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National Institute of Biological Sciences (NIBS), Beijing 102206, People's Republic of China
ABSTRACT
Epigenetic reprogramming is thought to play an important role in the development of cloned embryos reconstructed by somatic cell nuclear transfer (SCNT). In the present study, dynamic reprogramming of histone acetylation and methylation modifications was investigated in the first cell cycle of cloned embryos. Our results demonstrated that part of somatic inherited lysine acetylation on core histones (H3K9, H3K14, H4K16) could be quickly deacetylated following SCNT, and reacetylation occurred following activation treatment. However, acetylation marks of the other lysine residues on core histones (H4K8, H4K12) persisted in the genome of cloned embryos with only mild deacetylation occurring in the process of SCNT and activation treatment. The somatic cloned embryos established histone acetylation modifications resembling those in normal embryos produced by intracytoplasmic sperm injection through these two different programs. Moreover, treatment of cloned embryos with a histone deacetylase inhibitor, Trichostatin A (TSA), improved the histone acetylation in a manner similar to that in normal embryos, and the improved histone acetylation in cloned embryos treated with TSA might contribute to improved development of TSA-treated clones. In contrast to the asymmetric histone H3K9 tri- and dimethylation present in the parental genomes of fertilized embryos, the tri- and dimethylations of H3K9 were gradually demethylated in the cloned embryos, and this histone H3K9 demethylation may be crucial for gene activation of cloned embryos. Together, our results indicate that dynamic reprogramming of histone acetylation and methylation modifications in cloned embryos is developmentally regulated.
developmental biology, early development, embryo, epigenetic reprogramming, fertilization, histone acetylation, histone methylation, somatic cell nuclear transfer, TSA
Fertilization unites two highly specialized haploid genomes with markedly different chromatin structure within a single cell and forms a diploid zygote with totipotency. In a short period of time, the two haploid genomes undergo dramatic asymmetric chromatin remodeling to re-establish transcription activation of zygotic gene expression [1–3]. Protamines, highly compact paternal chromosomal complements, are replaced by maternally derived core histone octamers and by the oocyte-specific linker histone-H1FOO when fertilization occurs [4–6]. The maternal chromosomes, likewise, decondense upon the completion of meiosis.
Epigenetic modifications to the paternal and maternal genomes have been widely studied. Active DNA demethylation occurring in the paternal genome shortly after fertilization is independent of DNA replication. In the contrast, the maternal genome remains highly methylated and undergoes passive DNA demethylation following embryo development [3, 7]. However, the mechanism of this asymmetric reprogramming of DNA methylation in the process of fertilization is poorly understood. One recent study has proposed that PGC7/Stella (DPPA3) may play an important role in protecting the maternal genome from active demethylation [8]. However, the presence of DPPA3 in the paternal pronucleus is also detected, and what factors initiate the active DNA demethylation protected by DPPA3 in the paternal pronucleus is largely unknown. This asymmetric reprogramming of DNA methylation occurring in the parental genomes has been considered essential for establishing zygotic totipotency.
Histone modification—including acetylation, methylation, phosphorylation, and ubiquitination—is another important epigenetic modification to the chromatin and widely regulates gene transcription and silencing [9–11]. Histone H3K9 trimethylation and dimethylation are correlated to gene silencing while histone H3K4 methylation leads to initiation of gene transcription [12, 13]. Similar to the pattern of asymmetric DNA methylation in parental genomes, histone H3K9 trimethylation and dimethylation exhibit asymmetric modifications in the parental pronuclei, according to the recent studies [14, 15]. Shortly after fertilization, histone H3K9 is highly trimethylated and dimethylated in the maternal pronucleus while the paternal pronucleus exhibits a low level of tri- and dimethylation [14]. Histone acetylation is another important epigenetic modification to the chromatin in which the acetylation usually occurs on the lysine residues of core histones. Acetylation has the greatest potential for unfolding chromatin to recruit different transcriptional factors, and it is almost invariably associated with activation of gene transcription in cells [16–17]. Acetylation modification of core histones in oocytes and pre-implantation embryos has been investigated in recent studies [18–21]. Compared to asymmetric modification of DNA methylation and histone H3K9 tri- and dimethylation in parental pronuclei, histone acetylation modifications have been shown to be uniformly distributed in the parental pronuclei shortly after fertilization [20, 21].
