1 Department of Veterinary and Animal Sciences, University of Massachusetts,
Amherst, MA 01003, USA
2 Institute of Medical Biochemistry, University of Oslo, PO Box 1112 Blindern,
Oslo 0317, Norway
* Author for correspondence (e-mail: philippe.collas{at}basalmed.uio.no )
Accepted 18 April 2002
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Summary |
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Key words: AKAP95, Chromosome condensation, Condensin, Mitosis, Zygote
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Introduction |
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Condensation of somatic chromosomes at mitosis requires the highly
conserved 13S condensin complex (Hirano,
2000). The Xenopus and human condensin complex is made of
two structural maintenance of chromosomes (SMC) proteins
[chromosome-associated protein (CAP)-C and -E] and three non-SMC regulatory
proteins (CAP-D2, -G and -H) (Schmiesing
et al., 1998
; Schmiesing et
al., 2000
; Kimura et al.,
2001
). Condensins purified from Xenopus egg extracts
display ATPase activity, which introduces positive writhes in the DNA and
thereby may assist in condensation (Kimura
and Hirano, 1997
; Kimura et
al., 1999
). Targeting of condensins to chromatin at mitosis
correlates with phosphorylation of the non-SMC subunits
(Hirano et al., 1997
) and
phosphorylation of histone H3 (Kimura et
al., 1998
), and may involve additional factors
(Kimura et al., 1998
;
Schmiesing et al., 2000
;
Giet and Glover, 2001
).
The cAMP-dependent kinase (PKA or A-kinase) anchoring protein AKAP95 is
also essential for mitotic chromosome condensation. Human AKAP95 is a 95 kDa
zinc-finger protein of 692 amino acids, 89% identical to rat AKAP95
(Coghlan et al., 1994). AKAP95
is a component of the nuclear matrix-chromatin interface in interphase. Prior
to nuclear envelope breakdown at mitosis, AKAP95 is recruited to the chromatin
(Collas et al., 1999
). Studies
in mitotic HeLa cell extracts have shown that chromatin-bound AKAP95 acts as a
targeting protein for the condensin complex
(Steen et al., 2000
).
Chromatin- and condensin-binding domains of human AKAP95 have been mapped to
the C-terminal half of the protein within residues 387-450 and 525-569,
respectively (Eide et al.,
2002
). Chromatin-binding and chromosome condensation activities of
AKAP95 do not require PKA anchoring nor PKA activity, whereas maintenance of
condensed chromosomes throughout mitosis does
(Collas et al., 1999
).
Here, we present evidence that AKAP95 is implicated in a differential regulation of condensin targeting and condensation of maternal and paternal chromosomes in mouse zygotes. Our results suggest a concept whereby condensation of chromosomes in gametes, zygotes and somatic cells involves related but distinct mechanisms.
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Materials and Methods |
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Gametes, embryos and cells
MII oocytes and pronuclear (PN) embryos were collected from superovulated
B6D2 mice at 14 and 22 hours post-hCG injection, respectively. Cumulus cells
were dispersed with 1 mg/ml hyaluronidase, washed and dissolved in SDS sample
buffer or sedimented onto coverslips for immunofluorescence. Oocytes were
washed in Flushing Holding Media (FHM; Specialty Media) prior to use. PN stage
embryos were cultured in Potassium Simplex Optimized Media (KSOM; Specialty
Media).
Mature sperm were collected in phosphate buffered saline (PBS) from the epididymis of C57 males, washed and stored in liquid nitrogen without cryoprotectant. After thawing on ice, sperm were washed in FHM and held on ice until use. Sperm were also dissolved in SDS sample buffer or settled on coverslips for immunofluorescence. To prepare sperm nuclei, frozen-thawed sperm (without mid-piece and tail) were permeabilized for 15 minutes in 1% Triton X-100, washed in PBS and held on ice until use.
Oocyte activation
MII oocytes were parthenogenetically activated at 15 hours post-hCG in KSOM
containing 10 mM SrCl2 (Sigma) for 4 hours at 37°C. To inhibit
RNA polymerase II-mediated transcription, oocytes were activated with 10 mM
SrCl2 together with 5 µg/ml actinomycin D (Sigma) for 4 hours.
To inhibit protein synthesis, oocytes were activated for 4 hours with 10
µg/ml cycloheximide (Sigma). Activation was monitored by appearance of the
FPN at the end of the activation treatment. Activated oocytes were washed in
KSOM and processed for immunofluorescence or immunoblotting.
Micromanipulation
Intracytoplasmic sperm injection (ICSI)
MII oocytes were maintained in FHM at 37°C during ICSI. Sperm heads
were recovered from liquid nitrogen, thawed at 25°C, centrifuged at 2000
g and resuspended in 7% polyvinyl-pyrrolidone in PBS to
decrease stickiness. When required, sperm nuclei were labeled with 1 µg/ml
Hoechst 33342 prior to injection. Injections were performed at 19°C in FHM
containing 3 mg/ml BSA using a piezo-drill. Oocytes were injected at 15 hours
post-hCG, washed and cultured in KSOM. PN formation was detected by
phase-contrast microscopy 4-5 hours after ICSI.
Enucleation of the FPN
PN stage embryos collected from B6D2 females mated to CD1 males were
enucleated in FHM containing 3 mg/ml BSA and 5 µg/ml cytochalasin B. The
zona pellucida was penetrated with a piezo drill and the FPN (smaller than the
MPN) removed by aspiration. Embryos were washed and cultured in KSOM.
