Molecular Human Reproduction Vol.13, No.3 pp. 141–148*, 2007
Advance Access publication January 31, 2007
doi:10.1093/molehr/gal115
Histone acetylation and subcellular localization
of chromosomal protein BRD4 during mouse oocyte
meiosis and mitosis†
Takashi Nagashima1, Tetsuo Maruyama1,3, Masataka Furuya1, Takashi Kajitani1,
Hiroshi Uchida1, Hirotaka Masuda1, Masanori Ono1, Toru Arase1, Keiko Ozato2
and Yasunori Yoshimura1
1
3
To whom correspondence should be addressed at: Department of Obstetrics and Gynecology, Keio University School of Medicine, 35
Shinanomachi, Shinjuku, Tokyo 160-8582, Japan. E-mail: [email protected]
Most specific and general transcription factors (TFs) become dissociated from hypoacetylated mitotic chromosomes, which may
contribute to transcriptional silencing during mitosis. Only some chromosomal proteins, such as bromodomain containing protein
4 (BRD4), have a potential to associate with mitotic chromosomes in a histone acetylation-dependent manner. It remains to be fully
demonstrated whether similar displacement of nuclear factors takes place in meiotic oocytes whose chromosomes become globally
deacetylated. To address this, we here examined the subcellular localization of BRD4 in conjunction with the acetylation status of
histones in mouse oocytes. Immunofluorescence studies revealed that BRD4 preferentially localized to mitotic chromosomes in
early embryos. In contrast, not only endogenous BRD4 but also exogenous BRD4 overexpressed by mRNA microinjection
were displaced from meiotic chromosomes whose histones H3 and H4 were deacetylated. Treatment with trichostatin A (TSA),
an inhibitor of histone deacetylases, induced histone hyperacetylation of meiotic chromosomes from which endogenous BRD4,
however, remained dissociated. Finally, meiotic chromosomal localization of BRD4 could be achieved by BRD4 overexpression
together with TSA-induced histone hyperacetylation. These results indicate that, unlike mitosis, histone acetylation is necessary
but not sufficient for chromosomal localization of BRD4 during meiosis, suggesting that meiotic oocytes may have additional
mechanism(s) for displacement of chromosomal proteins and TFs.
Key words: BRD4/bromodomain/histone acetylation/meiosis/oocyte
Introduction
During the proliferation of somatic cells, the information on active
genes is transmitted precisely to daughter cells, thereby maintaining
the lineage-specific programme of gene expression in the subsequent
generations. In spite of the faithful transmission of genetic information, the whole genome becomes almost inert through the displacement of most specific and general transcription factors (TFs) from the
chromosomes during mitosis (Hershkovitz and Riggs, 1995; MartinezBalbas et al., 1995; Muchardt et al., 1996; Segil et al., 1996). To maintain the minimum information for lineage-specific gene programmes
over generations, there may exist mechanism(s) called ‘cell
memory’ by which certain epigenetic modifications remain as
markers of cell identity in the chromosomes during mitosis (Jeppesen,
1997; Turner, 2000, 2002). Histone modifications including
methylation, phosphorylation and acetylation have been proposed as
*N.B. An error was made in the initial online pagination of Molecular Human
Reproduction 13/3. The page span of this article was originally shown as 1– 8.
The publisher wishes to apologise for this error.
†
Presented in part at the 22nd Annual Meeting of the European Society of
Human Reproduction and Embryology.
candidate epigenetic markers of cell memory (Strahl and Allis,
2000; Turner, 2000, 2002).
It has been suggested that distinct histone acetylation patterns function as recognition codes for the recruitment of different TFs upon
transcriptional activation (Agalioti et al., 2002; Turner, 2002).
During mitosis, the activities of histone acetyltransferases and deacetylases are down-regulated (Kruhlak et al., 2001), and general hypoacetylation of core histones takes place together with the displacement
of TFs from mitotic chromosomes, which correlates with the steep fall
in transcriptional activities (Kruhlak et al., 2001). However, some
lysine residues on histones H3 and H4 remain acetylated on mitotic
chromosomes, which may serve as markers for transcriptional
memory (Kruhlak et al., 2001; Valls et al., 2005). Transcription
restarts at the end of telophase when chromatin-binding proteins and
TFs are reloaded onto chromatin (Prasanth et al., 2003). Thus,
histone acetylation has been proposed as an epigenetic marker for
the propagation of genomic information from one cell generation to
the next (Jeppesen, 1997; Turner, 2000; Jenuwein and Allis, 2001).
