Scriptaid – IC-87114 Inhibitor

Scriptaid improves the reprogramming of donor cells and enhances canine- porcine interspecies embryo development

Jin-Gu Noa,b,1, Tai-Young Hura,1, Minghui Zhaoa, Seunghoon Leea, Mi-Kyung Choia,
Yoon-Seok Nama, Dong-Hyun Yeoma, Gi-Sun Ima, Dong-Hoon Kima,⁎
a Department of Animal Biotechnology, National Institute of Animal Science, Wanju 55365, Republic of Korea
b Department of Biological Science, University of Sungkyunkwan, Suwon 16419, Republic of Korea

Abstract

Histone methylation, histone acetylation, and DNA methylation are the important factors for somatic cell nuclear transfer (SCNT). Histone deacetylase inhibitors (HDACi) and DNA methyltransferase inhibitors (DNMTi) have been used to improve cloning eﬃciency. In particular, scriptaid, an HDACi, has been shown to improve SCNT eﬃciency. However, no studies have been performed on canines. Here, we evaluated the eﬀects of scriptaid on histone modiﬁcation in canine ear ﬁbroblasts (cEFs) and cloned canine embryos derived from cEFs. The early development of cloned canine-porcine interspecies SCNT (iSCNT) embryos was also examined. cEFs were treated with scriptaid (0, 100, 250, 500, 750, and 1000 nM) in a medium for 24 h. Scriptaid treatment (all con- centrations) did not signiﬁcantly aﬀect cell apoptosis.

Treatment with 500 nM scriptaid caused a signiﬁcant increase in the acetylation of H3K9, H3K14, and H4K5. cEFs treated with 500 nM scriptaid showed signiﬁcantly decreased Gcn5, Hat1, Hdac6, and Bcl2 and increased Oct4 and Sox2 expression levels. After SCNT with canine oocytes, H3K14 acetylation was signiﬁcantly increased in the one- and two-cell cloned embryos from scriptaid- treated cEFs. In iSCNT, the percentage of embryos in the 16-cell stage was signiﬁcantly higher in the scriptaid- treated group (21.6 ± 2.44%) than in the control (7.5 ± 2.09%). The expression levels of Oct4, Sox2, and Bcl2 were signiﬁcantly increased in 16-cell iSCNT embryos, whereas that of Hdac6 was decreased. These results demonstrated that scriptaid aﬀected the reprogramming of canine donor and cloned embryos, as well as early embryo development in canine-porcine iSCNT, by regulating reprogramming and apoptotic genes.

1. Introduction

Somatic cell nuclear transfer (SCNT) has been developed as useful methyltransferase inhibitors (DNMTis) and histone deacetylase in- hibitors (HDACis). For example, 5-aza-2′-deoXycytidine (5-aza-dC) has been used as a DNMTi, and valproic acid, trichostatin A (TSA), oXam-
various animals have been cloned using SCNT [2]. Despite this success, the eﬃciency of this technique is rather low.

Epigenetic modiﬁcations are known to be critical for donor cell reprogramming. In fact, epigenetic abnormalities such as histone hypo- acetylation and DNA hyper-methylation may be responsible for the low cloning eﬃciency, and several approaches have been tried to rescue these epigenetic abnormalities. Although the mechanisms are not fully understood, several recent studies have shown that treatment with
various cell extracts [3–5] have positive eﬀects on epigenetic mod- iﬁcations, resulting in improved embryonic development and/or nu-
clear reprogramming. In addition, researchers have developed in- hibitors for enzymes that mediate epigenetic changes, such as DNA eroylanilide hydroXamic acid (SAHA), and scriptaid have been used as HDACis. In cows and mice, treatment of donor cells with 5-aza-dC has been shown to decrease the DNA methylation status and increase the blastocyst rate [6,7]. Diao et al. [8] had reported that treatment with RG108, a DNMTi, improved the blastocyst formation rates in pigs. Additionally, HDACis have been widely used to increase embryonic development for promoting SCNT cloning eﬃciency. Miyoshi et al. [9] had reported that valproic acid enhanced in vitro embryo development and Oct3/4 expression in miniature pig SCNT embryos. TSA also in- duced in vitro development of pig SCNT embryos; in a mouse study, TSA improved acetylation on histone 3 residues 9 and 14 and histone 4 residue 8 (H3K9, H3K14 and H4K8) [10,11]. Furthermore, SAHA and oXamﬂatin signiﬁcantly reduced apoptosis rates in mouse blastocysts, improved the full-term development of cloned mice, and increased the production of ntES cell lines [12]. Other HDACi reagents such as CBHA [13] and BiX-01294 [14] have also been reported to positively aﬀect epigenetic features, in vitro development of SCNT embryos, and cloning eﬃciency. Although the eﬀects of scriptaid treatment on donor nuclear reprogramming and in vitro development of embryo in some animals have been reported [15,16], its eﬀects on canine models have not been described.