Somatic cell nuclear transfer (SCNT) is a remarkable process in which the somatic cell gene expression can be converted into a totipotent state in the oocyte through an undiscovered mechanism. Epigenetic reprogramming of the somatic cell genome has been suggested as a key event occurring in the SCNT, with abnormal reprogramming of the epigenetic marks contributing to the inefficiency of somatic cell cloning [22, 23]. Abnormal expression of imprinted genes and X chromosome inactivation correlated with DNA methylation have been observed in cloned embryos [24–29]. As another important modification to the chromatin, histone modification of cloned embryos has been investigated in limited studies [6, 30–33]. Evidence from recent studies has shown that elevated levels of histone acetylation in cloned embryos can improve the development of cloned embryos, indicating that correct reprogramming of histone modification might be another important clue to improving the efficiency of somatic cell nuclear transfer [30, 31]. Our and other previous results have demonstrated that somatic linker histone H1 can be replaced by oocyte-specific linker histone H1FOO quickly after somatic cell nuclear transfer [6, 33]. In the present study, both the methylation and acetylation status of lysine residues on core histones H3 and H4 are investigated in cloned embryos treated with/without the histone deacetylase inhibitor Trichostatin A (TSA) to compare the similarity of cloned embryos to normal fertilized embryos produced by intracytoplasmic sperm injection (ICSI).
All inorganic and organic compounds were purchased from Sigma Chemical Company (St. Louis, MO) unless otherwise noted.
The SPF-grade hybrid mice B6D2F1 (C57BL/6 x DBA/2) were housed in the animal facility of the National Institute of Biological Sciences. All studies adhered to procedures consistent with the National Institute of Biological Sciences Guide for the care and use of laboratory animals.
Collection of Oocytes and Preparation of ICSI Embryos
Metaphase II oocytes were collected from the oviducts of B6D2F1 mice after sequential injection of 8–12-week-old females with pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG), as described in previous reports [34, 35]. Adherent cumulus cells were removed by hyaluronidase treatment, and the cumulus-cell-free oocytes were cultured in CZB medium supplemented with glucose [34, 36]. MII oocytes collected 14 h post hCG were used for SCNT, and the oocytes used for ICSI were cultured for another 4–5 h (18–19 h post hCG). To collect the embryos fertilized at different time points, ICSI was performed as in previous reports [37, 38]. Briefly, sperm were collected from epididymis of adult male B6D2F1 mice and washed in HCZB medium, then suspended in HCZB medium supplemented with 10% polyvinylpyrrolidone (PVP). ICSI was performed on the stage of an Olympus inverted microscope equipped with a Narishige micromanipulator. The MII oocytes were placed in a drop of HCZB medium and one sperm head was injected into a MII oocyte with the aid of a piezo-drill micromanipulator. The surviving oocytes were collected and cultured in KSOM medium.
SCNT was performed as described previously [39, 40]. Briefly, the spindle chromosome complex (SCC) with minimal volume of cytoplasm was removed by an enucleation pipette with an inner diameter of 10 µm attached to the piezo-drill micromanipulator. The cumulus cells (presumably G1 phase) from ovulated oocytes were employed as nuclear donors and the nuclei were injected into the enucleated oocytes one by one. The reconstructed oocytes were cultured in CZB medium for 1–3 h before activation treatment. The cloned constructs were activated by 6 h of culture in Ca2+-free CZB medium supplemented with 10 mM SrCl6 and 5 µg/ml of cytochalasin B [34]. Cloned embryos were cultured in KSOM at 37°C in an atmosphere of 5% CO2 in air.
The preparation and treatment of Trichostatin A (TSA) have been described previously [31]. Briefly, TSA was dissolved in DMSO and prepared as a 200-fold concentrated stock solution. The reconstructed oocytes were activated as above with supplementation of 5 nM TSA in the activation medium. Activated oocytes were cultured in KSOM containing 5 nM TSA for another 4 h and then transferred into KSOM without TSA for further culture.
To investigate the dynamic histone modifications in SCNT-cloned embryos compared to ICSI-produced embryos, four groups of samples, designated as Injection, Clone, Clone + TSA, and ICSI, were collected and processed for indirect immunocytochemistry staining.
MII oocytes and reconstructed oocytes following injection of somatic cell nuclei into enucleated oocytes at different time points (10 min, 30 min, 1 h, and 3 h) were collected as the Injection group. The immunocytochemical staining of these oocytes allowed us to visualize the dynamic changes of histone modifications in the condensed chromosomes formed from the injected somatic cell nucleus.
