are formed
and they are synapsed in meiotic nuclei in SCP3-deficient
spermatocytes (Pelttari et al.,
2001
). In addition, Rec8 was localized only on the sites of
chromatin loops adjacent to synaptonemal axes
(Fig. 5C), suggesting that
Rec8-containing cohesin cores determine the positions on chromatin loops at
which synaptonemal axes are conjugated with the replicated DNAs.
Cohesion and separation of homologous chromosomes in meiosis I and of
sister chromatids in meiosis II
In most organisms, the chiasmata and the arm cohesion distal to chiasmata
link homologous chromosomes together, allowing them to align on the spindle in
meiosis I. Cohesion along chromosome arms is lost during meiosis I, while
sister chromatids remain associated at centromeres until the onset of anaphase
II. Loss of arm cohesion is required for the resolution of chiasmata and thus
for separation of the homologs in meiosis I, whereas maintenance of cohesion
at centromeres is needed for sister chromatids to separate properly in meiosis
II (Miyazaki and Orr-Weaver,
1994). In mammals, localizations of SMC1ß and STAG3 have been
investigated to account for the meiosis-specific chromosome behavior. STAG3,
from its localization as above mentioned, can possibly account for the
selective loss of cohesion along chromosome arms in meiosis I but can not
account for the maintenance of cohesion between sister centromeres until
anaphase II. Conversely, SMC1ß remains chromatin-associated at the
centromeres up to metaphase II, but its dissociation from chromosome arms
occurs so early, in diplotene stage, that SMC1ß cannot maintain the
cohesion between homologs until metaphsae I
(Revenkova et al., 2001
). In
the present study, we showed that mammalian Rec8 is released along chromosome
arm regions both proximal and distal to chiasmata, when homologs separate from
each other in anaphase I, while Rec8 on inter-sister centromere regions is
maintained until anaphase II (Fig.
8). This spatially and timely selective loss of Rec8 from
chromosomes is concomitant with the two-step separation of chromosomes during
meiosis. Thus, Rec8 is the only molecule so far examined in mammals that can,
for itself, account for the meiosis-specific chromosome behavior in both
meiosis I and II.
|
Then, how is the selective loss of Rec8 from chromosome arms in meiosis I
and from centromeres in meiosis II regulated? In budding yeast, Rec8p is
cleaved by separin, and this cleavage is essential for progression into
anaphase I, since noncleavable mutations in Rec8p's potential separin cleavage
sites, as well as mutations in separin itself, arrest cells at metaphase I
(Buonomo et al., 2000).
Furthermore, securin, the inhibitor of separin, is destroyed at the onset of
anaphase in both meiosis I and II, probably through APC/Cdc20 pathway
(Salah and Nasmyth, 2000
). In
contrast to the reports for yeast, however, APC activity and securin
destruction are not required for the first meiotic division but are required
for the second meiotic division in Xenopus oocytes, because microinjection of
antibodies against APC activator or APC core subunit or microinjection of
destruction-box peptide or methylated ubiquitin, does not affect progression
through meiosis I and arrests the oocytes at metaphase II
(Peter et al., 2001
).
Therefore, it is assumed that segregation of homologous chromosomes and that
of sister chromatids are differentially regulated in higher eukaryotes. In
somatic cells of vertebrates, most of the arm regions of sister chromatids are
separated as chromosome condensation proceeds in prophase. Concomitantly, much
of cohesin dissociates from the arm regions, although a small amount of
cohesin persists predominantly in centromere regions until metaphase
(Losada et al., 1998
;
Waizenegger et al., 2000
). The
dissociation of cohesin from chromosomes in prophase is independent of
proteolytic cleavage, although the remaining cohesin at centromeres is finally
destroyed by proteolytic cleavage of the subunit SCC1/Rad21 by separin at the
metaphase-to-anaphase transition (Sumara
et al., 2000
; Waizenegger et
al., 2000
). Instead, the dissociation of cohesin from chromosome
arms in prophase seems to be regulated by phosphorylation of cohesin proteins
by mitotic kinases, Cdk1 kinase or polo-like kinase
(Losada et al., 2000
;
Alexandru et al., 2001
). The
two-step separation of chromosomes in the first and second meiotic divisions
is reminiscent of the two-step separation in prophase and anaphase in mitosis.
Therefore, the selective loss of Rec8 during meiosis may be caused by a
mechanism similar to that in mitotic division. However, it is notable that
loss of Rec8 from chromosome arms occurs at a different time from that of
mitotic cohesin in the cell cycle; Rec8 is released in anaphase during meiosis
I, while SCC1/Rad21 is released in prophase during mitosis. Therefore, in
mammals, it is possible that selective loss of Rec8 from the meiotic
chromosomes depends on the partner(s) in the cohesin complex; for example,
STAG3 (SA3)-associated Rec8 is released from chromosome arms at the onset of
anaphase I, while SAX (unidentified SA isoform)-associated Rec8 on centromeres
is maintained until the onset of anaphase II. Conversely, centromeric Rec8 may
be protected from dissociation by (an) unidentified molecule(s) such as
Mei-S332, which has been found on centromeres of meiotic chromosomes in
Drosophila (Kerrebrock et al.,
1995
). These hypotheses are merely speculative in vertebrates and
remain to be clarified.
In conclusion, we propose that Rec8 is an essential meiosis-specific cohesin and that its role in chromosome cohesion during meiosis is conserved from yeasts to mammals even though there may be several differences among species in its associated partners in cohesin complexes and in the mechanisms underlying its dissociation from chromosomes. The possible roles of Rec8 in the behavior of meiotic chromosomes are (1) maintenance of cohesion between homologous chromosomes until metaphase I by its association with chromosome arms distal to chiamata; (2) separation of homologous chromosomes in anaphase I by its selective dissociation from the chromosome arms; (3) maintenance of cohesion between sister chromatids until metaphase II by its association with centromere regions; and (4) separation of sister chromatids in anaphase II by its final dissociation from the centromere regions.
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References |
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