In the present study, we investigated whether scriptaid treatment of canine ear ﬁbroblasts (cEFs) could improve donor cell reprogramming and alter histone acetylation in canine SCNT embryos. We also ex- amined in vitro embryonic development of iSCNT embryos derived from scriptaid-treated and non-treated cEFs with porcine oocytes as recipient cytoplasts.

2. Materials and methods

2.1. Animals

All experiments carried out in accordance with the Guidelines for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee of the National Institute of Animal Science (NIAS), Korea (Approval No: NIAS-2015-145). All dogs were bred in the laboratory animal unit in the special canine re- search center of NIAS.

2.2. Cells and scriptaid treatment

The cEFs were derived from the ear tissue of a 5-year-old male German shepherd. The cells were cultured in an advanced Dulbecco’s modiﬁed Eagle’s medium (ADMEM; Thermo Fisher Scientiﬁc, Waltham,MA, USA) with 1% GlutaMAX™ (Thermo), 10% fetal bovine serum (FBS; Thermo Fisher Scientiﬁc), and 1% antibiotic-antimycotic (Thermo) at 38.5 °C in 5% CO2. cEFs were used at passages 2–4.Scriptaid (Enzo Life Sciences, Inc, NY, USA) was added in the culture medium for 24 h at concentrations of 0, 100, 250, 500, 750, and
1000 nM.

2.3. Donor cell preparation

cEFs were treated with 500 nM of scriptaid in the culture medium for 24 h. Scriptaid-treated and control cEFs were then cultured to conﬂuency for synchronization in the G0/G1 phase. The cells were rinsed in phosphate-buﬀered saline (PBS), singularized by 0.05% trypsin-ethylenediaminetetraacetic acid (trypsin-EDTA; Thermo) treat- ment, and then incubated in ADMEM containing 0.5% FBS at 38.5 °C in 5% CO2 for 1 h, until cell injection.

2.4. Canine oocyte collection and SCNT

Mature canine oocytes were surgically recovered by ﬂushing the oviducts using a ﬂushing medium (HEPES-buﬀered TCM-199; 10% FBS, 2 mM NaHCO3, 0.5% bovine serum albumin [BSA], and 1% penicillin perivitelline space of the oocytes using an injection medium (HEPES- buﬀered TCM-199 supplemented with 10% FBS and 100 μg/mL phy- tohemagglutinin). The oocyte-donor cell couplets were transferred into a fusion medium (260 mM D-mannitol, 0.15 mM MgSO4, 0.5 mM HEPES, and 0.05% BSA). Fusion was performed by applying a direct current twice at 0.1-s intervals (DC pulse of 34 V for 15 μs). Fusion was conﬁrmed by microscopic observation after 30 min. The fused oocytes were activated by 4 min of incubation with mSOF supplemented with 10 μM Ca-ionophore and incubated for 4 h with mSOF supplemented with 1.9 mM 6-dimethylaminopurine. Four hours after activation, the canine cloned embryos were cultured in mSOF medium. For scriptaid treatment, reconstructed canine embryos were cultured in mSOF medium contained 500 nM scriptaid for 24 h, then the embryos were cultured in mSOF medium for another 7 days. Cleavage rate and blas- tocyst formation were checked at 38 h and 192 h after activation, re- spectively.