The samples in the Clone group included the reconstructed oocytes at different time points (30 min, 1 h, 3 h, 6 h, and 10 h) during activation treatment. Different from the Clone group, TSA was supplemented in the activation medium in the Clone + TSA group, but all embryos were collected at the same time points. In the ICSI group, samples of the injected oocytes were collected at the same time points as indicated for the Clone and Clone + TSA groups. The immunocytochemistry staining of these oocytes allowed us to visualize the dynamic changes of histone modifications in the genomes of cloned and ICSI embryos, in which the chromosomes underwent transition from condensation to decondensation.
The method used for indirect immunofluorescence was adapted from Santos and colleagues with some modifications [14]. All steps were performed at room temperature, unless otherwise mentioned. The collected oocytes/embryos were fixed with 4% paraformaldehyde for at least 20 min, and then permeabilized for 30 min with 0.2% Triton X-100 in PBS. After three washings, all samples were incubated in a blocking solution (1% BSA and 0.05% Tween-20 in PBS) overnight at 4°C. Oocytes/embryos were incubated with the first antibodies to H3K9 acetylation (1:250), H3K14 acetylation (1:250) and H4K8 acetylation (1:400), H4K12 acetylation (1:400), H4K16 acetylation (1:250), di- (1:250) and trimethylation (1:400) of H3K9 for 1 h (all the first antibodies were purchased from Upstate). After three washings, the oocytes/embryos were incubated with a fluorescein isothiocyanate (FITC) conjugated goat anti-rabbit second antibody (Jackson ImmunoResearch, dilution 1:1000) for 1 h. Finally, the DNA was stained with 4', 6-diamidino-2-phenylindole (DAPI, 1 µg/ml) and all the samples were mounted in anti-fade solution. At least ten oocytes/embryos were processed for each separate sample and the experiments were replicated at least three times.
Stained embryos mounted on slides were observed on a LSM 510 META microscope (Zeiss, Germany) using a Plan Neofluar 63x/1.4 Oil DIC objective and excitation wavelengths of 488 nm and 405 nm. Each channel signal was collected sequentially. For each experiment, the same detector gain, amplifier offset, and pinhole parameters were used. All collected images were assembled using Adobe Photoshop software (Adobe Systems, San Jose, CA) without any adjustment of contrast and brightness to the images.
Dynamic Reprogramming of Acetylation on Lysine Residues 9 and 14 of Histone H3 in the First Cell Cycle of Cloned Embryos
The acetylation modification on lysine residues 9 and 14 in histone H3 of somatic cell nucleus underwent dramatic changes following injection of somatic cell nucleus into the enucleated oocyte and subsequent activation treatment. As shown in Figure 1A, H3K9 is not acetylated in the chromosomes of MII oocytes. H3K9 did not undergo acetylation modification in both parental genomes until 6 h after intracytoplasmic sperm injection, and both pronuclei exhibited equal H3K9 acetylation modification (ICSI panel, Fig. 1, P–T). Compared to MII oocytes, the somatic cells showed very high levels of H3K9 acetylation in the nucleus, which can be observed in the reconstructed oocyte 10 min after somatic cell nucleus injection (Fig. 1B). Following nuclear envelope breakdown and chromosome condensation, H3K9 acetylation was quickly eliminated from the somatic cell genome. No H3K9 acetylation could be observed in the somatic cell chromosomes 1 h after SCNT (Fig. 1, C–E). Following activation, the consequence of restoration of H3K9 acetylation in the pseudo-pronuclei was similar to the normal ICSI embryos except that the intensity of H3K9 acetylation in the cloned embryos was much weaker than in the normal embryos (Fig. 1, F–J), while similar intensity of H3K9 acetylation in the pseudo-pronuclei was achieved in the cloned embryos treated with TSA compared to normal embryos (Fig. 1, K–O). Similar to H3K9 acetylation in MII oocyte, H3K14 was not acetylated in the MII chromosomes, as shown in Fig. 2A. However, H3K14 acetylation occurred in both parental genomes much more quickly than H3K9 acetylation following ICSI. H3K14 underwent acetylation in both parental genomes when the oocyte was still at telophase II stage and the chromosomes remained condensed 3 h after ICSI (Fig. 2R). Acetylation of H3K14 in both parental genomes reached peak levels 10 h after ICSI (Fig. 2, S and T). Compared to normal embryos, acetylation of H3K14 in SCNT embryos showed dynamic modifications following nucleus injection and activation. As shown in Figure 2B–2E, H3K14 was highly acetylated in the somatic cell nucleus, and the intensity decreased along with the nuclear envelope breakdown and chromosome condensation following SCNT. The acetylation of H3K14 was removed 3 h after nucleus injection. Restoration of H3K14 acetylation in cloned embryos occurred following activation, and the H3K14 acetylation exhibited different patterns in cloned embryos with or without TSA treatment (Clone panel, Fig. 2, F–J; Clone + TSA panel, Fig. 2, K–O). Restoration of H3K14 acetylation occurred much more quickly in the cloned embryos treated with TSA when the chromosomes were separated into two groups 3 h after activation, whereas the untreated clones recovered H3K14 acetylation 6 h after activation and the chromosomes had already decondensed (Clone + TSA panel, Fig. 2M; Clone panel, Fig. 2I). Compared to normal embryos, the TSA-treated clones showed similar intensity of H3K14 acetylation while the intensity of H3K14 acetylation in untreated clones was much weaker.