In contrast to histone hypoacetylation during mitosis in somatic
cells, meiotic chromosomes become globally deacetylated in
oocytes (Kim et al., 2003). Meiosis-specific histone deacetylation is
# The Author 2007. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology.
All rights reserved. For Permissions, please email: [email protected]
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Department of Obstetrics and Gynecology, Keio University School of Medicine, Tokyo, Japan and 2Laboratory of Molecular Growth
Regulation, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, USA
T.Nagashima et al.
in TYH medium covered with mineral oil for more than 1 h at 378C under 5%
CO2 in humidified air for spermatozoal capacitation. MII stage oocytes were
obtained from ovulation-induced 4-week-old female BDF1 mice with PMSG,
as described above, followed 48 h later by human chorionic gonadotropin
(hCG, Mochida, Tokyo, Japan). After 12 h of hCG injection,
orulation-induced mice were killed at different times and the oviductal ampullae were broken to release the COCs at the MII stage of in vivo ageing. MII
stage oocytes were inseminated with capacitated spermatozoa
(1.0 106 cells ml21). After removing cumulus cells with 0.1% hyaluronidase, the emission of the second polar body and the formation of the pronuclei were observed with a stereoscopic microscope after 5 h of IVF and
culture in TYH medium. Embryos at the interphase of the 1-cell stage and
the interphase and metaphase of the 2-cell stage were obtained 5, 30 and
36 h after insemination and cultured in modified Whitten’s medium (Mitsubishi Kagaku Iatron) (Beckmann and Day, 1993) in the presence or
absence of 50 ng ml21 TSA, respectively. All animal studies were approved
by our Institutional Review Board(s).
Immunofluorescence
Oocytes, embryos and NIH3T3 cells were fixed with 4% paraformaldehyde/
phosphate-buffered saline (PBS) for 30 min at 378C and then permeabilized
with 0.2% Triton X-100/PBS for 15 min at room temperature (RT). After
blocking for non-specific staining with 5% (bovin serum albumin) BSA/PBS
for 1 h at RT, the cells were incubated with 1:400 polyclonal rabbit
anti-BRD4 antibody (Dey et al., 2000), 1:200 polyclonal rabbit anti-activating
protein 2 (AP-2) antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA),
or 1:200 polyclonal rabbit antibodies against acetylated lysine 9 and 14 on
histone H3 (AcH3/K9K14) or acetylated lysine 5 on histone H4 (AcH4/K5)
(Upstate Biotechnology, Lake Placid, NY, USA) for 1 h at RT or overnight
at 48C. The antibodies that bound to the cells were probed with Cy2 or Cy3
anti-rabbit IgG (Jackson Immuno Research Laboratosies, Bar Harbor, ME,
USA) for further 1 h at RT. Counterstaining was conducted with 2 mg ml21
Hoechst 33342 (Sigma– Aldrich) to visualize DNA by incubation for 20 min
at RT. Finally, the cells were mounted on glass slides and viewed with a
Leica DM IRE2 inverted microscope (Leica) using a 40 and 63 oil immersion objective. The captured images were processed using Adobe Photoshop
CS2TM software (Adobe Systems, San Jose, CA, USA). The experiment was
conducted three times with at least five oocytes and embryos per group.
Materials and methods
Preparation of mouse oocytes and embryos
Plasmid construction
Germinal vesicle (GV) stage oocytes were obtained from 4-week old female
BDF1 mice (Sankyo, Tokyo, Japan) as the cumulus–oocyte complex (COC).
Female mice were induced to ovulate with intraperitoneal injection of 5 IU
pregnant mare’s serum gonadotropin (PMSG, Teikokuzoki, Tokyo, Japan)
and were killed by cervical dislocation 48 h later. After removing the
ovaries and trimming fat and mesentery, they were recovered in M2
medium (Sigma– Aldrich, St Louis, MO, USA) (Fulton and Whittingham,
1978) for cell manipulation under a Leica M8 stereoscopic microscope
(Leica Microsystems, Heidelberger, Germany). After mechanical removal of
COCs by a 29-gauge needle, GV stage oocytes were pooled together after
dispersion and washed for removal of cumulus cells mechanically by
pipetting with a thin pipette in M2 medium containing 0.1% hyaluronidase
(Sigma–Aldrich). Then, denuded spherical oocytes of uniform size containing
intact GV stage oocytes were allocated to treatment groups and cultured in
Toyoda Yokoyama Hoshi (TYH) medium (Mitsubishi Kagaku Iatron,
Tokyo, Japan) (Toyoda et al., 1971) covered with mineral oil
(Sigma–Aldrich) at 378C under 5% CO2 in humidified air for in vitro maturation. To prevent germinal vesicle breakdown (GVBD), GV stage oocytes
were treated with 100 mg ml21 dibutyryl cyclic AMP (dbcAMP,
Sigma–Aldrich), a membrane permeable cyclic AMP analogue (Wassarman
et al., 1976), in combination with or without 50 ng ml21 trichostatin A
(TSA, Sigma–Aldrich), a potent histone deacetylase inhibitor (Yoshida
et al., 1995). GVBD, metaphase I (MI) and metaphase II (MII) stage
oocytes were collected 4, 9 and 17 h, respectively, after culture of GV
stage oocytes in the absence of 100 mg ml21 dbcAMP in combination with
or without 50 ng ml21 TSA. The fertilized embryos were prepared by in
vitro fertilization (IVF). Spermatozoa were dissected from the cauda epididymides of 8-week-old male BDF1 mice (Sankyo), minced slightly and cultured
EGFP-fused Brd4 fragments were excised from pC1-EGFP-Brd4 (Maruyama
et al., 2002) and cloned into pTRE (Clontech Laboratories, Palo Alto, CA,
USA) to generate pTRE-EGFP-Brd4. For mRNA microinjection experiments,
pcDNA3-Brd4-EGFP and pcDNA3-EGFP were constructed as follows. The
pN3-Brd4-EGFP (Maruyama et al., 2002) was digested with NotI and XhoI.