Peptide injection
PN stage embryos in FHM were microinjected into fully formed FPN or MPN
with 250 pg GST-AKAP95 peptide using a pulled glass capillary. Injections took
place at 8 hours post-ICSI, or 23 hours post-hCG when using normally
fertilized embryos. Embryos were washed and cultured in KSOM. Nuclei of
two-cell stage blastomeres were injected similarly except that the
peptide-containing injection solution contained 10 µg/ml of a 150 kDa
FITC-dextran (Sigma) as tracer.
Immunological procedures
Immunoblotting analysis of cumulus cells (30 µg protein), oocytes
(n=200), embryos (n=200) and sperm or sperm nuclei
(n=106) was performed essentially as described
(Collas et al., 1999) using
anti-AKAP95 polyclonal antibodies (1:250 dilution), anti-AKAP95 mAb47 (1:250),
anti-protamine Hup1N (1:500) or anti-hCAP-D2 antibodies (1:5000). For
immunofluorescence, oocytes and embryos were washed in PBS, fixed with 3%
paraformaldehyde for 15 minutes, washed in PBS, permeabilized with 0.1% Triton
X-100 for 15 minutes, washed in PBS/0.01% Tween-20 (TBST) and proteins blocked
in PBST/2% BSA. Primary and secondary antibodies were used at a 1:100 dilution
in PBST/BSA and incubated for 30 minutes. DNA was counterstained with 0.1
µg/ml Hoechst 33342 or 0.1 µg/ml propidium iodide (PI) as indicated.
Immunofluorescence analysis of methanol-fixed cumulus cells sedimented on
poly-L-lysine-coated coverslips was performed as described earlier
(Steen and Collas, 2001
).
Sperm were processed as described for oocytes after sedimentation onto
poly-L-lysine-coated glass coverslips. Observations were made on an Olympus
BX60 microscope and photographs taken with a JVC CCD camera and AnalySIS
software (Soft Imaging Systems).
In situ extraction of nuclear matrices was carried out as described
(Martins et al., 2000) after
affixing cumulus cells onto glass coverslips. Briefly, cells were extracted
with 0.1% Triton X-100 for 5 minutes, incubated for 5 minutes with ice-cold
cytoskeleton stabilization (CSK) buffer containing 0.1% Triton X-100 and
washed in CSK buffer. DNA was digested for 30 minutes with 1 mg/ml DNase I and
washed twice for 5 minutes in PBS. Resulting matrices were fixed with
-20°C methanol and processed for immunofluorescence.
RNA in situ hybridization
An anti-rat AKAP95 cDNA probe was generated using as a template a 732 bp
PCR product amplified from rat genomic DNA using the primers
5'-AGGTTGGCTGCTGAACAATTC-3' and
5'-GATGGCTATGACAGGTACTGG-3'. PCR conditions were denaturation at
94°C for 5 minutes, and 35 cycles of 94°C denaturation (30 seconds),
58°C annealing (30 seconds) and 72°C extension (1 minute). The probe
was biotinylated using a random-priming biotin labeling kit containing
biotin-16-dUTP (Amersham) (Collas and
Aleström, 1998).
Oocytes were fixed with 3% paraformaldehyde in PBS for 15 minutes, washed and post-fixed with methanol:acetic acid (3:1) for 10 minutes on ice. Fixed oocytes were permeabilized with 1% Triton X-100 for 15 minutes at room temperature. Samples were denatured at 70°C in 70% formamide/2x SSC for 5 minutes, dehydrated in ethanol series and air dried. The labeled DNA probe was allowed to hybridize overnight at 37°C. Post-hybridization washes were performed in 50% formamide/2x SSC (3 times, 5 minutes), all at 45°C, and twice for 3 minutes in PN buffer (0.2 M Na2HPO4, 6 mM NaH2PO4, pH 8.0, and 0.05% NP-40). Proteins were blocked for 5 minutes in PN buffer containing 5% dry milk (PNM buffer). The probe was revealed by successive 30-minute incubations at 37°C in avidin-TRITC (1:400 dilution in PNM buffer), biotin-conjugated anti-avidin antibodies (1:100 dilution in PNM buffer) and another layer of avidin-TRITC. Slides were washed in PN buffer for 5 minutes and mounted in antifade (1 mg/ml p-phenylenediamine dihydrochloride, 10% PBS, 90% glycerol) containing 0.2 µg/ml Hoechst 33342. When indicated, samples were treated with 1 mg/ml DNAse I (Sigma) or 100 µg/ml RNAse A (Amersham) for 30 minutes prior to hybridization.
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Results |
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Immunoblotting analysis of mouse epididymal sperm nuclei revealed no
AKAP95, even after reducing disulfide bonds with 100 mM DTT (resulting in
decondensed nuclear `halos') prior to SDS-PAGE
(Fig. 1C). Metaphase II (MII)
oocytes contained traces of AKAP95, as seen on an immunoblot of 200 oocytes
(Fig. 1C) and by overexposure
of immunofluorescently labeled oocytes
(Fig. 1D, MII, inset, arrow).
After fertilization, AKAP95 was strongly upregulated at the pronuclear (PN)
stage (Fig. 1C,D). Notably,
AKAP95 was restricted to the FPN usually smaller than the MPN
and was absent from polar bodies (Fig.