2.5. In vitro maturation of porcine oocytes

Porcine ovaries were obtained from a local slaughterhouse. Cumulus oocyte complexes (COCs) were aspirated from 3 to 6-mm antral follicles using an 18-gauge needle syringe. The COCs surrounded by compact cumulus cells were collected and washed until the medium was clear in a TALP-HEPES medium. The collected COCs were trans- ferred to a TCM-199 medium supplemented with 10% porcine follicular ﬂuid (pFF), 0.1% polyvinylalcohol, 3.05 mM d-glucose, 0.91 mM sodium pyruvate, 75 μg/mL penicillin G, 50 μg/mL streptomycin,0.57 mM cysteine, 0.5 μg/mL LH, 0.5 μg/mL FSH, and 10 ng/mL epi- dermal growth factor. The miXture was then incubated for 40–42 h at 38.5 °C in a 5% CO2 incubator. After in vitro maturation, denuded porcine MII oocytes were stained with 10 μg/mL Hoechst 33342. Enucleation was performed under an epiﬂuorescence microscope.

2.6. Interspecies nuclear transfer (iSCNT) and embryo culture

Donor cEFs treated with 500 nM of scriptaid and the untreated controls were transferred into the perivitelline space of the enucleated porcine oocytes. The couplets were transferred into a fusion medium (0.1 mM MgCl2·6H2O, 1.0 mM CaCl2·2H2O, 0.3 M d-mannitol, and
0.5 mM HEPES) and fused by applying two pulses of direct current (140 V/mm and 50 μs). The fused embryos were activated with 5 μg/ mL cycloheximide (CHX) and 5 μg/mL cytochalasin D (CD) for 4 h at 38.5 °C. Activated embryos were then cultured in a PZM-3 medium
(10 mM KCl, 108 mM NaCl, 0.4 mM MgSO4·7H2O, 0.35 mM KH2PO4, 0.2 mM sodium pyruvate, 25.07 mM NaHCO3, 1 mM L-glutamine, 2 mM Ca-(lactate)2·5H2O, 20 mL/L BME-essential amino acids, 5 mM hypotaurine, 0.05 mg/mL gentamycin, 10 mL/L MEM nonessential amino acids, and 40 mg/mL fatty acid-free BSA) and incubated in 5% CO2 and 5% O2 at 38.5 °C for 7 days. Embryos developed to the 2-, 4-, 8-, and 16-cell stages were observed at 24, 48, 72, and 96 h after fusion, respectively.

2.7. Immunostaining

Scriptaid-treated cells were washed thrice, ﬁXed in 4% expected to be the day when the progesterone concentration reached 10–15 ng/mL. To obtain in vivo matured oocytes, we collected the oo- cytes by aseptic laparotomy, as previously described [17]. Cumulus cells were removed from oocytes by repeated pipetting in a holding medium (HEPES-buﬀered TCM-199 supplemented with 10% FBS). Oocytes in the metaphase II stage were stained with 10 μg/mL Hoechst 33342 (Thermo) for 5 min at 38.5 °C. The stained oocytes were transferred into droplets of HEPES-buﬀered TCM-199 supplemented with 10% FBS and 5 μg/mL cytochalasin B (Sigma, St. Louis, MO, USA), and enucleation was performed under an epiﬂuorescence microscope. Scriptaid-treated and untreated donor cells were transferred into the ormaldehyde for 40 min at room temperature, and washed thrice again. Permeablilization was carried out with a permeabilization solution (0.2% Triton X-100) for 20 min at room temperature. After washing thrice in PBS, the cells were blocked in PBS containing 4% BSA for 30 min at room temperature. The primary antibodies, which included anti-acetyl H3K9 (Abcam, Cambridge, MA, USA), anti-acetyl H3K14 (Abcam), and anti-acetyl H4K5 (Abcam), were diluted at 1: 100 ratio in PBS containing 4% BSA. Samples were treated with these primary an- tibodies, incubated for 2 h at room temperature, and rinsed with PBS. Secondary goat anti-rabbit IgG Alexa Fluor 488 (Thermo) was diluted at 1: 100 ratio in the blocking solution and used as the secondary antibody. After incubation with the secondary antibody for 20 min at room temperature, the samples were mounted and counterstained using the Vecta-shield Mounting Solution containing 4′6-diamidino-2-phe- nylindole (Vector Laboratory,
Burlingame, CA, USA). Constructed em- bryo staining was performed using the same methods, but with PBS containing 0.3%
polyvinylpyrrolidone (PBS/PVP) as the washing of iSCNT embryos were performed using the Fastlane cell cDNA kit (Qiagen). Each experiment was performed on two iSCNT embryos in the 16-cell stage. The mRNA expression was quantiﬁed using the RG SYBR green PCR miX (Qiagen), and ampliﬁcation was performed using the Rotor Gene Cycler 6000 device (Corbett, San Francisco, CA, USA). PCR conditions and primer sequences are described in Table 1. The relative expression levels were calculated using the delta–delta CT method.Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control.

