Histone exchange activity and its correlation with histone
acetylation status in porcine oocytes
Tsutomu Endo, Aoi Imai, Takuma Shimaoka, Kiyoshi Kano and Kunihiko Naito
Laboratory of Applied Genetics, Graduate School of Agriculture and Life Science, University of Tokyo,
Bunkyo-ku, Tokyo 113-8657, Japan
Correspondence should be addressed to K Naito; Email: email@example.com
T Endo is now at Department of Biology, Howard Hughes Medical Institute, Whitehead Institute, Massachusetts Institute of
Technology, 9 Cambridge Center, Cambridge, Massachusetts 02142, USA
In mammalian oocytes, histone H3 and histone H4 (H4) in the chromatin are highly acetylated at the germinal vesicle (GV) stage,
and become globally deacetylated after GV breakdown (GVBD). Although nuclear core histones can be exchanged by cytoplasmic free
histones in somatic cells, it remains unknown whether this is also the case in mammalian oocytes. In this study, we examined the histone
exchange activity in maturing porcine oocytes before and after GVBD, and investigated the correlations between this activity and both
the acetylation profile of the H4 N-terminal tail and the global histone acetylation level in the chromatin. We injected Flag-tagged
H4 (H4-Flag) mRNA into GVoocytes, and found that the Flag signal was localized to the chromatin. We next injected mRNAs of mutated
H4-Flag, which lack all acetylation sites and the whole N-terminal tail, and found that the H4 N-terminal tail and its modification were
not necessary for histone incorporation into chromatin. Despite the lack of acetylation sites, the mutated H4-Flag mRNA injection
did not decrease the acetylation level on the chromatin, indicating that the histone exchange occurs partially in the GV chromatin.
In contrast to GVoocytes, the Flag signalwas not detected on the chromatin after the injection of H4-Flag protein into the second meiotic
metaphase oocytes. These results suggest that histone exchange activity changes during meiotic maturation in porcine oocytes, and that
the acetylation profile of the H4 N-terminal tail has no effect on histone incorporation into chromatin and does not affect the global level
of histone acetylation in it.
Reproduction (2011) 141 397–405
Chromatin is a complex in which DNA is wrapped
around an octamer of four core histones, called H2A,
H2B, H3, and H4. The N-terminal tails of these nuclear
core histones, especially H3 and H4, undergo various
modifications including acetylation, methylation, and
phosphorylation (Wu & Grunstein 2000). These post-
translational modifications play crucial roles in the
regulation of chromatin structure and transcription
(Strahl & Allis 2000, Turner 2002). H3 and H4 can be
acetylated at each of the four lysine residues (K) on their
N-terminal tails, and the acetylation status is determined
by histone acetyltransferase (HAT) and histone deacetyl-
ase (HDAC) activities (Strahl & Allis 2000). In mamma-
lian somatic cells, acetylation levels of most lysine
residues on H3 and H4 are maintained throughout
mitosis (Kruhlak et al. 2001), suggesting that histone
acetylation works as a cellular memory mark that
transmits gene expression patterns from parent cells to
the daughter cells. On the other hand, immunocyto-
chemical analysis has revealed in mammalian oocytes
that H3 and H4 in the entire chromatin are highly
acetylated during the germinal vesicle (GV) stage, and
become globally deacetylated after GV breakdown
(GVBD; Kim et al. 2003, Endo et al. 2005). This
chromatin-wide deacetylation during meiotic matu-
ration is thought to be involved in the construction of
meiotic chromosomes (De La Fuente et al. 2004,
Akiyama et al. 2006) and the erasure of cell memory
for genome reprogramming (Nagashima et al. 2007). The
treatment of mouse and porcine oocytes with an HDAC
inhibitor, trichostatin A, prevented the chromatin-wide
the involvement of HDAC activity in this process (Kim
et al. 2003, Endo et al. 2005).
It is also known that chromatin structure and
transcriptional states can be affected by exchanging
nuclear core histones with free histones present in the
cytoplasm. In mammalian somatic cells, H3 has three
types of variants – H3.1, H3.2, and H3.3 (Loyola &
Almouzni 2007) – and histone chaperones incorporate
these H3 variants and H4 into the chromatin as an
H3–H4 heterodimer (De Koning et al. 2007). H3.1 and
q 2011 Society for Reproduction and Fertility
ISSN 1470–1626 (paper) 1741–7899 (online)
Online version via www.reproduction-online.org
H3.2 are generally incorporated into chromatin during
the S phase in a replication-coupled manner, whereas
H3.3 can be incorporated replication independently into
the transcriptionally active regions of chromatin at
interphase (Loyola & Almouzni 2007). A recent study
has shown that core histones were exchanged with free
histones at detectable levels even in the highly
condensed chromosome during mitosis, and that the
elevation of histone exchange activity during mitotic exit
was correlated with the increase in acetylation level at
some lysine residues in the entire chromatin (Chen et al.