1D, PN). In subsequent stages of development in vitro, AKAP95 was
localized in the nucleus of each blastomere and on condensed chromosomes in
mitotic cells (Fig. 1D,
arrows), reminiscent of earlier findings in somatic cells
(Collas et al., 1999).
Synthesis of AKAP95 correlated with activation of the oocyte. Parthenogenetic activation of MII oocytes with 10 mM SrCl2 elicited FPN formation and pronuclear accumulation of AKAP95 (Fig. 1E, +SrCl2). SrCl2-induced AKAP95 synthesis was verified by western blot (Fig. 1E, insert). AKAP95 labeling was essentially absent from oocytes activated with 10 µg/ml of the protein synthesis inhibitor, cycloheximide (Fig. 1E, +CHX), indicating that accumulation of AKAP95 in the FPN results from protein synthesis. However, AKAP95 was detected after activation with 10 mM SrCl2 together with 5 µg/ml of the RNA polymerase II inhibitor, actinomycin D (Fig. 1E, +SrCl2+Act.D). Therefore, oocyte activation induces strong upregulation of AKAP95 translation from a maternal store of mRNA.
AKAP95 mRNA is localized near the meiotic spindle
To demonstrate the presence of AKAP95 mRNA in the MII oocyte and to provide
some explanation to account for targeting of AKAP95 to the FPN after
fertilization (Fig. 1D), we
visualized AKAP95 mRNA in MII oocytes by in situ RNA hybridization. A 730 bp
fragment of the AKAP95 coding region amplified by PCR was used to generate a
biotinylated oligonucleotide probe. Hybridization of MII oocytes and detection
of the probe with TRITC-conjugated avidin revealed a concentration of AKAP95
mRNA around the meiotic spindle, as judged by DNA staining with Hoechst 33342
(Fig. 2A). The signal
disappeared when oocyte mounts were treated with RNAse A prior to
hybridization (Fig. 2B). DNAse
treatment did not eliminate the RNA hybridization signal, despite the
disappearance of most of the MII chromosome Hoechst labeling
(Fig. 2C). Thus, MII oocytes
contain AKAP95 mRNA, arguing that AKAP95 protein synthesis upon oocyte
activation is translationally regulated. Moreover, RNA hybridization data
reveal AKAP95 mRNA exclusively in the vicinity of the meiotic spindle. To our
knowledge, this is the first report of a highly restricted mRNA localization
in a mammalian oocyte. By analogy to eggs of non-mammalian species
(Alarcon and Elison, 2001) (see
Discussion), this may enable efficient targeting of the AKAP95 protein to the
FPN.
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Localization of mCAP-D2, a putative subunit of the mouse condensin
complex, in mouse gametes
Distribution of the condensin complex in mouse gametes was examined using
an affinity-purified antibody against hCAP-D2/CNAP1, a non-SMC subunit of the
human condensin complex. Immunoblotting analysis of mouse cumulus cells
revealed a single anti-hCAP-D2-reactive band of 150 kDa, the expected apparent
Mr of hCAP-D2. Anti-hCAP-D2 reactivity also occurred with
MII oocytes and in PN stage embryos, but not with sperm, even after enhancing
antigen accessibility in decondensed halos with DTT
(Fig. 3A). In unfertilized
oocytes, anti-hCAP-D2 labeling was restricted to the MII chromosomes
(Fig. 3B, arrow) and
co-localized with the faint AKAP95 staining (not shown). Thus, the antibody
crossreacted with a putative homologue of hCAP-D2/CNAP1 in mouse cumulus cells
and oocytes. The mouse protein was designated mCAP-D2.
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AKAP95 is targeted to the female pronucleus and to maternal
chromosomes at first mitosis
To unequivocally demonstrate targeting of AKAP95 to the FPN in mouse
zygotes, sperm was pre-labeled with the DNA stain, Hoechst 33342, and
introduced into MII oocytes by ICSI. Resulting PN stage zygotes were analyzed
by immunofluorescence using the anti-AKAP95 polyclonal antibody. Total
(maternal and paternal) DNA was counterstained with PI. Dual DNA staining
unequivocally discriminated the FPN (labeled red only) and the MPN (labeled
blue), and indicated that AKAP95 was localized exclusively in the FPN
(Fig. 4A, green and yellow
labeling). Examination of similar Hoechst- and PI-labeled embryos at mitosis
showed that anti-AKAP95 antibodies decorated a set of chromosomes not labeled
with Hoechst (Fig. 4B), illustrating the restriction of AKAP95 to maternal chromosomes. At the
two-cell stage, AKAP95 decorated both chromosome complements (not shown).
Together, these results indicate that AKAP95 is specifically targeted to the
FPN and to condensed maternal chromosomes at first mitosis.
|
Both maternal and paternal chromosomes recruit mCAP-D2
To assess the presence of mCAP-D2 on condensed maternal and paternal
chromosomes at first mitosis, mitotic zygotes produced by ISCI of
Hoechst-labeled sperm were examined by total DNA staining with PI and by
immunofluorescence using the anti-hCAP-D2 antibody.
Fig. 4C shows that mCAP-D2 was
detected on both parental chromosome sets. Double immunolabeling with
anti-AKAP95 mAb47 demonstrated mCAP-D2 and AKAP95 co-localization on female
chromosomes (Fig. 4D, yellow
label), and mCAP-D2 localization on male chromosomes despite the absence of
AKAP95. We concluded from these observations that mCAP-D2 is targeted to both
chromosome complements at first mitosis.