2.10. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay

Scriptaid-treated cEFs were washed thrice in PBS and ﬁXed with 4% paraformaldehyde for 40 min at room temperature. The cells were again washed thrice, permeabilized with a permeabilization solution (0.2% Triton X-100) for 40 min at room temperature, and then washed again with a washing buﬀer. They were then incubated for 1 h at 38.5 °C in the dark with an In Situ Cell Death Detection Kit, Fluorescein (Roche Diagnostics Corp; Indianapolis, IN, USA). Finally, the cEFs were washed thrice with PBS, and then mounted and nuclear stained using Vecta-shield containing DAPI.

2.11. Statistical analysis

Diﬀerences between control cells and treated cells were measured by two-way Student’s t-tests. The embryo development data were analyzed using χ2 tests. Diﬀerences with p value < 0.05 were Because high concentrations of most HDACis have induced apop- tosis in cells, we evaluate the toXicity of scriptaid in cEFs. The cEFs were treated with various concentrations (0–1000 nM) of scriptaid for 24 h, and apoptosis was evaluated using the TUNEL assay (Fig. 1). Our results showed no signiﬁcant diﬀerences in apoptosis rates between the scriptaid-treated and control groups, even with high concentrations of scriptaid (1000 nM). Fig. 1. Eﬀect of scriptaid treatment on apoptosis of canine ear ﬁbroblasts (cEFs). cEFs were treated with diﬀerent concentrations of scriptaid (0, 100, 250, 500, 750, and 1000 nM) for 24 h in a culture medium. Terminal deoXynucleotidyl transferase dUTP nick end labeling (TUNEL) assay was used to determine donor cell apoptosis. All experiments were replicated more than thrice. Fig. 2. Eﬀects of scriptaid treatment on histone acetylation levels in canine ear ﬁbroblasts (cEFs). cEFs were treated with diﬀerent concentrations of scriptaid (0, 100, 250, 500, 750, and 1000 nM) for 24 h in a culture medium. (A) Fold changes of H3K9 acetylation levels at indicated scriptaid concentrations. (B) Fold changes of H3K14 acetylation levels at indicated scriptaid concentrations. (C) Fold changes of H4K5 acetylation levels at indicated scriptaid concentrations. Signal intensities were calculated using the Image J software. DAPI signal was used as the internal control. * and ** indicate p < 0.05 and p < 0.01, respectively. All experiments were replicated more than thrice. 3.2. Eﬀect of scriptaid on H3K9, H3K14, and H4K5 acetylation in cEFs Using immunocytochemistry, we evaluated H3K9, H3K14, and H4K5 acetylation in scriptaid-treated cells. We found that H3K9 and H3K14 acetylation levels were signiﬁcantly increased (p < 0.01) in cells treated with 500 nM scriptaid (Fig. 2A and B), compared to control (untreated) cells. H4K5 acetylation was also increased signiﬁcantly (p < 0.05) in cells treated with scriptaid, compared with the control cells (Fig. 2C). Therefore, we chose 500 nM scriptaid for all subsequent experiments. To verify these results, we performed western blotting, which conﬁrmed the signiﬁcant increase in H3K9, H3K14, and H4K5 acetylation in cEFs treated with 500 nM scriptaid (Fig. 3A, B, and C). 3.3. Eﬀect of scriptaid on expression of reprogramming- and apoptosis- related genes in cEFs Next, we tested the expression levels of genes encoding histone acetyltransferases, histone deacetylases, apoptosis-related proteins, and pluripotency-related proteins using RT-PCR. As shown in Fig. 4, the expression levels of both Gcn5 and Hat1 were signiﬁcantly decreased (p < 0.05) in scriptaid-treated cells, compared with control cells (Fig. 4A). The expression level of the histone deacetylase gene, Hdac6, was also signiﬁcantly decreased (p < 0.05) following scriptaid treat- ment (Fig. 4A). In contrast, scriptaid did not aﬀect the expression of Hdac1 in cEFs (Fig. 4A). Although there was no diﬀerence in apoptosis measured by the TUNEL assay, Bcl2 expression was signiﬁcantly decreased (p < 0.05) in scriptaid-treated cells. However, the expres- sion of Bax did not signiﬁcantly diﬀer between the groups (Fig. 4B). The Oct4 and Sox2 expression levels were signiﬁcantly increased (p < 0.05) in scriptaid-treated cells (Fig. 4C). However, Nanog ex- pression was not detected in either group of cells. 