pcDNA3 (Invitrogen, Carlsbad, CA, USA) was digested with ApaI. Digested
fragments containing Brd4-EGFP and pcDNA3 were both treated with T4
DNA polymerase (Takara, Tokyo, Japan) to make blunt-ends and ligated for
the construction of pcDNA3-Brd4-EGFP. The pN3-EGFP (Clontech) and
pcDNA3 were both digested with NotI and XhoI and ligated for the construction of pcDNA3-EGFP.
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Generation of stable transfectants capable of inducing EGFP-BRD4
under DOX control
Doxycycline (DOX, Sigma– Aldrich)-inducible BRD4 expression was
performed as described elsewhere (Maruyama et al., 2002). Along with the
procedure, NIH3T3 stable transfectants (doxEGFP-BRD4) were generated
following transfection of pTk-hygro and pTRE-EGFP-Brd4 into cells that
had been transfected with pTet-On vector (Clontech).
Immunoblot
doxEGFP-BRD4 cells were first inoculated on 60 mm dishes at 4.0 104 cells dish21 and cultured in Dulbecco’s modified Eagle medium
(DMEM; Sigma–Aldrich) supplemented with 10% fetal calf serum (FCS)
and 1% antibiotic– antimycotic mixture (Gibco, Grand Island, NY, USA) at
378C under 5% CO2 in humidified air. Once confluent, the cells were washed
twice in PBS and maintained in DMEM with or without DOX (2 mg ml21)
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thought to contribute to transcriptional silencing and erasing of cell
memory, which may be required for the reprogramming of the
oocyte genome to allow the remarkable transformation from differentiated oocytes into the totipotent embryos of the next generation
(Schultz et al., 1999; Kim et al., 2003). However, it remains to be
demonstrated whether non-histone chromosomal proteins, as well as
TFs, are displaced from meiotic chromosomes in mouse oocytes.
Bromodomains are conserved protein modules that bind to acetylated histones and are present in a number of chromatin-binding
proteins, including histone acetyltransferases, general TFs and chromatin remodelling factors (Zeng and Zhou, 2002). BRD4 is a mammalian bromodomain protein that belongs to the conserved bromodomain
and extraterminal domain (BET) family (Dey et al., 2000) whose
members carry two tandem bromodomains and an additional
extraterminal domain (Florence and Faller, 2001). Previous analysis
with fluorescence loss in photobleaching showed that BRD4
dynamically interacts with acetylated histones H3 and H4 in living
interphase cells (Dey et al., 2003). A unique and notable feature of
BRD4 is that it behaves as a non-histone chromosomal protein,
being tightly associated with chromosomes during mitosis when
many other nuclear factors are released into the cytoplasm (Dey
et al., 2000). Similar to interphase cells, localization of BRD4 to
mitotic chromosomes is attributed at least in part to its binding to
acetylated histones.
In this study, taking advantage of the unique property of BRD4, we
here examined the acetylation status of histones in conjunction with
the subcellular localization of BRD4 to address whether meiotic
oocytes displace chromosomal proteins as well as TFs from their
chromosomes through regulation of histone acetylation and deacetylation. We here provide evidence suggesting that not only histone deacetylation but also additional mechanism(s) are required for the
displacement of chromosomal proteins, including BRD4, during
mouse oocyte meiosis.