AKAP95 is required for condensation of maternal chromosomes
Targeting of AKAP95 to maternal chromosomes at mitosis suggests that the
protein may be involved in the condensation process. To test this hypothesis,
we investigated the effect of displacing endogenous AKAP95 in the FPN by
microinjecting a competitor GST-AKAP95 fusion peptide encompassing the
chromatin-binding domain (residues 387-450), but not the condensin-binding
domain (extending beyond residue 450), of human AKAP95
(Eide et al., 2002). This
GST-fusion peptide was referred to as AKAP95(387-450). Zygotes were produced
by ICSI of Hoechst-labeled sperm in order to distinguish male and female
pronuclei. The FPN or the MPN was microinjected with 250 pg of AKAP95(387-450)
(Fig. 5A), embryos were allowed
to reach mitosis, fixed and DNA conformation and AKAP95 localization were
examined. The anti-AKAP95 polyclonal antibody was used to label endogenous
AKAP95, while anti-AKAP95 mAb47 was used to detect the AKAP95(387-450)
peptide, not recognized by the polyclonal antibody (T. Eide, K. Tasken and
P.C., unpublished).
|
As expected from out previous data, only the FPN contained AKPA95 at the
time of peptide injection (Fig.
5A). Injection of AKAP95(387-450) into the MPN promoted binding of
the peptide to male chromatin, as detected with mAb47
(Fig. 5B, inset, `M'), but did
not affect AKAP95 localization on female (`F') chromosomes nor condensation of
either chromosome complement (Fig.
5B). Similar results were obtained with 500 pg peptide to
compensate for the volume difference between FPN and MPN (not shown).
Remarkably, however, injection of 250 pg AKAP95(387-450) into the FPN
completely inhibited condensation of the female chromatin
(Fig. 5C, `F'). The female
chromatin was devoid of endogenous AKAP95, as shown by the lack of polyclonal
anti-AKAP95 labeling (Fig. 5C).
However, mAb47 immunolabeling indicated that the peptide was bound to the
female chromatin and thereby displaced endogenous AKAP95 from maternal
chromosomes (Fig. 5C, inset,
`F'). In contrast to female chromatin, male chromosome condensation was not
affected by injection of AKAP95(387-450) into the FPN
(Fig. 5C, `M'). As a result,
these embryos simultaneously displayed condensed male chromosomes and a
decondensed maternal chromatin mass. Labeling of these embryos with an
antibody against B-type lamins, a marker of the nuclear envelope, indicated
that AKAP95(387-450) did not prevent female pronuclear envelope breakdown
(data not shown). Thus, the peptide inhibited maternal chromosome condensation
per se. Additionally, injection of the FPN with 250 pg AKAP95(387-692), which
binds both chromatin and condensins (Eide
et al., 2002) and is detected by the anti-AKAP95 polyclonal
antibody (Steen et al., 2000
),
allowed condensation of female, and male, chromosomes
(Fig. 5D), as did a
mock-injection with peptide buffer (data not shown). Lastly, FPN injection of
the N-terminal domain of human AKAP95 (AKAP95[1-195]), which does not bind
chromatin (Eide et al., 2002
),
or of an AKAP95(387-450) peptide mutated in the zinc finger to abrogate
chromatin binding (Eide et al.,
2002
), did not impair male or female chromosome condensation (data
not shown). These controls indicate that inhibition of condensation of female
chromatin following FPN injection of AKAP95(387-450) is not due to steric
hindrance, but rather to a specific inhibitory effect of the peptide.
Collectively, these results indicate that AKAP95(387-450) displaces endogenous AKAP95 from maternal chromatin. This correlates with inhibition of mitotic maternal chromosome condensation whereas paternal chromosome condensation remains unaffected. The chromatin- and condensin-binding peptide AKAP95(387-692) has no inhibitory effect, suggesting that the AKAP95(387-450) peptide acts as a dominant negative. These observations suggest an essential role of AKAP95 in the condensation of maternal chromosome in mitotic zygotes.
AKAP95(387-450) abolishes recruitment of mCAP-D2 to maternal
chromosomes
Human AKAP95 was recently proposed to act as a targeting molecule for the
condensin complex, a five-subunit structure required for chromosome
condensation (Steen et al.,
2000). To account for the failure of maternal chromosomes
condensation after FPN injection of AKAP95(387-450), decondensed female
chromatin and condensed male chromosomes obtained at mitosis after FPN
injection with AKAP95(387-450) (Fig.
5C) were labeled using anti-hCAP-D2 antibodies. mCAP-D2 was not
detected on decondensed female chromatin
(Fig. 6A, `F'), whereas male
chromosomes (`M') were labeled (Fig.
6A, `M'). mCAP-D2 targeting to paternal or maternal chromosomes
was not abolished by injection of AKAP95(387-450) into the MPN (not shown),
nor by FPN injection of AKAP95(387-692) or AKAP95(1-195)
(Fig. 6B). Therefore,
displacement of endogenous AKPA95 by the competitor AKAP95(387-450) peptide
correlated with the lack of mCAP-D2 association with maternal chromosomes.