3.4. Eﬀect of scriptaid on H3K14 acetylation in canine cloned embryos To examine the changes in histone acetylation in canine cloned embryos derived from scriptaid-treated cEFs, H3K14 acetylation in canine cloned embryos at 4 h (1-cell stage) and 24 h (2-cell stage) after fusion was analyzed by immunostaining, and signal intensities were calculated. Intensities of the DNA signals were set as internal controls. As shown in Fig. 5, signiﬁcantly higher (p < 0.05) acetylation of H3K14 was detected in canine cloned embryos derived from scriptaid- treated cells in both 1-cell and 2-cell stages, compared to the control cells. 3.5. Eﬀect of scriptaid on early embryo development in iSCNT and SCNT embryos It was diﬃcult to obtain a suﬃcient number of in vivo matured canine oocytes; even among the ones obtained, only a few had the high quality required for SCNT experiments. In order to overcome the pro- blem of limited oocytes, iSCNT is can be used as an alternative. In this study, we used in vitro matured porcine oocytes collected from porcine ovaries. The in vitro maturation rate of the porcine oocytes was 81.8%. The embryonic developmental potential of cells treated with 500 nM scriptaid was then examined using iSCNT embryos. As shown in Table 2, no diﬀerences were found between the two groups in terms of cleavage rates (2-cell stage) and development (4–8-cell stage). However, the percentage of cells that developed to the 16-cell stage was signiﬁcantly higher in the scriptaid-treated group (21.6 ± 2.44%) than the control group (7.5 ± 2.09%). There was no embryo developed to higher stage in iSCNT embryos. In SCNT embryos, scriptaid sig- niﬁcantly enhanced total cell number of 8 days embryos (32.14 ± 26.28 vs. 14.57 ± 11.79, p = 0.043. Fig. 6). Fig. 3. Eﬀects of scriptaid treatment on histone acetylation levels in canine ear ﬁbroblasts (cEFs). cEFs were treated with scriptaid (500 nM) or left untreated (control) for 24 h in a culture medium. (A) Western blot and fold changes of H3K9 acetylation at indicated scriptaid concentrations. (B) Western blot and fold changes of H3K14 acetylation at indicated scriptaid concentrations. (C) Western blot and fold changes of H4K5 acetylation at indicated scriptaid concentrations. Band intensities were calculated using the Image J software. β-actin intensities were used as internal controls. * and ** indicate p < 0.05 and p < 0.01, respectively. All experiments were replicated more than thrice. Fig. 4. Eﬀects of scriptaid treatment on histone modiﬁcation, apoptosis, and pluripotency-related gene expression in canine ear ﬁbroblasts (cEFs). cEFs were treated with scriptaid (500 nM) or left untreated (control) for 24 h in a culture medium. (A) Fold changes of histone modiﬁcation-related gene expression. (B) Fold changes of apoptosis-related gene expression.(C) Fold changes of pluripotency-related gene expression. * and ** indicate p < 0.05 and p < 0.01, respectively. Delta-delta CT methods were used for data analysis. GAPDH was used as the internal control. Values shown are the means ( ± S.D.) from more than 3 individual experiments for each group. 3.6. Eﬀect of scriptaid on expression of reprogramming- and apoptosis- related genes in iSCNT embryos To estimate the gene expression patterns in iSCNT embryos, we tested the mRNA levels of pluripotency-related genes (Oct4, Sox2, and Nanog), acetylation-modifying enzymes (Gcn5, Hat1, Hdac1, and Hdac6), and apoptosis-related genes (Bax and Bcl2) in iSCNT embryos at the 16-cell stage. Among the pluripotency-related genes, the ex- pression levels of Oct4 and Sox2 were signiﬁcantly increased in the scriptaid-treated group (iSCNT embryos derived from cEFs treated with 500 nM scriptaid), compared with the controls. Among the acetylation- modifying enzyme genes, the expression level of the histone deacetylase gene, Hdac6, was signiﬁcantly decreased in the scriptaid-treated group; however, the other genes (Gcn5, Hat1, and Hdac1) showed no sig- niﬁcant diﬀerences between the two groups. Among the apoptosis-re- lated genes, Bcl2, an anti-apoptotic gene, showed signiﬁcantly higher expression in the scriptaid-treated group than the control group (Fig. 7). 