Histone acetylation and BRD4 during meiosis and mitosis
Time-lapse video microscopy of living cells
Prior to microscopic analysis, doxEGFP-BRD4 cells were allowed to attach
and spread on glass bottom 60-mm dishes at 4 104 cells per dish and
grown for 12 h in DMEM supplemented with 10% FCS and 1% antibiotic–
antimycotic mixture at 378C under 5% CO2 in humidified air. Furthermore,
doxEGFP-BRD4 cells were cultured for the synchronization in the presence
of 0.1% FCS for 24 h. On the following morning, fresh culture medium
without phenol red supplemented with 10% FCS was added with DOX to
induce EGFP-BRD4, and the cells were cultured for 12 h at 378C in a CO2
tissue culture incubator. After 12 h of treatment, doxEGFP-BRD4 cells
during mitosis were placed under a time-lapse video microscope in a 378C
under 5% CO2 humidified air box.
Images were captured using a Leica TCS-SP2 confocal laser scanning unit
(Leica) attached to a DM IRE2 inverted microscope using a 63 oil immersion
objective. Filters and lightpaths were controlled with a filter wheel and shutters.
To minimize bleaching of fluorescence, EGFP-BRD4 fluorescence images
were taken at 2 min intervals by recording for 1– 4 s using a photometric
digital camera controlled by Leica confocal software, version 2.5. Time-lapse
series were subsequently converted to 8-bit images and assembled using Adobe
Photoshop CS2TM software.
Preparation and verification of mRNA for microinjection
For the linearization of plasmids, pcDNA3-EGFP was digested with EcoRV,
and pcDNA3-Brd4-EGFP was digested with NotI. These digested plasmids
were subjected to proteinase K treatment to eliminate RNase by mixing 5 mg
purified digested plasmids, 20 ml proteinase K, 2.5 ml of 10% SDS and incubating the mixture at 508C for 30 min. Then, an in vitro transcription reaction was
carried out using a mMESSAGE mMACHINE T7 Kit (Ambion, Austin, TX,
USA) according to the manufacturer’s protocols. The constructed mRNA for
EGFP and BRD4-EGFP were added with a poly(A) tail of at least 150 nucleotides in length to the 30 termini of mRNA, using the Poly (A) Tailing Kit
(Ambion) according to the manufacturer’s protocols. The addition of the
poly(A) tail was confirmed by agarose-formaldehyde gel electrophoresis.
The translation of EGFP and Brd4-EGFP mRNA were confirmed by in vitro
translation assays using TNT Quick Coupled Transcription/Translation
Systems (Promega, Madison, WI, USA) according to the manufacturer’s protocols. As in vitro translation reaction, mixtures of purified 1 mg EGFP or
Brd4-EGFP mRNA, 20 ml of reticulocyte lysate, 10 mg of RNase A and
50 mCi of L-[35S] methionine (GE Healthcare Bio-Sciences, Piscataway, NJ,
USA) were incubated at 308C for 90 min. One microlitre of the incubated
mixture was separated by 18% and 6% SDS–PAGE gels for the detection of
EGFP and BRD4-EGFP, respectively, and the bands were visualized by
autoradiography.
Microinjection of mRNA
The linearized EGFP or Brd4-EGFP mRNA in PBS were microinjected directly
into the cytoplasm of fully grown mouse GV stage oocytes at a concentration of
1.5 mg ml21 using NWO-202 micromanipulators and IM-300 microinjectors
(Narishige, Tokyo, Japan). In each experiment, oocytes were injected within
45 min after oocyte collection. The microinjected GV stage oocytes were
treated and collected in a similar way as described above. The experiment
was conducted three times, with at least five embryos per group.
Results
Histone acetylation and subcellular localization of BRD4
during mitosis in fibroblast NIH3T3 cells
We first examined the acetylation status of histones H3 and H4 in conjunction with the cellular localization of BRD4 in mitotic NIH3T3
cells. Indirect immunofluorescence using specific antibodies against
AcH3/K9K14, AcH4/K5 and BRD4 revealed that BRD4 formed
a faint, diffuse coat over mitotic chromosomes (Figure 1C) whose
H3/K9K14 was acetylated (Figure 1A) but H4/K5 was deacetylated
Figure 1. Effects of trichostatin A (TSA) on the acetylation status of histones H3 and H4 and the subcellar localization of bromodomain containing protein a
(BRD4) in mitotic NIH3T3 cells. NIH3T3 cells were treated without (A– C) or with TSA (D –F) for 12 h and immunostained with antibodies against AcH3/
K9K14 (A and D), AcH4/K5 (B and E) or BRD4 (C and F) that were visualized by Cy2- (A, B, D and E) or Cy3- (C and F) labelled secondary antibodies.
DNA was stained with Hoechst 33342. Bars (A– F), 5 mm.