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AKAP95(387-692) restores condensation of maternal chromatin
To further demonstrate a requirement of AKAP95 for female chromatin
condensation, we determined whether the chromatin- and condensin-binding
AKAP95(387-692) peptide would restore condensation of decondensed maternal
chromosomes. Cytoplasmic injection of a 20x mass excess (5 ng) of
AKAP95(387-692) in embryos containing a decondensed female chromatin mass (as
in Fig. 5C) restored
recruitment of mCAP-D2 to female chromosomes and condensation
(Fig. 6C). These chromosomes
were seen either distant from the already condensed paternal chromatin or
closely apposed to male chromosomes as in a normal mitosis. Cytoplasmic
injection of AKAP95(1-195) (not shown) or AKAP95(387-450) was ineffective in
rescuing female chromatin condensation
(Fig. 6D). Immunolabeling with
the anti-AKAP95 polyclonal antibody suggested association of AKAP95(387-692)
with female chromosomes (Fig.
6C). We concluded that restoration of binding of a functional
AKAP95 peptide to decondensed maternal chromosomes rescued mCAP-D2 targeting
to chromatin and condensation. This suggests that chromatin association of
AKAP95 is necessary for condensin recruitment to maternal chromosomes and
condensation. In contrast, condensins appear to associate with paternal
chromatin independently of AKAP95.
PKA anchoring to AKAP95 is dispensable for maternal chromosome
condensation
We previously reported that AKAP95 function in chromatin condensation in
mitotic extract and in vivo was independent of PKA anchoring to AKAP95, as
shown by microinjection of Ht31, a well-characterized AKAP-binding competitor
peptide (Carr et al., 1991;
Collas et al., 1999
). To
determine whether AKAP95 function in zygotic maternal chromatin condensation
required PKA anchoring, the FPN was injected with 750 nM Ht31, or 750 nM of a
control Ht31-P mutant which does not bind PKA
(Carr et al., 1991
). This Ht31
concentration, adjusted to account for the
15-fold increase in volume
between a somatic nucleus and a FPN, was shown to disrupt PKA-AKAP95 anchoring
(Collas et al., 1999
). Neither
peptide affected maternal chromosome condensation at mitosis (data not shown).
Thus, as in mitotic cells, PKA anchoring to AKAP95 is probably not required
for AKAP95 function in zygotic chromatin condensation.
Paternal chromosome condensation is AKAP95-independent
The lack of AKAP95 labeling of male chromosomes led us to determine whether
AKAP95 was required for condensation of male chromosomes at first mitosis.
Endogenous pronuclear AKAP95 was removed by non-invasive enucleation of the
FPN from normally fertilized pronuclear zygotes. The resulting androgenetic
embryos harbored only the MPN devoid of detectable AKAP95
(Fig. 7A). These androgenetic
embryos progressed through mitosis (Fig.
7B) and cleaved to at least two cells
(Fig. 7C). Mitotic androgenotes
displayed condensed chromosomes devoid of detectable AKAP95
(Fig. 7B). At the two-cell
stage, however, AKAP95 staining was detected in the nucleus of each blastomere
(Fig. 7C; yellow labeling).
Thus, AKAP95 is clearly dispensable for condensation and segregation of
paternal chromosomes. Nevertheless, paternal genes are capable of transcribing
AKAP95 at the two-cell stage in androgenotes, suggesting that the protein
might be necessary from this stage of development onwards.
|
Association of AKAP95 with chromatin is required for cleavage to the
four-cell stage
Requirements for AKAP95 anchoring to chromatin for condensation of two-cell
stage chromosomes and cleavage was determined. The nucleus of a blastomere of
a normal fertilized two-cell stage embryo
(Fig. 8, left panel) was
microinjected with 250 pg of the AKAP95(387-450) peptide together with a
150-kDa FITC-conjugated dextran to trace the peptide-injected blastomere
(Collas et al., 1999). The
second blastomere was mock-injected with peptide buffer. AKAP95(387-450)
inhibited chromosome condensation and cleavage of the injected blastomere in
14/15 embryos (Fig. 8, Mitosis,
arrow). Chromosomes of mock-injected blastomeres, however, condensed normally
(Fig. 8, Mitosis, arrowhead)
and blastomeres cleaved at least twice in these embryos
(Fig. 8, `8-16-cell').
Additional mock injections of 250 pg of the functional AKAP95(387-692) peptide
or of the non-chromatin binding AKAP95(1-195) peptide in a similar number of
embryos were not inhibitory (Fig.
8, Mock-injected). These results indicate that AKAP95 is
implicated in chromatin condensation and cleavage in early mouse embryos.
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Discussion |
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At fertilization, AKAP95 is translated from a maternal pool of mRNA upon
oocyte activation, assembles in the FPN and associates with maternal chromatin
at mitosis. What restricts AKAP95 to the FPN remains speculative. Recombinant
AKAP95 binds sperm chromatin in vitro (our unpublished results) and in vivo,
thus differential AKAP95 targeting is unlikely to result from differences in
chromatin composition between the parental genomes. Pronuclear accumulation of
AKAP95 may be regulated at the level of the nuclear import machinery or at the
level of the female pronuclear envelope; however, these possibilities remain
to be examined. Alternatively, a putative AKAP95 docking protein might be
absent or non-functional in the MPN. Our unpublished immunoprecipitation
studies suggest that AKAP95 interacts in interphase HeLa nuclei with the
nuclear matrix protein, NuMA (Compton et
al., 1992). Whereas mouse MPNs are devoid of NuMA
immunoreactivity, FPNs display variable amounts of NuMA (our unpublished
results). Thus, NuMA may constitute a possible AKAP95 anchor specific for the
FPN.
A third attractive possibility is that the specific localization of the
AKAP95 protein may be a consequence of the site of AKAP95 translation.