4. Discussion The SCNT technique has been used for several years now; however, the cloning eﬃciency is still low, likely due to abnormal epigenetic modiﬁcations [18–20]. In recent years, many methods have been pro- posed to improve donor cell nuclear reprogramming and in vitro development of SCNT embryos in various species. Various reagents have been used for improving the cloning eﬃciency. In this study, we investigated whether scriptaid treatment could improve the repro- gramming of cEFs and the in vitro development of iSCNT embryos from cEFs. Fig. 5. H3K14 acetylation in cloned canine embryos. Cloned canine embryos were derived from donor cells treated with scriptaid (500 nM) or left untreated (control). (A) Immunostaining of H3K14 acetylation at 4 h after fusion and (B) fold changes in canine SCNT embryos. (C) Immunostaining of H3K14 acetylation at 24 h after fusion and (D) fold changes in canine SCNT embryos. Signal intensities were calculated using the Image J software. DAPI signal was used as the internal control. * indicates p < 0.05. All experiments were replicated more than thrice. Scale bar = 25 μm. Fig. 6. Eﬀect of scriptaid on canine SCNT embryo development in vitro. (A) Cleavage of canine SCNT embryos in control (Ctrl) and scriptaid (SCR) treatment group. (B) The embryos were stained with 10 μg/mL DAPI at 192 h after activation and (C) total cell number of embryos was statistic. Values represent the mean ( ± SEM) of three separate experiments, * indicate p < 0.05. Scale bar = 100 μm. Scriptaid is a commonly used, non-toXic HDACi that induces tran- scriptional activity and gene expression [21]. Recent studies have shown that scriptaid can improve the cloning eﬃciency and regulate epigenetic modiﬁcations. The eﬀects of scriptaid vary among species. Zhao et al. had reported that 500 nM of scriptaid enhanced the cloning eﬃciency of NIH miniature pigs by improving histone acetylation [16]. Additionally, scriptaid treatment has been shown to be eﬀective at 5–500 nM in cows [22,23] and at 250 nM in rabbits [24]. Therefore, in this study, we evaluated the eﬀects of scriptaid at 0–1000 nM in cEFs, and found no diﬀerences in the apoptosis rates in scriptaid-treated and control groups, conﬁrming that scriptaid was a non-toXic reagent, consistent with prior reports [21]. Epigenetic modiﬁcations are important for chromatin assembly and activation and transcriptional silencing [25]. In particular, acetylation of the lysine residues on histones 3 and 4 is strongly related to chro- matin unfolding and transcription factor recruitment, resulting in the transcriptional activation of genes. Wang et al. had reported that acetylation of H3K9 and H3K14 was lower in SCNT embryos than natural embryos, and that this eﬀect could be rescued by treatment with an HDACi [11]. Moreover, hyperacetylation of H4K5 is related to gene activation, and re-acetylation of H4K5 increases embryonic develop- ment in mouse and rabbit SCNT embryos [13]. These ﬁndings indicated that the acetylation of H3K9, H3K14, and H4K5 could aﬀect SCNT embryo development, and that epigenetic reprogramming could be improved by treatment with an HDACi. In this study, we treated cEFs with scriptaid for donor cell reprogramming. The results of im- munostaining and western blotting showed that the acetylation of H3K9, H3K14, and H4K5 increased after scriptaid treatment. We also found that acetylation was signiﬁcantly increased after treatment with 500 nM scriptaid, indicating that scriptaid may have potential appli- cations in donor cell reprogramming in canine embryos, similar to other species. Fig. 7. Relative mRNA expression in canine-porcine iSCNT embryos at the 16-cell stage. Cloned 16-cell stage iSCNT embryos were derived from cEFs treated with scriptaid (500 nM) or left untreated for 96 h of in vitro culture. Fold changes of pluripotency-related genes (Oct4, Sox2, and Nanog), acetylation-modifying enzymes (Gcn5, Hat1, Hdac1, and Hdac6), and apoptosis-related genes (Bax and Bcl2). * and ** indicate p < 0.05 and p < 0.01, respectively. Delta-delta CT methods were used for data ana- lysis. GAPDH was used as the internal control. Values represent the mean ( ± S.D.) from ﬁve embryos from each group. Recent studies have shown that scriptaid aﬀects cell cycle arrest, apoptosis [26,27], nascent mRNA production [15], ribosomal RNA activation [28], and telomerase activity [29], thereby modulating gene expression. Therefore, to estimate the eﬀects of scriptaid on donor cell reprogramming, we determined the expression of acetyltransferase, deacetylase, apoptosis-related, and pluripotency-related genes. GCN5 has been reported to possess acetyltransferase