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to induce EGFP-BRD4. After 8 h of treatment, doxEGFP-BRD4 cells
were washed twice in cold PBS and lysed with 200 ml RIPA buffer
[20 mmol l21 Tris–HCl (pH 7.5), 150 mmol l21 NaCl, 1 mmol l21 EDTA,
1% Na-deoxycholate, 0.1% sodium dodecyl sulphate (SDS), 1 mmol l21
Na3VO4, 50 mmol l21 NaF and 1 mmol l21 Na2MoO4] containing a
protease inhibitor cocktail (Roche Molecular Biochemicals, Mannheim,
Germany). After three cycles of stirring for 15 s and chilling for 5 min at
48C, the cell lysates were centrifuged at 17 000 g for 10 min at 48C and
stored immediately at 2808C until electrophoresis. The protein concentration
was measured using a DC protein assay kit (BioRad, Hercules, CA, USA). In
a typical experiment, 8 mg of cell lysates were mixed with RIPA buffer plus
2 SDS sample buffer and heated at 958C for 5 min. The heated samples
were separated on 6% SDS– polyacrylamide gel electrophoresis (PAGE) for
2 h at 125 V and subsequently transferred onto a polyvinylidene digluoride
(PVDF) membrane (Immobilon P; Millipore, Bedford, MA, USA) for 2 h at
52 V using transfer buffer made by mixing 50 mM Tris aminomethane,
40 mM glycine, 0.04% SDS and 20% methanol. The membrane was incubated
in TBS-T [20 mM Tris–HCl, 100 mM NaCl (pH 7.6) and 0.1% Tween-20]
containing 5% BSA (Sigma– Aldrich) for 1 h at RT to block non-specific
binding sites. After three 10-min washes in TBS-T, the membranes were incubated overnight with a primary antibody against EGFP (1:1000 dilution; Clontech) in TBS-T containing 1% BSA. After overnight incubation, membranes
underwent three 10-min washes in TBS-T and were incubated for 1 h with a
matching horse-radish peroxidase (HRP)-conjugated secondary antibody
(1:10 000 dilution; Jackson Immuno Research Laboratories) in TBS-T containing 1% BSA. After three 10-min washes in TBS-T, bound antibodies were
detected using an enhanced chemiluminescence (ECL) plus detection kit
(Amersham Biosciences Co., Piscataway, NJ, USA) according to the manufacturer’s instructions and exposed to X-ray films (Eastman Kodak, Rochester,
NY, USA).
T.Nagashima et al.
Figure 2. Acetylation status of histones H3 and H4 and subcellular localization of bromodomain containing protein 4 (BRD4) in mouse oocytes and
early embryos. Oocytes at germinal vesicle (GV) (A, H, O), GV breakdown
(B, I, P), metaphase I (C, J, Q) and metaphase II stages (D, K, R) and fertilized
oocytes at 1-cell (E, L, S), 2-cell (F, M, T) and 2-cell metaphase (G, N, U)
stages were collected, fixed and immunostained with antibodies against
AcH3/K9K14 (A– G), AcH4/K5 (H-N) or BRD4 (O–U), all of which were
visualized by Cy2- (A– N) or Cy3- (O – U) labelled secondary antibodies.
DNA was stained with Hoechst 33342. For a clear view, the metaphase
chromosome is enlarged on the right lower corner of each corresponding
panel. Bar, 70 mm.
Figure 3. Effects of trichostatin A (TSA) on the acetylation status of histones
H3 and H4 and the subcellular localization of BRD4 in mouse oocytes and early
embryos. Oocytes at germinal vesicle (GV) (A, H, O), GV breakdown (B, I, P),
metaphase I (MI) (C, J, Q) and MII stages (D, K, R) and fertilized oocytes at
1-cell (E, L, S), 2-cell (F, M, T) and 2-cell metaphase (G, N, U) stages were
treated with TSA, collected, fixed and immunostained with antibodies
against AcH3/K9K14 (A– G), AcH4/K5 (H–N), or BRD4 (O–U), all of
which were visualized by Cy2- (A–N) or Cy3- (O–U) labelled secondary antibodies. DNA was stained with Hoechst 33342. For a clear view, the metaphase
chromosome is enlarged on the right or left lower corner of each corresponding
panel. Bar, 70 mm.