Polarized mRNA localization and site of translation is a well known mechanism
elaborated in (usually large) eggs of several non-mammalian species and in
some differentiated somatic cells to ensure efficient protein function at a
specific locus. Localized RNAs establish axial pattern formation and act as
cell fate determinants during development in, for example, Caenorhabditis
elegans (Kemphues, 2000),
Drosophila (Lasko et al.,
1999
), zebrafish (Suzuki et
al., 2000
) and Xenopus
(King et al., 1999
;
Alarcon and Elinson, 2001
).
Localized RNAs also contribute to maintaining the specialized characteristics
of differentiated somatic cells such as neurons, oligodentrocytes and
fibroblasts (Bashirullah et al.,
1998
). Our RNA in situ hybridization results show a highly
restricted concentration of AKAP95 mRNA near the meiotic spindle in mature
mouse oocytes. Whether the AKAP95 message is associated with spindle
microtubules, and the fate of the mRNA during resumption of meiosis remain
undetermined at present. It would be interesting to determine whether the RNA
remains in the oocyte cytoplasm upon second polar body emission; in particular
since no AKAP95 protein was detected in polar bodies (see, for example,
Fig. 1D,E and
Fig. 2A). Upon formation of the
FPN, AKAP95 translation may take place concomitant with, or shortly after,
disassembly of the meiotic spindle. The protein may either be engulfed in the
nucleus by the targeted nuclear membrane precursors or selectively imported in
the FPN. This remains to be investigated. In any event, restriction of AKAP95
mRNA to the meiotic spindle may provide a regulatory mechanism for efficient
AKAP95 targeting to the FPN upon activation.
Perhaps as a consequence of association with the FPN, AKAP95 specifically
translocates to maternal chromosomes at first mitosis. This observation is
reminiscent of the prompt redistribution of AKAP95 from the nuclear matrix to
chromatin prior to, or during, somatic nuclear envelope breakdown in mitotic
extract (Steen et al., 2000).
Thus, AKAP95 may simply not be available to paternal chromatin. This view is
supported by the binding of AKAP95 peptides to male chromatin when made
available by co-incubation with sperm chromatin (our unpublished results) or
by injection into the oocyte cytoplasm or into the MPN (this paper). High
affinity association of AKAP95 with maternal chromosomes may prevent its
release and binding to the closely apposed paternal chromosomes; we are
currently exploring this possibility. Selective targeting of AKAP95 to
maternal chromatin at first mitosis may have implications on the extent or
kinetics of female chromosome condensation. For example, maternal chromosomes
appear more compact than paternal chromosomes in mitotic zygotes
(Donahue, 1972
;
Kaufman, 1973
;
Dyban and Sorokin, 1983
). A
correlation between the extent of compaction and resolution of HeLa
chromosomes in mitotic extract and the amount of AKAP95(387-692) peptide bound
to chromatin was shown previously, and this paralleled increasing amounts of
hCAP-D2 recruited to the chromosomes
(Steen et al., 2000
). Thus,
the different extent of compaction of male and female chromosomes at first
mitosis may relate to the absence or presence of AKAP95.
A second issue is how mCAP-D2 and, potentially, the entire condensin
complex, are recruited to paternal chromatin independently of AKAP95.
Condensins may be targeted to male chromosomes via a putative loading factor
distinct from AKAP95 (Kimura et al.,
2001) or by binding DNA directly. Condensin association with
chromatin has been proposed to involve a binding of SMCs to DNA stabilized by
the non-SMC subunits (Hirano and Hirano,
1998
; Akhmedov et al.,
1998
; Kimura et al.,
2001
). Condensins may also have a higher affinity for DNA
modifications (Howlett and Reik,
1991
) or histone alterations
(Adenot et al., 1997
) that
characterize male chromatin. Both these modifications are expected to alter
chromatin conformation. The higher affinity of SMCs for DNA sequences (such as
AT-rich regions) with a propensity to form secondary structures
(Kimura and Hirano, 1997
;
Akhmedov et al., 1998
) supports
this hypothesis.
A question that remains to be addressed is how condensation of foreign
chromatin, such as that of a somatic nucleus, is regulated in the mature
oocyte cytoplasm following somatic nuclear transplantation (`cloning'). A
consistent phenomenon in nuclear transfer embryos is the premature
condensation of the transplanted chromatin into a metaphase-like conformation
in the MII oocyte cytoplasm (e.g. Collas
and Robl, 1991). As AKAP95 is present in somatic cell nuclei
(Fig. 1), it is anticipated
that it remains associated with the condensing chromosomes. Whether it is
required for condensation is not yet known; however, studies of somatic
chromosome condensation in mitotic HeLa cell extracts suggest that AKAP95
function is necessary for condensation to take place
(Collas et al., 1999
). It
would be interesting to examine somatic chromosome condensation requirements
in a mammalian meiotic egg extract.
Genetic, nuclear transplantation and pathological evidence suggests that
normal mammalian development requires the contribution of maternal and
paternal genomes (McGrath and Solter,
1984; Cattanach and Kirk,
1985
; Surani et al.,
1986
; Lalande,
1996
). These may function in distinct entities during
preimplantation development when extensive epigenetic remodeling and nuclear
reprogramming take place. This view is supported by the topological separation
of the two genomes at first mitosis (this paper) and up to the four-cell
stage, as shown by BrdU labeling of sperm and differential heterochromatin
staining in mouse interspecific hybrid embryos
(Mayer et al., 2000
).