activity at lysines 9, 14, and 18 of histone H3, and to modulate gene expression [30]. HAT1 also functions as an acetyltransferase, and acts mainly on histone H4, par- ticularly lysine residues 5 and 12 [31]. Conversely, both HDAC1 and HDAC6 act as histone deacetylases, functioning as transcriptional re- pressors in combination with cofactors [32]. Paul et al. had reported that global histone acetylation of H3 and H4 was enhanced following HDACi treatment [33]. In this study, we showed that the expression of Gcn5 and Hat1 was reduced in scriptaid-treated cEFs. Moreover, the expression of Hdac6 was also reduced by scriptaid treatment. Thus, both acetyltransferases and deacetylases were reduced following scriptaid treatment; these results indicated that alterations in H3K9, H3K14, and H4K5 acetylation may be regulated by a competition be- tween acetyltransferases and deacetylases, and that the downregulation of deacetylase activity may have been caused by a histone modiﬁcation induced by scriptaid treatment. Our analysis of Bax and Bcl2, which regulate apoptosis [34], showed that Bcl2 expression was reduced fol- lowing scriptaid treatment, although Bax expression was not altered. However, scriptaid treatment caused no changes in apoptosis rates, suggesting that apoptotic functions were not substantially modulated. Additionally, the treatment of cEFs with scriptaid signiﬁcantly en- hanced the expression of pluripotency genes such as Oct4 and Sox2, but did not alter the expression of Nanog. In this study, Oct4 and Sox2 were found to be expressed in both scriptaid-treated cEFs and control cEFs. These results showed that partially diﬀerentiated ﬁbroblasts share mesenchymal phenotypes with adult stem cells [35], indicating that the control cEFs used in our experiments were not fully diﬀerentiated, as they were derived from a primary cell culture. Oct4 and Sox2 are es- sential for the establishment and maintenance of pluripotency [36]. Pesce and Scholer had found that the expression levels of Oct4, Sox2, and Nanog were lower in SCNT embryos than IVF embryos; both Oct4 and Sox2 synergistically activated transcription by binding to each other [37]. A lack of inner cell mass (ICM) was also reported when Oct4 was reduced [38]. Thus, our data indicated that the enhanced expres- sion of pluripotency genes following scriptaid treatment might con- tribute to donor cell reprogramming and embryo development in canine-porcine iSCNT embryos. For the successful development of embryos, epigenetic modiﬁca- tions such as histone acetylation, histone methylation, and DNA me- thylation are essential [39]. In the present study, we showed that the acetylation of H3K9, H3K14, and H4K5 was induced by scriptaid treatment in donor cells. Our immunostaining data also showed that H3K14 acetylation signiﬁcantly increased during the 1-cell and 2-cell stages; thus, donor cell reprogramming following scriptaid treatment may aﬀect reprogramming in canine cloned embryos. A previous report had shown that H3K14 acetylation was essential for the expression of embryonic development-related genes [40]. Additionally, cloned mouse embryos that showed reduced H3K14 acetylation were rescued by treatment with the HDACi, TSA [11]. These results indicated that the induction of H3K14 acetylation might contribute to the normal devel- opment of cloned embryos, which is also consistent with our data. Generally, SCNT is performed with donor cells and oocytes from the same species. In order to estimate the embryonic development of SCNT in a canine model, both matured oocytes and donor cells should be prepared from canines. However, it is diﬃcult to obtain a suﬃcient number of oocytes, because of the limitations of in vivo matured oocytes and the low in vitro maturation rate [41,42], especially in the canine in vitro culture (IVC) system. iSCNT embryos have been produced for various species, including chickens, mice, cattle, and canines, using pig oocytes as recipients [43,44], and cloned iSCNT embryos have been successfully developed. Thus, iSCNT is an excellent alternative for studying canine embryonic development and reprogramming. In this study, we estimated the embryonic development in canine-porcine iSCNT embryos from scriptaid-treated and untreated cEFs as donor cells. Although, complete development was not achieved, our data showed that