Journal of Reproduction and Development, Vol. 58, No 5, 2012 —Original Article— The Expression and Nuclear Deposition of H3.1 in Murine Oocytes and Preimplantation Embryos Machika Kawamura1), Tomohiko Akiyama1)#, Satoshi Tsukamoto2), Masataka G. Suzuki1) and Fugaku Aoki1) 1)Department of Integrated Biosciences, Graduate School of Frontier Sciences, University of Tokyo, Chiba 277-8562, Japan 2)Laboratory of Animal and Genome Science Section, National Institute of Radiological Sciences, Chiba 263-8555, Japan #Present: Laboratory of Genetics, NIH Biomedical Research Center, Baltimore, MD 21224, U.S.A. Abstract. Differentiated oocytes acquire totipotency through fertilization. During this transition, genome-wide remodeling occurs, which leads to change in expression. However, the mechanism that underlies this global change in chromatin structure has not been fully elucidated. Histone variants play a key role in defining chromatin structure and are implicated in inheritance of epigenetic information. In this study, we analyzed the nuclear localization and expression of H3.1 to elucidate the role of this histone variant in chromatin remodeling during oogenesis and preimplantation development. Analysis using Flag-tagged H3.1 transgenic mice revealed that Flag-H3.1 was not present in differentiated oocytes or early preimplantation embryos before the morula stage, although Flag-H3.1 mRNA was expressed at all stages examined. In addition, the expression levels of endogenous H3.1 were low at the stages where H3.1 was not present in chromatin. These results suggest that H3.1 is not incorporated into chromatin due to the inactivity of the histone chaperone and low mRNA expression level. The significance of the dynamics of H3.1 is evaluated in terms of chromatin remodeling that takes place during development. Key words: H3.1, H3 variants, Oocyte, Preimplantation embryo, Reprogramming (J. Reprod. Dev. 58: 557–562, 2012)

ifferentiated gametes, eggs and sperm, acquire totipotency is incorporated into chromatin by histone regulator A (HIRA) and Dthrough fertilization [1, 2]. During this developmental process, DAXX-ATRX [9, 11–13]. Since each H3 variant is associated with genome-wide chromatin remodeling takes place that alters the gene different histone modifications and genome regions, this suggests that expression pattern and begins differentiation [1–3]. This chromatin each variant serves a unique role in chromatin structure [6, 7, 10]. remodeling is essential in erasing previous epigenetic markers Although H3.3 and its role in chromatin remodeling have been and forming and transmitting new ones to the next generation [1, extensively studied in different model organisms, the role of H3.1 is 4]. However, the mechanism that underlies this global change in not well known. For example, the dynamics of H3.3 during oogenesis chromatin structure during development has not been fully elucidated. and preimplantation development have been studied in mice [10, Recent studies have shown that in addition to histone modifications 14]. Although H3.3 is incorporated in oocytes, this histone variant and transcription factors, histone variants play a key role in defining rapidly disappears and then reappears just after fertilization [10]. and altering chromatin structure [5]. consist of 4 different Defects in heterochromatin formation have been reported in embryos , H2A, H2B, H3 and H4. Notably, the association between H3 expressing mutated H3.3 [14]. In addition, in Arabidopsis, H3.3 is variants and chromatin remodeling has been extensively studied. In present in both gametes but rapidly disappears after fertilization, mammals, there are five variants, i.e., centromere-specific and only the paternal H3.3 remains in the zygote [15]. Finally, CENP-A, testis-specific H3.1t and three major H3 variants, H3.1, H3.3 is associated with decondensation of the paternal genome after H3.2 and H3.3 [6–9]. H3.1 and H3.2, which differ by 1 amino acid, fertilization in Drosophila melanogaster [16]. Consequently, these are canonical histones expressed and incorporated into chromatin in altogether suggest that H3.3 is essential for chromatin remodeling a DNA replication-dependent manner. Although H3.1 and H3.2 are and for acquiring a totipotent characteristic in 1-cell embryos, but associated with heterochromatin, they differ in histone modifications little is known about the role of H3.1 in chromatin remodeling before [6–8]. In contrast, H3.3 is expressed and incorporated into chromatin and after fertilization. throughout the cell cycle and is associated with euchromatin [6, 8, In this study, we analyzed the nuclear localization and expression 10]. H3.1 is deposited into chromatin via the histone chaperone of H3.1 to elucidate the role of this histone variant in chromatin called chromatin assembly factor 1 (CAF-1) [9–11], whereas H3.3 remodeling during oogenesis and preimplantation development. H3 variants are similar in amino acid sequence, and currently, there are no antibodies that recognize each variant. Therefore, we generated Received: April 20, 2012 Flag-tagged H3.1 transgenic mice that ubiquitously express Flag-H3.1 Accepted: May 15, 2012 Published online in J-STAGE: June 14, 2012 to analyze the presence of Flag-H3.1 in the chromatin. In addition, ©2012 by the Society for Reproduction and Development the expression of H3.1 genes was analyzed. Finally, the biological Correspondence: F Aoki (e-mail: [email protected]) significance and mechanisms concerning nuclear incorporation of 558 KAWAMURA et al.