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Dynamic Reprogramming of Acetylation on Lysine Residues 8, 12, and 16 of Histone H4 in the First Cell Cycle of Cloned Embryos
Acetylation of lysine residues 8, 12, and 16 on histone H4 of the somatic cell nucleus exhibited dramatic differences after injection into enucleated MII oocytes. As shown in Figures 3 and 4, both H4K8 and H4K12 acetylation showed similar patterns in control MII oocytes and ICSI embryos. Both H4K8 and H4K12 were acetylated in the chromosomes of MII oocytes, and such acetylation in the maternal genome was maintained in the process of fertilization without deacetylation occurring (Fig. 3A; Fig. 3, P–T; Fig. 4A; Fig. 4, P–T). H4K8 and H4K12 acetylation was quickly restored in the paternal genome following the replacement of protamines with histones, and the intensity of acetylation in both pronuclei was equally distributed (Fig. 3, Q–T; Fig. 4, Q–T). However, H4K12 acetylation exhibited dynamic localization following pronuclei formation, and intense dispersal of H4K12 acetylation was observed in the periphery of the nucleus (Fig. 4T), which is in agreement with a previous report [21]. Similar to the control oocytes and ICSI embryos, acetylation of H4K8 and H4K12 was present consistently in the somatic cell nucleus and cloned embryos (Fig. 3, F–J; Fig. 4, F–J). However, the acetylation intensity of both lysine residues in cloned embryos treated with TSA was much higher than in untreated clones (Fig. 3, K–O; Fig. 4, K–O). Moreover, localization of H4K12 acetylation observed in the perinuclear area of TSA-treated clones resembled that in the control ICSI embryos, while the untreated clones showed equal distribution of H4K12 acetylation in the pseudo-pronuclei (Fig. 4O; Fig. 4J). The pattern of H4K16 acetylation in cloned embryos and control embryos was similar to the pattern of H3K9 acetylation described above. As shown in Figure 5, H4K16 was not acetylated in the MII oocyte, and both parental genomes recovered acetylation by 6 h after ICSI and exhibited equal distribution in both pronuclei with strong intensity at 10 h after ICSI (Fig. 6A; Fig. 6, P–T). In contrast, H4K16 was acetylated in the somatic cell nucleus and was deacetylated following injection of the somatic cell nucleus into the enucleated MII oocyte within 1 h (Fig. 6, B–E). H4K16 acetylation was recovered in the genome of cloned embryos following activation, similar to the situation in ICSI embryos except that the TSA-treated clones more closely resembled the normal embryos with similar H4K16 acetylation intensity present in the pronuclei (Clone panel, Fig. 6, F–J; Clone + TSA panel, Fig. 6, K–O).