Histone acetylation and subcellular localization of BRD4
in mouse oocytes and early embryos
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We then examined the acetylated status of histones H3 and H4 together
with the intracellular distribution of BRD4 in mouse oocytes and early
embryos. Indirect immunofluorescence using specific antibodies against
AcH3/K9K14, AcH4/K5 and BRD4 revealed that both H3/K9K14 and
H4/K5 became acetylated and BRD4 was preferentially localized in the
nucleus of GV stage oocytes (Figure 2A, H and O). Also, BRD4 was
present in the nucleus of 1-cell and 2-cell stage embryos (Figure 2S
and T), and in the condensed chromosomes of 2-cell metaphase stage
embryos (Figure 2U), all whose H3/K9K14 and H4/K5 were acetylated
(Figure 2E–G and L–N). In contrast, H3/K9K14 and H4/K5 became
deacetylated, and BRD4 was dissociated from condensed chromosomes
and dispersed into the cytoplasm in GVBD (Figure 2B, I and P), MI
(Figure 2C, J and Q) and MII stage oocytes (Figure 2D, K and R).
The histone acetylation and deacetylation patterns presented here are
consistent with the previous results, except for H4/K5, which has
been reported to be deacetylated during mitosis in early embryos
(Kim et al., 2003). Wee et al. (2006) have recently demonstrated that
H4/K5 become acetylated during mitosis in bovine IVF embryos
being consistent with our present data.
As BRD4 binds to acetylated histones H3 and H4, it is reasonable to
speculate that the dissociation of BRD4 from meiotic chromosomes
may be attributable to the deacetylation of histones H3 and H4
during meiosis. To test this, we examined whether TSA-induced
histone hyperacetylation can localize BRD4 to meiotic chromosomes.
As shown in Figure 3A, E– H and L–N, treatment with TSA provoked
comparable or increasing levels of histone H3/K9K14 and H4/K5
acetylation in comparison with non-treated oocytes and early
embryos (Figure 2A, E– H and L –N). Consistent with this finding,
immunofluorescence signals for BRD4 were comparable or more prominent in TSA-treated GV oocytes, 1-cell, 2-cell and 2-cell metaphase
embryos (Figure 3O and S –U), as compared with non-treated oocytes
and embryos (Figure 2O and S –U). Notably, meiotic chromosomes in
oocytes treated with TSA were strongly immunoreactive with specific
antibodies against AcH3/K9K14 (Figure 3B –D) and AcH4/K5
(Figure 3I–K), indicating that treatment with TSA successfully
induced histone hyperacetylation in meiotic oocytes. However, in
spite of the histone hyperacetylation (Figure 3B –D and I –K),
BRD4 was dissociated from condensed chromosomes and dispersed
into the cytoplasm during meiosis (Figure 3P –R), indicating that
(Figure 1B). These patterns of histone acetylation were consistent with
a previous report (Kruhlak et al., 2001). BRD4 has been originally
reported to tightly associate with mitotic chromosomes in P19 embryonal carcinoma cells (Dey et al., 2000); however, subsequent studies
demonstrated that the chromosomal localization of BRD4 depends
on the acetylation status of histones, and therefore its localization patterns are variable among cell lines (Dey et al., 2003; McPhillips et al.,
2005). Treatment with TSA increased the levels of AcH3/K9K14
(Figure 1D) and AcH4/K5 (Figure 1E) concomitant with an increase
in the amount of chromosomally bound BRD4 (Figure 1F), which
was in agreement with the previous report (Dey et al., 2003). Peptidebinding assays show that BRD4 preferentially binds to acetylated H3/
K9K14 and H4/K5 peptides (Dey et al., 2003), which consistently
accounts for the spatio-temporal behaviours of BRD4 in NIH3T3
cells as presented here. Thus, the chromosomal localization of
BRD4 was highly dependent on the level of histones H3 and H4 acetylation in mitosis.
Histone acetylation and BRD4 during meiosis and mitosis
histone acetylation is not the sole determinant of chromosomal localization of BRD4 in meiotic oocytes.
Subcellular localization of AP-2 in mitotic NIH3T3 cells
and meiotic oocytes
Subcellular localization of EGFP-BRD4 during mitosis
in living NIH3T3 cells
Displacement of BRD4 from meiotic chromosomes raises another
possibility that there may be unknown BRD4-binding factor(s) that
inhibit the association of BRD4 with meiotic chromosomes, presumably by competing out BRD4. Indeed, BRD4 is known to interact
with several proteins including signal-induced proliferation-associated
protein 1 (SPA-1), replication factor C (RFC-140, the largest subunit),
positive transcription elongation factor in transcription b (P-TEFb)
and bovine papillomavirus E2 protein (Maruyama et al., 2002;
Farina et al., 2004; Jang et al., 2005; Yang et al., 2005). In support
of this hypothesis, besides histone hyperacetylation by TSA, overexpression of BRD4 also leads to the efficient and tight association of
BRD4 to chromatin (Dey et al., 2003). To address the spatio-temporal
behaviours of overexpressed BRD4, we generated NIH3T3 stable
clones in which the expression of EGFP-BRD4 was induced by
DOX (Figure 5A). Time-lapse video imaging of the stable clone
Figure 4. Subcellular localization of activating protein 2 (AP-2) in mitotic
NIH3T3 cells and mouse meiotic oocytes. Mitotic NIH3T3 cells (A) and
meiotic oocytes (B) were immunostained with anti-AP-2 antibody that was
visualized by Cy3-lablled secondary antibody. DNA was stained with
Hoechst 33342. For a clear view, the metaphase chromosome is enlarged on
the left upper corner of each panel. Bars, 5 mm (A) and 70 mm (B).
