Effects of histone methyltransferase inhibitor chaetocin on histone H3K9 methylation of cultured ovine somatic cells and development of preimplantation cloned embryos

Accepted Manuscript Title: Effects of Histone Methyltransferase Inhibitor Chaetocin on Histone H3K9 Methylation of Cultured Ovine Somatic Cells and Development of Preimplantation Cloned Embryos Authors: Yu-Mei Zhang, En-En Gao, Qian-Qian Wang, Hao Tian, Jian Hou PII: DOI: Reference:

S0890-6238(17)30600-7 https://doi.org/10.1016/j.reprotox.2018.06.006 RTX 7681

To appear in:

Reproductive Toxicology

Received date: Revised date: Accepted date:

2-9-2017 11-6-2018 13-6-2018

Please cite this article as: Zhang Y-Mei, Gao E-En, Wang Q-Qian, Tian H, Hou J, Effects of Histone Methyltransferase Inhibitor Chaetocin on Histone H3K9 Methylation of Cultured Ovine Somatic Cells and Development of Preimplantation Cloned Embryos, Reproductive Toxicology (2018), https://doi.org/10.1016/j.reprotox.2018.06.006 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Effects of Histone Methyltransferase Inhibitor Chaetocin on Histone H3K9

Methylation

of

Cultured

Ovine

Somatic

Cells

and

Development of Preimplantation Cloned Embryos

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Yu-Mei Zhang, En-En Gao, Qian-Qian Wang, Hao Tian, Jian Hou

State Key Laboratory of Agrobiotechnology and College of Biological Science, China

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Agricultural University, Beijing, China

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To whom correspondence should be addressed: Jian Hou, State Key Laboratory of

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Agrobiotechnology and College of Biological Science, China Agricultural University,

Chaetocin down-regulates H3K9 di- and trimethylation levels in cultured

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

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Highlights

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Beijing 100193, China. Telephone: 86-10-62733355-17; E-mail: [email protected]

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ovine cells.

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Chaetocin suppresses the expression of H3K9 methyltransferases

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Chaetocin-treated cells can be used as donor cells for somatic cell nuclear transfer.

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Chaetocin impairs the development of embryos

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Abstract

Aberrant hypermethylation of histone H3 lysine 9 (H3K9) is a key barrier to the

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development of cloned embryos by somatic cell nuclear transfer (SCNT). The objective of this study was to assess the effects of chaetocin, an inhibitor of H3K9

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methyltransferase SUV39 H, in regulating the H3K9 methylation in ovine SCNT embryos. Treatment of sheep fetal fibroblast cells with chaetocin specifically

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decreased the levels of H3K9 di-and trimethylation, and down-regulated the

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expression of H3K9 methyltransferases, SUV39H1/2 and G9A. Cloned embryos from

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chaetocin-treated cells could develop to the blastocyst stage at a similar rate to those

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derived from non-treated cells. However, direct treatment of SCNT or in vitro

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fertilized embryos with chaetocin impaired the embryonic development. These results suggest that although chaetocin is a potential agent for modulating H3K9 methylation

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in cells, it may have an adverse effect on the development of embryos.

Key words: somatic cell nuclear transfer, histone H3K9 methylation, chaetocin,

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SUV39H, G9A, sheep, embryo

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1. Introduction

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Although animal cloning by somatic cell nuclear transfer (SCNT) has been succeeded in many species, its efficiency is still low. Incomplete epigenetic

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reprogramming is thought to be one of the main factors affecting the development of

SCNT embryos [1]. Regulation of epigenetic modification with relevant agents may

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facilitate the epigenetic reprogramming and benefit the development of SCNT

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embryos. For example, several histone deacetylase inhibitors, such as trichostatin A

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(TSA) [2-5], valproic acid (VPA) [6] and Scriptaid [7, 8], have been shown to

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enhance in vitro and full-term development of SCNT embryos by regulating histone

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acetylation. DNA demethylation agents like 5-aza-2'-deoxycytidine (5-aza-dC) could also improve the developmental competence of cloned embryos by modifying DNA

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methylation of the donor genome [9-12]. Therefore, treatment of SCNT embryos or

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somatic cells with epigenetic modification agents could be an easy and convenient way to improve the cloning efficiency. In addition to histone acetylation and DNA methylation, histone methylation is