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Dynamic Reprogramming of Histone H3K9 Tri- and Dimethylation in Cloned Embryos
As shown in Figure 6, H3K9 trimethylation in the maternal genome was observed in the MII oocyte and asymmetrically distributed in parental genomes following intracytoplasmic sperm injection (Fig. 6A; Fig. 6, P–T). The maternal genome exhibited H3K9 trimethylation while the paternal genome never developed such trimethylation marks on H3K9 even after chromosomal decondensation occurred. Compared to control MII oocytes and ICSI embryos, H3K9 trimethylation exhibited slight differences in the SCNT reconstructed oocytes and cloned embryos. H3K9 trimethylation was present in the somatic cell nucleus and did not diminish after injection (Fig. 6, B–E). Following activation and pseudo-pronuclei formation, H3K9 trimethylation marks were gradually removed and no asymmetric distribution was observed in the two (or more) pseudo-pronuclei (Fig. 6, F–J). Moreover, TSA treatment showed no effect on the distribution and disappearance of H3K9 trimethylation in cloned embryos (Fig. 6, K–O). Similar to the pattern of H3K9 trimethylation observed in both control and cloned embryos, dimethylation of H3K9 was asymmetrically distributed in parental genomes of ICSI-produced embryos and equally distributed in the genomes of cloned embryos. As shown in Figure 7, H3K9 dimethylation was observed in the genome of MII oocytes and asymmetrically distributed in the parental genomes following ICSI (Fig. 7A; Fig. 7, P–T). No H3K9 dimethylation was detected in the paternal genome, and dimethylation marks persisted in the maternal genome until 6 h after ICSI (Fig. 7S). Disappearance of H3K9 dimethylation marks in the maternal genome occurred 10 h after ICSI, and both pronuclei displayed no H3K9 dimethylation marks (Fig. 7T). H3K9 dimethylation in the somatic cell nucleus could be observed following SCNT, and the intensity of dimethylation decreased along with chromosome condensation (Fig. 7, B–E). Dimethylation marks persisted in the cloned embryos following activation and disappeared at 10 h after activation treatment (Fig. 7, F–J). Both TSA-treated and untreated cloned embryos showed no difference in persistence and disappearance of H3K9 dimethylation marks (Fig. 7, K–O).
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In the present study, we investigated the dynamic changes of histone acetylation and methylation modifications in the first cell cycle of somatic cell nuclear transfer embryos compared with embryos produced by intracytoplasmic sperm injection. We found that the oocyte could reprogram the histone modifications to a certain degree, comparable to ICSI-produced embryos, which might facilitate the gene expression or silencing in the cloned embryos to support further development. Moreover, reprogramming of histone acetylation in the cloned embryos could be improved by treating them with a histone deacetylase inhibitor, TSA, which might account for the improved development of cloned embryos treated with TSA. In contrast to fertilized embryos, histone H3K9 tri- and dimethylation did not exhibit asymmetric distribution in pseudo-pronuclei of somatic-cell-cloned embryos.
Histone acetylation is an important epigenetic modification to the chromatin in which the acetylation usually occurs on the lysine residues of core histones. Acetylation has the greatest potential for unfolding chromatin to recruit different transcriptional factors, and it is almost invariably associated with activation of gene transcription [9, 16, 17]. Changes in acetylation on specific lysine residues in histone H3, H4 during mouse oocyte meiosis, and early embryo development have been studied previously [18–21]. It has been shown that the core histones of oocytes recovered from ovaries in which the oocytes are arrested at prophase of meiosis I are highly acetylated and that deacetylation occurs quickly with resumption of spontaneous maturation and chromosome condensation. Most lysine residues of core histones are deacetylated in the condensed chromosomes of MII oocytes, except H4K8 is acetylated [18]. In the present study, we found that H4K12 is also acetylated in the MII oocyte, which is different from a previous report, and this difference might be due to the aged oocytes used in the previous study [18]. When somatic cell nuclei were injected into the enucleated MII oocytes to reconstruct cloned embryos, we found that acetylated core histones in the somatic cell genome underwent two different fates. The first occurred in three lysine residues of histone H3 and H4 (H3K9, H3K14, H4K16) in which the acetylated lysine residues were quickly deacetylated upon somatic cell nuclear transfer and full deacetylation could be observed 3 h after SCNT. Restoration of acetylation in these lysine residues of core histones occurred along with activation of reconstructed oocytes. This deacetylation and re-acetylation of histone lysine residues in the process of SCNT must be critical for regulation of gene expression and establishment of totipotency of cloned embryos. Since the somatic gene expression program has to be turned off before embryonic gene expression is established, histone deacetylation occurring after somatic cell nuclear injection would facilitate somatic gene silencing. Reestablishment of histone acetylation in cloned embryos after activation mimicked the program in normal embryos, which could be important for initiating embryonic gene activation in cloned embryos.