H3.1 are discussed. Tween 20 (MP Biomedicals, Solon, OH, U.S.A.) overnight at 4 C. After being washed in 1% BSA/PBS three times, they were incubated Materials and Methods with the Alexa Fluor 488-conjugated mouse IgG secondary antibody diluted 1:100 with 1% BSA/PBS containing 0.2% Tween 20 for 1 h at Transgenic mouse room temperature. The cells were then mounted on VECTASHIELD The pCAGGS vector [17], which contains the CAG promoter (Vector Laboratories, Burlingame, CA, U.S.A.), an anti-bleaching and a 3’UTR region of rabbit β-globin, was used to generate CAG reagent, and were observed using a confocal laser scanning microscope promoter-driven Flag-tagged H3.1 transgenic mice. For microinjection, (Carl Zeiss 510, Oberkochen, Germany). the transgene was diluted to 4 ng/μl in Tris/EDTA buffer (pH7.5) and microinjected into the pronucleus of C57 BL/6 1-cell embryos (Japan Reverse transcription PCR (RT-PCR) SLC, Shizuoka, Japan). One day after microinjection, normal 2-cell For RT-PCR analyses to detect the expression of Flag-H3.1 embryos were collected and transferred to day 0.5 pseudopregnant transgene, 10 oocytes/embryos were placed in 400 μl ISOGEN ICR (MCH) females (Japan SLC). Thirty-four mice were screened, (Nippon Gene, Tokyo, Japan). RNA was extracted by following the and seven of them were positive for the transgene. The founder manufacturer’s protocol (Nippon Gene), and reverse transcription transgenic mouse was backcrossed twice with C57 BL/6 female was carried out with an Oligo dT Primer using a PrimeScript RT- mice (Japan SLC). The pups were subjected to genotyping of the PCR Kit (TaKaRa, Shiga, Japan). PCR was carried out using Ex transgene by PCR. Taq DNA Polymerase (TaKaRa) and 0.4 oocytes/embryos cDNA in a tube as a template. Flag-H3.1 was detected with the follow- Oocyte collection ing primers: 5’ATGACGACGATAAGGGAGGA3’ (forward) and Growing oocytes were collected from nine-day-old mice. The 5’CTCGGTCGACTTCTGGTAGC3’ (reverse). PCR conditions were ovaries were dissected in HEPES-buffered KSOM (potassium as follows: 95 C for 30 sec (denaturation), 57 C for 30 sec (annealing) simplex optimized medium) [18]. The ovaries were further dissected and then 72 C for 30 sec (elongation). Fifty pg of Rabbit α-globin after trypsin treatment to obtain additional growing oocytes. The mRNA (Sigma-Aldrich) was added into the samples before RNA growing oocytes were placed in α-minimum essential medium extraction as an external control and was detected with the following (Gibco BRL, Grand Island, NY, U.S.A.) supplemented with 5% primers: 5’GTGGGACAGGAGCTTGAAAT3’ (forward) and 5’ fetal bovine serum (Sigma-Aldrich, St. Louis, MO, U.S.A.) and 10 GCAGCCACGGTGGCGAGTAT3’ (reverse). The PCR conditions ng/μl epidermal growth factor (Invitrogen, Carlsbad, CA, U.S.A.) were: 95 C for 20 sec (denaturation), 58 C for 30 sec (annealing), until use for immunocytochemistry or RT-PCR analysis. Full-grown and 72 C for 30 sec (elongation). oocytes were collected from three- to five-week-old mice 48 h after For real-time PCR analyses to examine the expression levels of injection of 5 IU pregnant mare’s serum gonadotropin (PMSG; Aska endogenous histone H3 variants, 40 oocytes/embryos from three- Pharmaceutical, Tokyo, Japan). week-old ddY mice (Japan SLC) were placed in 400 μl ISOGEN (Nippon Gene), and RNA was extracted. The contaminated DNA In vitro fertilization and embryo culture was digested with DNase I (Promega, Madison, WI, U.S.A.) for 40 To obtain mature metaphase II (MII) oocytes, three- to five-week- min at 37 C, and then RNA was purified with phenol/chloroform or old mice were superovulated by injection of 5 IU of human chorionic ISOGEN. Primers were based on the common sequences of several gonadotropin (hCG; Aska Pharmaceutical) 48 h after injection of genes encoding H3.1 as well as to the individual sequences of H3.1 PMSG. The oocytes were collected in human tubal fluid (HTF) genes (Table 1). The primers of common sequences in each of H3.2 medium [19] from the ampulla of the oviduct 16 h after injection and H3.3 were also prepared. Two-step PCR reactions were carried of hCG. Sperm was collected from ICR mice (Japan SLC). In vitro out with the following conditions using a Thermal Cycler Dice Real fertilization was carried out in the HTF medium by fertilizing the Time System (TaKaRa): 95 C for 5 sec (denaturation) and 60 C for MII stage oocytes with the sperm, which had been preincubated 30 sec (annealing/elongation). After PCR, the samples were subjected for 2 h, with a final concentration of 200,000–1,000,000 sperm / to agarose gel electrophoresis to confirm that only a band with the ml. The oocytes were washed in the KSOM medium [20] 6 h after expected size was observed. insemination, and the fertilized oocytes with 2 pronuclei were selected. Embryos at various developmental stages were collected Results according to the following time schedule after fertilization: 1-cell embryos at 10 h, 2-cell embryos at 28–30 h, 4-cell embryos at 45 Flag-H3.1 is absent from chromatin before fertilization h, morulae at 72 h, and blastocysts at 96 h. We have previously shown that when Flag-tagged H3.1 mRNA was microinjected, Flag-H3.1 was not incorporated into the Immunocytochemistry nuclei before fertilization or during early preimplantation development, The oocytes and embryos were fixed in 3.7% paraformaldehyde although the protein was efficiently translated [10]. These results (Wako Pure Chemical Industries, Osaka, Japan) with 0.2% Triton showed that the de novo incorporation of endogenous H3.1 into X-100 (Sigma-Aldrich) for 40 min at room temperature and washed chromatin does not occur during these periods, but it is still unknown with PBS containing 1% BSA (1% BSA/PBS) three times. The cells whether or not H3.1 is present in the chromatin because preexisting were then incubated with anti-Flag mouse IgG primary antibody H3.1 that had been incorporated into chromatin before fertilization (Sigma-Aldrich) diluted 1:100 with 1% BSA/PBS containing 0.2% may remain there after fertilization and during early preimplantation H3.1 IN OOCYTES AND EMBRYOS 559