Complementarity of genome function is reflected by distinct patterns of
expression of maternally and paternally derived alleles of imprinted genes
(Tilghman, 1999
). In addition,
relative to the FPN, the MPN displays earlier initiation of replication and
transcription (Bouniol-Baly et al.,
1997
), greater transcriptional activity
(Adenot et al., 1997
;
Aoki et al., 1997
) and an
enrichment in hyperacetylated histone H4
(Adenot et al., 1997
). The FPN
harbors AKAP95, a major component of the nuclear matrix and chromatin
interface. Interestingly, AKAP95 is found in hypoacetylated chromatin in mouse
cumulus cells and fibroblasts, as shown in chromatin immunoprecipitation
studies using an anti-acetylated histone H4 antibody (our unpublished
results). This suggests that at least a fraction of AKAP95 is enriched in
transcriptionally silent chromatin. Collectively, these observations raise the
possibility that AKAP95 is involved in imposing a repressive chromatin
structure, or acts as an anchoring protein for chromatin remodeling complexes
or transcriptional regulators. It is tempting to speculate that implications
of AKAP95 association with the maternal genome after fertilization may extend
beyond a role in the regulation of mitotic chromosome condensation.
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Adenot, P., Mercier, Y., Reinsch, S. and Thompson, E. M.
(1997). Differential H4 acetylation of paternal and maternal
chromatin precedes DNA replication and differential transcriptional activity
in pronuclei of 1-cell mouse embryos. Development
124,4615
-4625.
Akhmedov, A. T., Frei, C., Tsai-Pflugfelder, M., Kemper, B.,
Gasser, S. M. and Jessberger, R. (1998). Structural
maintenance of chromosomes protein C-terminal domains bind preferentially to
DNA with secondary structure. J. Biol. Chem.
273,24088
-24094.
Alarcon, V. B. and Elinson, R. P. (2001). RNA
anchoring in the vegetal cortex of the Xenopus oocyte. J.
Cell Sci. 114,1731
-1741.
Aoki, F., Worrad, D. M. and Schultz, R. M. (1997). Regulation of transcriptional activity during the first and second cell cycles in the preimplantation mouse embryo. Dev. Biol. 181,296 -307.[Medline]
Bashirullah, A., Cooperstock, R. L. and Lipshitz, H. D. (1998). RNA localization in development. Annu. Rev. Biochem. 67,335 -394.[Medline]
Bouniol-Baly, C., Nguyen, E., Besombes, D. and Debey, P. (1997). Dynamic organization of DNA replication in one-cell mouse embryos: relationship to transcriptional activation. Exp. Cell Res. 236,201 -211.[Medline]
Carr, D. W., Stofko-Hahn, R. E., Fraser, I. D., Bishop, S. M.,
Acott, T. S., Brennan, R. G. and Scott, J. D. (1991).
Interaction of the regulatory subunit (RII) of cAMP-dependent protein kinase
with RII-anchoring proteins occurs through an amphipathic helix binding motif.
J. Biol. Chem. 266,14188
-14192.
Cattanach, B. M. and Kirk, M. (1985). Differential activity of maternally and paternally derived chromosome regions in mice. Nature 315,496 -498.[Medline]
Ciemerych, M. A. and Czolowska, R. (1993). Differential chromatin condensation of female and male pronuclei in mouse zygotes. Mol. Reprod. Dev. 34, 73-80.[Medline]
Coghlan, V. M., Langeberg, L. K., Fernandez, A., Lamb, N. J. and
Scott, J. D. (1994). Cloning and characterization of AKAP95,
a nuclear protein that associates with the regulatory subunit of type II
cAMP-dependent protein kinase. J. Biol. Chem.
269,7658
-7665.
Collas, P. and Aleström, P. (1998). Nuclear localization signals enhance germline transmission of a transgene in zebrafish. Transgenic Res. 7, 303-309.[Medline]
Collas, P. and Robl, J. M. (1991). Relationship between nuclear remodeling and development in nuclear transplant rabbit embryos. Biol. Reprod. 45,455 -465.[Abstract]
Collas, P., le Guellec, K. and Tasken, K.
(1999). The A-kinase anchoring protein, AKAP95, is a multivalent
protein with a key role in chromatin condensation at mitosis. J.
Cell Biol. 147,1167
-1180.
Compton, D. A., Szilak, I. and Cleveland, D. W. (1992). Primary structure of NuMA, an intranuclear protein that defines a novel pathway for segregation of proteins at mitosis. J. Cell Biol. 116,1395 -1408.[Abstract]
Donahue, R. P. (1972). Fertilization of the mouse oocyte: sequence and timing of nuclear progression to the two-cell stage. J. Exp. Zool. 180,305 -318.[Medline]
Dyban, A. P. and Sorokin, A. V. (1983). Comparison of the size of paternal and maternal homologous chromosomes during the first 2 cleavage divisions in mouse embryos. Ontogenez 14,238 -246.[Medline]
Eide, T., Coghlan, V., Orstavik, S., Holsve, C., Solberg, R., Skalhegg, B. S., Lamb, N. J., Langeberg, L., Fernandez, A., Scott, J. D. et al. (1998). Molecular cloning, chromosomal localization, and cell cycle-dependent subcellular distribution of the A-kinase anchoring protein, AKAP95. Exp. Cell Res. 238,305 -316.[Medline]
Eide, T., Carlson, C. R., Tasken, K. A., Hirano, T., Tasken, K.
and Collas, P. (2002). Distinct but overlapping domains of
AKAP95 are implicated in chromosome condensation and condensin targeting.