2005). In addition, it has been proposed that free
histones carry a distinct set of histone modifications
before their incorporation into the chromatin and, after
their incorporation, affect the chromatin’s final epige-
netic state (Loyola et al. 2006, Loyola & Almouzni 2007).
For example, upon gene activation, H3 carrying the
repressive methyl K9 is replaced by H3.3 unmethylated
at K9 in human somatic cells (Loyola et al. 2006). These
reports indicate that high histone exchange activity
causes the dynamic change in histone modification in
the entire chromatin, and that the incorporation of the
free histones into the chromatin might be affected by
the modification profile of their histone tails. Although
the general feature of histone exchange has been
reported in somatic cells, histone exchange activity in
meiotic mammalian oocytes is poorly understood.
Recently, it has been reported that at least H3.3 is
detected on the chromatin at the GV stage in mouse
oocytes (Torres-Padilla et al. 2006), implying the
presence of histone exchange activity during this stage
in mammalian oocytes. Although the chromatin become
more condensed upon GVBD through second meta-
phase (M2), the oocyte cytoplasm at M2 stagehas unique
chromatin remodeling activity that replaces the prota-
mines in sperm chromatin with free histones upon
fertilization (McLay & Clarke 2003). Thus, it is possible
that core histones in oocyte chromatin are dynamically
replaced with the free histones by the oocyte cytoplasm
after GVBD. This replacement may also be involved in
chromatin-wide histone deacetylation during meiotic
maturation. However, histone exchange activity and its
correlation with the N-terminal acetylation and global
histone acetylation levels in the chromatin have not yet
been examined in mammalian oocytes.
In this study, we attempted to analyze the histone
exchange activity before and after GVBD and to
investigate its correlation with histone acetylation status
using porcine oocytes maturing in vitro. Since H3.1,
H3.2, and H3.3 are known to be incorporated into
the chromatin as an H3–H4 heterodimer by histone
chaperones, the amount of H4 incorporation into
chromatin can be regarded as an indicator of total
histone exchange activity. Therefore, we examined
the incorporation of Flag-tagged H4 (H4-Flag) into the
chromatin by injection of H4-Flag mRNA or protein into
the oocyte cytoplasm. To examine whether or not the
acetylation status of the free histones affects their histone
exchange activity, we generated two mutated H4-Flag
constructs, in which all four acetylatable lysines on the
N-terminal tail were substituted with glutamines or in
which amino acids 1–20 on the N-terminal tail were
deleted. We investigated the degree of histone incorpor-
ation into chromatin and subsequent global histone
acetylation levels in the chromatin after injection of
these mutated H4-Flag mRNAs into the GV oocytes.
Global histone deacetylation during in vitro maturation
of porcine oocytes
In our in vitro maturation system, porcine oocytes
undergo GVBD at approximately 24 h of culture, and
they reach the first meiotic metaphase (M1) at 30 h and
M2 at 48 h of culture. Immunostaining examples of
acetylated H4K8 (H4acK8) are shown in Fig. 1. A high
level of histone acetylation was maintained during
the GV stage until 20 h of culture, and the level was
remarkably decreased at the M1 and M2 stages.
Although the acetylation signals of the first polar bodies
were stronger than those of the M2 chromosomes, the
acetylation level of M2 chromosomes was comparable
with that of M1 chromosomes. This confirms the finding
of our previous report (Endo et al. 2005), indicating that
the global level of histone deacetylation occurs after
GVBD in porcine oocytes.
H4-Flag mRNA injection into GV-stage oocytes
GV-stage oocytes were injected with H4-Flag mRNA,
and the localization of H4-Flag protein was then
examined by immunostaining (Fig. 2). There was no
Flag signal on the chromatin just after injection
Figure 1 Global histone deacetylation during in vitro maturation of
porcine oocytes. Porcine oocytes were cultured for 0, 30, or 48 h and
immunostained with an antibody specific to lysine 8-acetylated histone
H4 (H4acK8). Each sample was counterstained with Hoechst 33342 to
visualize the DNA (lower panels). GV, non-cultured and 20 h cultured
oocytes at the GV stage; M1, 30 h cultured oocytes at the first
metaphase; M2, 48 h cultured oocytes at the second metaphase.
Arrowheads indicate the first polar bodies. Scale bar indicates 30 mm.
T Endo and others
Reproduction (2011) 141 397–405www.reproduction-online.org
This work is supported bya Grant-in-Aid for Scientific Research
(no. 19380155) to K Naito and by a Research Fellowship for
Young Scientists (grant no. 19-2573) to T Endo, both from the
Japan Society for the Promotion of Science.
We are grateful to Dr K Chida of the University of Tokyo, for
the generous gift of HEK 293 cells. We also thank Dr FAoki and
Dr T Akiyama for their helpful suggestions on this work.
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Received 31 March 2010
First decision 28 April 2010
Revised manuscript received 7 December 2010
Accepted 14 January 2011
Histone exchange activity in porcine oocytes
www.reproduction-online.orgReproduction (2011) 141 397–405