Table 1. Primers used for detection of endogenous H3.1 genes Target gene Ref Seq accession no. Sequences A. Common PCR primers Forward 5’-TGCAGGAGGCCTGTGA-3’ H3.1 Reverse 5’-TGGATGTCCTTGGGCATG-3’ Forward 5’-TGCAGGAGGCGAGCGA-3’ H3.2 Reverse 5’-TGGATGTCCTTGGGCATG-3’ Forward 5’-ATCCACCGGTGGTAAAGC-3’ H3.3 Reverse 5’-CTTCACCCCTCCAGTAGAGG-3’ B. Individual PCR primers Forward 5’-GTGCTCACCAGCTTGCTTTC-3’ Hist1h3a NM_013550.4 Reverse 5’-GTAGCGGTGAGGCTTCTTCAC-3’ Forward 5’-CACTTTTCCCTACGGTTACTTGC-3’ Hist1h3g NM_145073.2 Reverse 5’-CTTGGTGGCCAGCTGCTTG-3’ Forward 5’-GGAAGCTCGGGTGTTACC-3’ Hist1h3h NM_178206.2 Reverse 5’-CTTGGTGGCCAGCTGCTTG-3’ Forward 5’-CTTTATTCACCTTTCTAGTGTACTGAG-3’ Hist1h3i NM_178207.2 Reverse 5’-CTTGGTGGCCAGCTGCTTG-3’

Fig. 1. Nuclear deposition of Flag-H3.1 in the oocytes and preimplantation embryos of Flag-tagged H3.1 transgenic mice. (A) Flag-H3.1 was detected by immunocytochemistry with ant-Flag antibody in growing (GO) and full-grown oocytes (FGO) and preimplantation embryos. Scale bar= 10 μm. (B) Expression of Flag-H3.1 mRNA was examined by RT-PCR in growing (GO) and full-grown oocytes (FGO), 1-cell embryos (1-C) and blastocysts (Bl) obtained from transgenic mice and FGO from wild-type mice (Control). Rabbit α-globin mRNA was added into each sample to control the RNA extraction and reverse transcription during the process of RT-PCR assay.