The second fate occurred in the other two lysine residues of histone H4 (H4K8 and H4K12) in which acetylation persisted in the somatic cell nucleus, condensed chromosomes, and pseudo-pronuclei of SCNT embryos. In this process, only mild deacetylation occurred in condensed chromosomes after nuclear injection, and this deacetylation allowed the transferred somatic genome to exhibit an intensity of acetylation similar to the oocyte chromosomes. The same outcome was achieved through a different process in which the oocyte could reprogram the acetylation of lysine residues to mimic the situation of normal MII oocytes and fertilized embryos. Our results indicate that specific histone deacetylase and histone acetylase must co-exist in the MII oocyte and coordinate with other reprogramming factors to remodel the somatic cell nucleus at the epigenetic level to mimic normal embryo development. Moreover, we have found that TSA treatment of cloned embryo can improve development, possibly by increasing the intensity and improving the localization of acetylation in pseudo-pronuclei of cloned embryos. Results of previous reports have indicated that localization of acetylation of specific lysine residues might be important for embryo development, especially zygotic gene activation [20, 21]. H4K12 acetylation localized in the nuclear periphery has been observed in both ICSI-produced and TSA-treated cloned embryos but not in untreated clones in the present study. This result agrees with a previous study in which localization in the nuclear periphery of H4K12 acetylation in 1- and 2-cell-stage embryos has been observed and has been suggested as essential for embryonic gene activation [21].
Histone methylation includes methylation of arginine and lysine residues, and it plays an important role in regulating transcription, genome integrity, and epigenetic inheritance. Recently, histone methylation has been found to be crucial for early embryo development and for maintaining pluripotency of embryonic stem cells [41, 42]. Differential methylation of H3R26 (arginine residue 26 of H3) in blastomeres of 4-cell-stage mouse embryos has been observed in a recent study, and the blastomere with a high level of H3R26 methylation showed great potential for developing into an inner cell mass (ICM) [41].
Methylation of lysine residues in core histones contributes to both active and repressive chromatin functions. In particular, methylation of H3K4 and H3K36 is associated with gene activation, whereas methylation of H3K9 and H3K27 is generally correlated with gene silencing. Histone lysine methylation has been considered a static modification to chromatin, but recently several histone demethylases have been characterized [43–47]. Binding of HP1 with H3K9 trimethylation has been proved essential for heterochromatin maintenance, and methylation of H3K9 plays an important role in X chromosome inactivation in female cells [48–50]. In recent studies, histone H3K9 tri- and dimethylation has been investigated in early embryo development, and the results have demonstrated that asymmetric H3K9 tri- and dimethylation modifications exist in the parental pronuclei after fertilization [14]. Similar to asymmetric DNA methylation observed in parental pronuclei, the maternal pronucleus exhibits high levels of H3K9 tri- and dimethylation whereas the paternal pronucleus never produces H3K9 tri- or dimethylation during pronucleus formation. Active DNA demethylation has been observed in the paternal genome during fertilization, and although the mechanism is poorly understood, it deserves further investigation into whether the lysine residue demethylated histone modification (specifically undetected H3K9 tri- and dimethylation) plays a role in the process of active DNA demethylation. In the present study, histone H3K9 tri- and dimethylation in the somatic cell genome was gradually demethylated in the SCNT embryos following activation, and this demethylation might correlate with embryonic gene activation required for further development. Unlike fertilized embryos, no asymmetric H3K9 tri- or dimethylation distribution in pseudo-pronuclei was observed in cloned embryos, perhaps due to the different chromatin structure existing between somatic cell genome and oocyte chromatin. Our results also indicate that a specific histone demethylase must be present in the oocyte through which the H3K9 tri- and dimethylation marks could be erased, and indeed we have detected that mRNA of a specific H3K9 demethylase was highly expressed in MII oocytes and zygotes (unpublished data). It would be pivotal to characterize this histone H3K9 demethylase in oocytes because it might assist us in uncovering the mechanism of active DNA demethylation during fertilization in future studies.
In summary, our present study investigated the dynamic changes of H3K9 methylation and acetylation of different lysine residues on core histones H3 and H4 in somatic-cell-cloned embryos. Our findings suggest that oocytes can reprogram the histone acetylation through different programs to mimic the histone acetylation modifications in normal embryos. Treatment of cloned embryos with TSA could improve the histone acetylation reprogramming. Demethylation of H3K9 tri- and dimethylation occurring in the process of cloning might be crucial for further development of cloned embryos. Whether this histone H3K9 demethylation is correlated with active DNA demethylation needs to be investigated further.
ACKNOWLEDGMENTS
We thank the lab members for helpful comments on the manuscript.
FOOTNOTES
1This work was supported by National High Technology Project 863 (2005AA210930). 
Correspondence: 2Shaorong Gao, National Institute of Biological Sciences, NIBS, #7 Science Park Road, Zhongguancun Life Science Park, Beijing 102206, P.R. China. FAX: 86 10 8072 7535; e-mail: gaoshaorong{at}nibs.ac.cn
Received: 30 May 2007.
First decision: 27 June 2007.
Accepted: 16 August 2007.
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