Figure 5. Time-lapse video imaging of EGFP-bromodomain containing protein
4 (BRD4) in doxycycline (DOX)-inducible NIH3T3 stable clones during mitosis.
NIH3T3 stable clones (doxEGFP-BRD4) capable of inducing EGFP-BRD4
under the control of DOX were established and treated with DOX for 8 h. (A)
Western blot analysis of endogenous BRD4 and EGFP-BRD4 derived from nontreated or DOX-treated doxEGFP-BRD4 using antibodies against BRD4 and
EGFP. (B) Mitotic doxEGFP-BRD4 treated with DOX were placed under the
time-lapse video microscope in a 378C under 5% CO2 humidified air box.
Fluorescence and phase contrast pictures were taken every 2 min for 16 min.
Bar, 5 mm.
treated with DOX revealed that EGFP-BRD4 was entirely present
on fully condensed mitotic chromosomes (Figure 6B), which is in
agreement with the previous report that exogenous BRD4 is also
exclusively associated with mitotic chromosomes (Dey et al., 2000,
2003).
Subcellular localization of BRD4-EGFP during meiosis
in mouse oocytes
We hypothesized that overexpression of BRD4 would overcome the
possible sequestration of BRD4 and thereby facilitate its localization
to meiotic chromosomes, as observed in mitotic chromosomes
(Figure 5). To test this, we first prepared mRNA for EGFP or
BRD4-EGFP followed by the addition of a long poly(A) tail
(Figure 6A) and overexpressed them by microinjection into the cytoplasm of mouse GV stage oocytes. Figure 6B shows that these
poly(A) þ mRNA were successfully translated in vitro. As shown in
Figure 6C, EGFP alone was distributed diffusely in the nucleus and
cytosol in GV stage oocytes. In contrast, BRD4-EGFP was exclusively
located in the nucleus (Figure 6Da). However, BRD4-EGFP was dissociated from condensed chromosomes during meiosis, as determined
by confocal microscopy (Figure 6Db –d), which was similar
to EGFP alone (Figure 6C, lower two panels). Thus, either histone
hyperacetylation or overexpression of BRD4 alone is insufficient to
localize BRD4 to meiotic chromosomes.
Finally, to overcome the resistance of BRD4 to meiotic chromosomal localization, the GV stage oocytes overexpressing BRD4-EGFP
by mRNA microinjection were co-incubated with TSA and collected
at the GVBD, MI and MII stages. Confocal microscopy of those TSAtreated living oocytes revealed that BRD4-EGFP was predominantly
located in the nucleus at the GV stage (Figure 6De) and that it
became tightly associated with condensed chromosomes during
meiosis (Figure 6Df–h). We also performed immunofluorescence
using an anti-BRD4 antibody followed by incubation with a Cy3
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Besides some chromosomal proteins including BRD4, at least two
TFs, p67SRF (Gauthier-Rouviere et al., 1991) and AP-2 (MartinezBalbas et al., 1995) were found to remain associated with the condensed metaphase chromosomes. The AP-2 TF family is required
for multiple aspects of mouse embryo development (Eckert et al.,
2005), most of whose isoforms are expressed in dynamic patterns
before and after fertilization of the oocyte (Winger et al., 2006).
We, therefore, examined the subcellular localization of AP-2 in
mitotic NIH3T3 cells and meiotic oocytes. As shown in Figure 4A,
AP-2 remained preferentially associated with mitotic chromosomes,
being consistent with the previous report (Martinez-Balbas et al.,
1995). In contrast, AP-2 was dispersed in the cytoplasm at MI and
MII stages (Figure 4B). Thus, meiotic oocytes are able to dissociate
not only BRD4 but also AP-2, a mitotic chromsome-associated TF,
from meiotic condensed chromosomes.