the number of embryos that reached the 16-cell stage was signiﬁcantly higher in the scriptaid-treated group than in the untreated group. Similar results were observed in canine-canine SCNT embryos which displayed a higher cell number after scriptaid treatment. For the normal development of cloned embryos, embryonic genome activation (EGA), which is essential for the maternal to embryonic transition, is considered an important factor [45]. Recent studies have shown con- ﬂicting results for the necessity of Oct4 in EGA. In mice, Oct4 was found to be not essential for EGA, as knockout of maternal Oct4 had no eﬀect on the oﬀspring [46]; however, in non-mammalian vertebrates, Oct4 has been shown to regulate EGA [47,48]. These reports suggest that the roles of Oct4 on EGA are diﬀerent between species, which may explain the controversy. Furthermore, there has been some evidence that Sox2 contributes to EGA [49]; the inhibition of Sox2 expression prevented post-EGA development [50]. Although we did not observe embryonic development after the 16-cell stage, our results showed that the upre- gulation of Oct4 and Sox2 by scriptaid treatment might enhance early embryo development. Some reports have shown that both Oct4 and Sox2 are essential for maintaining pluripotency through synergistic interactions [51,52], as well as for maintaining the fate of the ICM. HDAC6 is known to be a class IIb HDAC that regulates α-tubulin acetylation, and the acetylation of α-tubulin is important for microtubule stability and function [53]; Hou et al. had reported that the acetylation of α-tubulin could be increased by suppressing Hdac6 expression [54]. The Bcl2 family members are known to protect cells from apoptosis, which is programmed cell death [34]. In the present study, the ex- pression levels of Oct4, Sox2, and Bcl2 were found to be increased, while that of Hdac6 was found to be decreased, in iSCNT derived from scriptaid-treated cEFs. Although the mechanisms remain unclear, the results of our in vitro development and gene expression studies on iSCNT embryos demonstrated that scriptaid treatment could improve the in vitro development of iSCNT embryos by regulating the expression of histone modiﬁcation-, apoptosis-, and pluripotency-related genes. The Bcl2 displayed a disparity expression in cEF and iSCNT embryos after scriptaid treatment. Similar results have been reported on altera- tion of apoptosis related genes in donor cells and SCNT embryos fol- lowing other HDACi treatment [55,56]. These results agreed with the data in the present study. However, responses of Bcl2 to HDACi in diﬀerentiated cells are diﬀerent according to cell type. Previous reports demonstrated that HDACi activate both extrinsic and intrinsic apoptosis pathway and resulted in inducing cell apoptosis and reducing pro- liferation in sveral cancer cell line with use of other HDACi, such as sodium butyrate, TSA and MS-275 [57,58].The diﬀerent expression of Bcl2 in cEF and iSCNT embryos in the present study might result by diﬀerent cell type and diﬀerent cell culture medium. The environment of cell and nuclear might be the main reason for the disparity ex- pression,but further exploring is needed. A previous study had reported that the treatment of donor cells with SAHA increased H3K9 acetylation, but did not aﬀect the oﬀspring birth rate [59]. We showed that scriptaid treatment altered cellular repro- gramming and early embryonic development in canine models; addi- tional embryo transfer studies are needed to clarify the eﬀects of scriptaid treatment on the oﬀspring.In summary, we found that scriptaid treatment of cEFs induced the reprogramming of donor cells, as well as cloned canine embryos. We also observed enhanced early embryonic development in iSCNT embryos derived from scriptaid-treated cEFs. Scriptaid, which is an HDACi, causes these eﬀects by regulating the expression of repro- gramming- and apoptosis-related genes. In this study, we could not perform in vitro culture and embryo transfer of the cloned canine em- bryos due to a lack of canine IVC systems and the limited canine oo- cytes. The eﬀects of scriptaid on the cloning eﬃciency in canine models and the in vitro development of cloned embryos should be further in- vestigated after establishing a canine embryo IVC system. 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