EMBO Rep. 3,433
-437.
Giet, R. and Glover, D. M. (2001).
Drosophila aurora B kinase is required for histone H3 phosphorylation
and condensin recruitment during chromosome condensation and to organize the
central spindle during cytokinesis. J. Cell Biol.
152,669
-682.
Hirano, M. and Hirano, T. (1998). ATP-dependent
aggregation of single-stranded DNA by a bacterial SMC homodimer.
EMBO J. 17,7139
-7148.
Hirano, T. (2000). Chromosome cohesion, condensation, and separation. Annu. Rev. Biochem. 69,115 -144.[Medline]
Hirano, T., Kobayashi, R. and Hirano, M. (1997). Condensins, chromosome condensation protein complexes containing XCAP-C, XCAP-E and a Xenopus homolog of the Drosophila Barren protein. Cell 89,511 -521.[Medline]
Howlett, S. K. and Reik, W. (1991). Methylation levels of maternal and paternal genomes during preimplantation development. Development 113,119 -127.[Abstract]
Kaufman, M. H. (1973). Timing of the first cleavage division of haploid mouse eggs, and the duration of its component stages. J. Cell Sci. 13,553 -566.[Medline]
Kemphues, K. (2000). PARsing embryonic polarity. Cell 101,345 -348.[Medline]
Kimura, K. and Hirano, T. (1997). ATP-dependent positive supercoiling of DNA by 13S condensin: a biochemical implication for chromosome condensation. Cell 90,625 -634.[Medline]
Kimura, K., Hirano, M., Kobayashi, R. and Hirano, T.
(1998). Phosphorylation and activation of 13S condensin by Cdc2
in vitro. Science 282,487
-490.
Kimura, K., Rybenkov, V. V., Crisona, N. J., Hirano, T. and Cozzarelli, N. R. (1999). 13S condensin actively reconfigures DNA by introducing global positive writhe: implications for chromosome condensation. Cell 98,239 -248.[Medline]
Kimura, K., Cuvier, O. and Hirano, T. (2001).
Chromosome condensation by a human condensin complex in Xenopus egg
extracts. J. Biol. Chem.
276,5417
-5420.
King, M. L., Zhou, Y. and Bubunenko, M. (1999). Polarizing genetic information in the egg: RNA localization in the frog oocyte. Bioessays 21,546 -557.[Medline]
Lalande, M. (1996). Parental imprinting and human disease. Annu. Rev. Genet. 30,173 -195.
Lasko, P. (1999). RNA sorting in
Drosophila oocytes and embryos. FASEB J.
13,421
-433.
Martins, S. B., Eide, T., Steen, R. L., Jahnsen, T.,
Skålhegg, B. S. and Collas, P. (2000). HA95 is a
protein of the chromatin and nuclear matrix regulating nuclear envelope
dynamics. J. Cell Sci.
113,3703
-3713.
Mayer, W., Smith, A., Fundele, R. and Haaf, T.
(2000). Spatial separation of parental genomes in preimplantation
mouse embryos. J. Cell Biol.
148,629
-634.
McGrath, J. and Solter, D. (1984). Inability of mouse blastomere nuclei transferred to enucleated zygotes to support development in vitro. Science 226,1317 -1319.[Medline]
Schmiesing, J. A., Ball, A. R. J., Gregson, H. C., Alderton, J.
M., Zhou, S. and Yokomori, K. (1998). Identification of two
distinct human SMC protein complexes involved in mitotic chromosome dynamics.
Proc. Natl. Acad. Sci. USA
95,12906
-12911.
Schmiesing, J. A., Gregson, H. C., Zhou, S. and Yokomori, K.
(2000). A human condensin complex containing hCAP-C-hCAP-E and
CNAP1, a homolog of Xenopus XCAP-D2, colocalizes with phosphorylated
histone H3 during the early stage of mitotic chromosome condensation.
Mol. Cell. Biol. 20,6996
-7006.
Stanker, L. H., Wyrobek, A., McKeown, C. and Balhorn, R. (1993). Identification of the binding site of two monoclonal antibodies to human protamine. Mol. Immunol. 30,1633 -1638.[Medline]
Steen, R. L. and Collas, P. (2001).
Mistargeting of B-type lamins at the end of mitosis: implications on cell
survival and regulation of lamins A/C expression. J. Cell
Biol. 153,621
-626.
Steen, R. L., Cubizolles, F., le Guellec, K. and Collas, P.
(2000). A-kinase anchoring protein (AKAP)95 recruits human
chromosome-associated protein (hCAP)-D2/Eg7 for chromosome condensation in
mitotic extract. J. Cell Biol.
149,531
-536.
Surani, M. A., Barton, S. C. and Norris, M. L. (1986). Nuclear transplantation in the mouse: heritable differences between parental genomes after activation of the embryonic genome. Cell 45,127 -136.[Medline]
Suzuki, H., Maegawa, S., Nishibu, T., Sugiyama, T., Yasuda, K. and Inoue, K. (2000). Vegetal localization of the maternal RNA encoding and EDEN-BP/Bruno-like protein in zebrafish. Mech. Dev. 93,205 -209.[Medline]
Tilghman, S. M. (1999). The sins of the fathers and mothers: genomic imprinting in mammalian development. Cell 96,185 -193.[Medline]
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