T.Nagashima et al.
secondary antibody. As shown in Figure 6E, EGFP fluorescence
signals (green) were consistent with immunofluorescence signals for
BRD4 (red), indicating that EGFP fluorescence was derived from
the exogenously introduced BRD4-EGFP. Notably, meiotic condensed chromosomes preferentially displayed immunofluorescence
signals for BRD4 in TSA-treated, but not untreated, oocytes overexpressing BRD4-EGFP (Figure 6E). Thus, BRD4 overexpression in
combination with TSA treatment was required for meiotic chromosomal localization of BRD4.
Discussion
The biological significance of meiosis-specific histone deacetylation
remains elusive. Epigenetic regulation of chromatin configuration
and gene expression by histone acetylation (Jeppesen, 1997; Turner,
146
2000; Jenuwein and Allis, 2001) supports the idea that meiosisspecific histone deacetylation may contribute to transcriptional
silencing and the erasing of cell memory, which may be required to
ensure genome remodelling and reprogramming for the transformation from differentiated oocytes into totipotent zygotes (Li, 2002;
Kim et al., 2003; Sarmento et al., 2004). On the contrary, Rybouchkin
et al. (2006) have demonstrated that meiotic deacetylation of somatic
histones is not important for further development of nuclear transfer
embryos, casting doubts on the assumptions that meiotic deacetylation
has reprogramming significance. This may not, however, eliminate the
possible biological significance of histone deacetylation in the
reprogramming during mouse oocyte meiosis, because the natural
reprogramming events are not equivalent to those that take place in
nuclear transfer embryos. Recently, De La Fuente et al. (2004) have
reported that exposure to TSA during meiotic maturation induces
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Figure 6. Effects of overexpression of bromodomain containing protein 4 (BRD4)-EGFP and histone hyperacetylation on the subcellular localization of BRD4 in
mouse oocytes. (A) In vitro transcription was conducted using plasmids encoding EGFP or BRD4-EGFP, followed by addition of a long poly (A) tail. The EGFP or
BRD4-EGFP transcripts with or without a long poly (A) tail were subjected to 8% agarose formaldehyde gel electrophoresis and the bands were visualized by ethidium bromide staining. (B) EGFP and BRD4-EGFP mRNA with a long poly (A) tail were in vitro translated, and the products were separated by 18% and 6%
SDS-PAGE gels for the detection of EGFP and BRD4-EGFP proteins, respectively. The bands were visualized by autoradiography. Messenger RNA of EGFP
(C) or BRD4-EGFP (D) with a long poly (A) tail was microinjected into the cytoplasm of fully-grown GV-stage oocytes. The microinjected GV-stage oocytes
were then cultured for 4 h (GVBD stage), 9 h (MI stage) and 17 h (MII stage) in the absence or presence of trichostatin A as indicated. Confocal microscopic
images of the living oocytes were captured. (E) These microinjected cells were also fixed and immunostained with anti-BRD4 antibody, followed by incubation
with Cy3-labeled secondary antibodies. Cy3 and EGFP fluorescence signals were detected by fluorescence microscopy. DNA was stained with Hoechst 33342.
For a clear view, the metaphase chromosome is enlarged on the right or left lower corner of each corresponding panel. Bars, 70 mm.
Histone acetylation and BRD4 during meiosis and mitosis
with the acetylated histones, entrap BRD4 and thereby inhibit the
chromosomal interaction of BRD4. In this context, we postulate that
overexpression of BRD4 enough to compete out the BRD4-binding
factor(s) together with sufficient levels of histone hyperacetylation
may allow BRD4 to localize to the meiotic chromosomes. Alternatively, it is conceivable that a possible post-translational modification
of BRD4 such as (de)phosphorylation may contribute to the transient
displacement during meiosis.
In summary, we demonstrate that histones H3 and H4 become deacetylated concomitant with the displacement of mitotic chromosomal
protein BRD4 from chromosomes during mouse oocyte meiosis.
Although BRD4 binds to mitotic chromosomes in dose-dependent
and histone acetylation-dependent manner in somatic cells, neither
overexpression nor histone hyperacetylation could localize BRD4 to
the meiotic chromosomes. Both are required for the meiotic chromosomal localization of BRD4. These results indicate that, unlike
mitosis, histone acetylation is necessary but not sufficient for chromosomal localization of BRD4 during meiosis, suggesting that meiotic
oocytes may have additional mechanism(s) for displacement of
mitotic chromosomal proteins from condensed chromosomes.
Acknowledgements
We thank Mamoru Tanaka and Maruyama’s lab members for helpful advice,
Rei Sakurai for technical assistance, Shino Kuwabara for her secretarial
work and Fugaku Aoki for helpful comments on immunofluorescence
studies. This study was supported, in part, by Grant-in-Aids from the Japan
Society for the Promotion of Science (JSPS) (to T.M. and to Y.Y.) and by
grants from the Keio Health Counseling Center (to T.M.).
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