development. Therefore, we generated Flag-tagged H3.1 transgenic of mRNA microinjection [10], Flag-H3.1 was not detected until mice that ubiquitously express Flag-H3.1 in all tissues to analyze the morula stage after fertilization, although Flag-H3.1 mRNA was whether Flag-H3.1 is present during oogenesis and preimplantation expressed in all examined stages from the growing stage of oocytes development. In these mice, Flag-H3.1 was absent in the nuclei of to the blastocyst stage (Fig 1B). These results strongly suggest that both growing and full-grown oocytes (Fig. 1A). Similar to the results H3.1 is not incorporated into chromatin due to the absence and/ 560 KAWAMURA et al. or inactivity of a histone chaperone before fertilization and early preimplantation development.

The expression level of endogenous H3.1 is low at the stages where Flag-H3.1 is not present in chromatin Flag-H3.1 was absent from the nucleus before the morula stage (Fig. 1A). In order to investigate the expression of endogenous H3 variants in oocytes and preimplantation embryos, we performed real-time PCR and examined overall expression of H3.1, H3.2 and H3.3. Each of the H3 variants is encoded by several genes. First, we investigated the overall expression of H3.1, H3.2, and H3.3 by designing a common primer that recognizes all the genes for each variant (Fig. 2). The result showed that H3.1 transcript was expressed at low levels in oocytes and early preimplantation embryos, whereas its nuclear deposition was not observed in the transgenic mice expressing the Flag-H3.1. The expression increased from the morula stage, which is consistent with the increase in the nuclear deposition of Flag-H3.1. In contrast, H3.2 and H3.3 was expressed at high levels in full-grown oocytes and 1-cell stage embryos. Next, we examined the expression of individual H3.1 genes. H3.1 is coded by 4 genes, Hist1h3a (NM_013550.4), Hist1h3g (NM_145073.2), Hist1h3h (NM_178206.2), and Hist1h3i (NM_178207.2), included Fig. 2. Overall expression of endogenous H3 variant transcripts in oocytes and preimplantation embryos. The expression of H3 variants was in the same histone cluster on 13 [21, 22]. However, its analyzed by real-time PCR in full-grown oocytes (FGO) and individual expression in mammalian preimplantation embryos has embryos at various preimplantation stages. Since each variant has not been reported. Here, we investigated individual gene expression several genes, a set of common primers that recognizes all the using primers that specifically recognize each gene. Similar expression genes for each variant was used. Four independent experiments were performed, and the averaged values are shown relative to patterns were observed for all 4 genes, with the expression level being that of FGO. Bars represent the SEM. low in full-grown oocytes to 4-cell stage embryos and increasing from the morula stage (Fig. 3), suggesting that all H3.1 genes are regulated by a common mechanism. Taken together, the nuclear incorporation of H3.1 may be limited due to its low expression level detected at this stage in the transgenic embryos. Next, according along with the absence of the histone chaperone activity. to a previous study, the endogenous expression of H3.1 was not detected in preimplantation mouse embryos [23]. Although there is Discussion no description about which genes were examined in that study, we could detect all of the genes encoding H3.1 (Fig. 3). It is likely that In this study, we found that Flag-H3.1 is absent from chromatin the expression was not detected because a small amount of embryo in early preimplantation embryos (Fig. 1A), which is consistent cDNA was used for analysis in the previous study, i.e., 0.05 embryos with our previous finding that Flag-H3.1 was not incorporated into per tube in the PCR assay [23], whereas we used 4 embryos per tube. the nucleus in these embryos [10]. Furthermore, our results showed After fertilization, Flag-H3.1 was detected from the 4-cell stage, that Flag-H3.1 was also absent in the chromatin of differentiated and thus may be essential for heterochromatin formation. It is oocytes, both growing and full-grown oocytes. To our knowledge, known that heterochromatin formation changes dynamically during this is the first study to analyze the dynamics of Flag-H3.1 during preimplantation development [24]. Before the 4-cell stage, hetero- oogenesis. We have also shown that the expression level of H3.1 chromatin is detected only in the peripheral region of the nucleolus, was low at the stages in which the presence of Flag-H3.1 was not and thus there are few chromocenters [24, 25]. Heterochromatin detected in chromatin (Fig. 2). Consequently, these results suggest formation progressively takes place from the 4-cell to the blastocyst that the presence of H3.1 in chromatin is regulated by its expression stage. This pattern of heterochromatin formation is similar to that of level along with the activity of a histone chaperone(s). Flag-H3.1 nuclear deposition, suggesting that H3.1 is involved in Our results may be regarded as both consistent and inconsistent heterochromatin formation. Indeed, it has been suggested that H3.1 with previous reports for several reasons. First, we previously is associated with constitutive heterochromatin [6]. Moreover, in the analyzed the dynamics of Flag-H3.1 by microinjection of Flag H3.1 embryos expressing mutated CAF-1, a histone chaperone for H3.1, mRNA into embryos and found that Flag-H3.1 was incorporated heterochromatin formation was delayed during preimplantation from the 4-cell stage [10]. However, the analysis using transgenic development [24], supporting our hypothesis that H3.1 is responsible mice revealed that Flag-H3.1 is present in chromatin from the morula for the formation of heterochromatin in the late preimplantation stage. stage (Fig. 1A). Since nuclear incorporation of H3.1 would have just We have previously shown by microinjecting Flag-H3.1 mRNA started at the 4-cell stage, a significant amount of H3.1 would not into embryos that the Flag-H3.1 protein was not incorporated into yet be accumulated in the nucleus. Therefore, Flag-H3.1 was not the nucleus after fertilization when genome reprogramming occurs H3.1 IN OOCYTES AND EMBRYOS 561

Fig. 3. Individual expression patterns of endogenous H3.1 genes. The expression of H3.1 genes was analyzed by real- time PCR using specific primers for individual genes in full-grown oocytes (FGO) and the embryos at various preimplantation stages. Three independent experiments were performed, and averaged values are shown relative to that of FGO. Bars represent the SEM.

to transform the differentiated oocytes into totipotent embryos, and development, embryos gradually lose their totipotency and finally we suggested that the absence of H3.1 is essential for the genome start to differentiate at the morula/blastocyst stage [1, 27]. The reprogramming to establish totipotency [10]. However, as described incorporation of H3.1 occurs coincidentally with this process, in the Results section, those experiments could not exclude the pos- suggesting that H3.1 is associated with differentiation. Similarly, sibility that the H3.1 that had been incorporated into the chromatin the cell lines derived from differentiated tissues are rich in H3.1, during oogenesis still remained there after fertilization. In the whereas embryo-derived cell lines have high levels of H3.3 [7]. present study, we could ensure the actual deposition of the histone Therefore, the lack of H3.1 would be a preparatory state for oocytes variant into chromatin by analyzing the transgenic mice ubiquitously to acquire totipotency soon after fertilization. expressing Flag-H3.1 and obtained results that strongly support We found that the expression levels differ among H3 variants in that H3.1 is absent from the chromatin after fertilization. We also oocytes and preimplantation embryos. Notably, H3.1 is expressed at previously examined the dynamics of histone variant replacement to low level in oocytes and increases from the morula stage, whereas investigate their involvement in genome remodeling in the somatic H3.2 and H3.3 are highly expressed in oocytes (Fig. 2). Although nucleus transplanted into the enucleated oocyte [26]. Soon after the genes that code for H3.3 are separately located in the genome, those transplanted oocytes were activated, all three H3 variants derived that code for H3.1 and H3.2 are located as histone clusters. In the from the somatic cell were removed from the chromatin, and then case of all four genes that code for H3.1, all are localized in the major oocyte-derived H3 variants were incorporated. At this time, H3.1 histone cluster HIST1 on Chromosome 13 [21, 22]. More than half was incorporated as well as other H3 variants, although H3.1 was not of the genes encoding H3.2 are also localized in the same cluster usually incorporated in the 1-cell stage embryos. This incorporation on Chromosome 13. Since the expressions of these H3.1 and H3.2 may be due to the presence of a somatic cell-derived histone chaperone genes thus seem to be regulated in the identical mechanism, the or a factor involved in the nuclear import of H3.1. Moreover, this difference in the expression patterns between H3.1 and H3.2 may anomalous incorporation of H3.1 would be responsible for the be due to a difference in stability of mRNA. The stability of histone failure in genome reprogramming resulting in the low success rate mRNA is regulated by the stem-loop binding protein (SLBP) [28, of somatic nuclear cloning. 29]. In addition, factors associated with histone mRNA processing The lack of H3.1 in differentiated oocytes may facilitate the such as the 100 kDa zinc finger protein (ZFP100) may influence the acquisition of totipotency after fertilization. During preimplantation stability of mRNA [29, 30]. 562 KAWAMURA et al.

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