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also an important epigenetic mark. Abnormal hypermethylation of histone 3 lysine 9 (H3K9) has been observed in cloned bovine, ovine and porcine embryos [13-15]. Two small molecular chemicals, BIX-01294 and UNC0638, which specifically inhibit the activity of H3K9 methyltransferase G9A, have been used to reduce the H3K9 3

dimethylation (me2) levels in SCNT embryos in several species [14, 16-20]. Recent studies identified H3K9 trimethylation (me3) in donor cell genome as a major barrier to efficient reprogramming by SCNT in mice [21, 22]. Down-regulation of H3K9me3 levels through injecting mRNA of H3K9 lysine-specific demethylase (KDM) into

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SCNT embryos or silencing the expression of H3K9 methyltransferase SUV39H1/2 in donor cells, could greatly improve the development of mouse and human SCNT

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embryos [21-23]. However, no chemical inhibitors that are against the SUV39H1/2 methyltransferase have been tested in SCNT procedure so far.

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Chaetocin, a fungal mycotoxin originally isolated from Chaetomium minutum,

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was reported as the first of inhibitors of lysine-specific histone methyltransferases

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[24]. Chaetocin was found to be specific for inhibiting the activity of histone

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methyltransferase SUV39 family, such as SUV39H1 and G9a [24, 25]. SUV39H1

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catalyzes H3K9 di - to tri-methylation on pericentric heterochromatin, while G9A is responsible for mono- to dimethylation of H3K9 at euchromatic regions [26, 27].

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Thus, chaetocin may be a potential agent that can be used to modulate H3K9

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methylation in SCNT embryos. Chaetocin-mediated pharmacological inhibition of SUV39H1 has been shown to

be a promising therapeutic strategy for inhibiting the growth of human cancer cells

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[28-30]. However, its potential influences on other mammalian somatic cells or embryos are less known. In the present study, we evaluated the effects of chaetocin on H3K9 methylation in sheep somatic cells for SCNT and found that chaetocin could efficiently down-regulate the H3K9me2 and H3K9me3 levels. Chaetocin-treated cells 4

could be used for producing cloned embryos, but direct treatment of embryos with chaetocin impaired the development of embryos.

2. Materials and methods

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All chemicals and regents were purchased from Sigma-Aldrich Chemical

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Company (St. Louis, MO, USA) unless otherwise stated.

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2.1. Cell culture and chaetocin treatment

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Primary sheep fetal fibroblast (SFF) cells were derived from a 90-day-old sheep

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fetus. All procedure of animal experiments was in accordance with the animal care

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policies of China Agricultural University and was approved by the Animal Ethics

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Committee at the university. Fetus was recovered by laparotomy and a small skin piece was cut, rinsed and minced into pieces (1 mm3). Minced pieces were digested

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with 0.25% trypsin in phosphate-buffered saline (PBS) for 1 h and cells were

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collected by filtration and centrifugation. Cells were cultured in D-MEM/F-12 medium (Gibco, Invitrogen Corporation, Grand Island, NY, USA) supplemented with 10% (v/v) fetal bovine serum (FBS; HyClone, Logan, UT, USA), at 37℃ in a

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humidified atmosphere of 5% CO2. Cells at 10 to 15 passages were used as donor cells for nuclear transfer. Stock solutions of chaetocin were dissolved in DMSO at 10 mM and stored at -80℃. Various concentrations (5, 10, 15 and 20 nM) of chaetocin diluted with the cell 5

culture medium were added to the cells. The cells were treated with chaetocin for 48 h and then used for immunofluorescent staining, western blotting or nuclear transfer according to the experimental design.

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2.2 Cell viability assay

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Cell viability was assayed with the Cell Counting Kit-8 (Beyotime, Jiangsu,

China). SFF cells were seeded into 96-well plate at a density of 1×103 cells/well.

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After 12 h, the cells were treated with different concentrations of chaetocin for 48 h

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and then 10 μl WST-8 (Beyotime) were added into each well. After 1 h-incubation at

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37℃, the absorbance at 450 nm wave length was read by a microplate reader (Tecan

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Group Ltd., Zurich, Switzerland). All experiments were performed on three

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independent cell cultures. Values for each treatment group were normalized to the untreated cells that were set at 100% viability.

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2.3. In vitro fertilization

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The procedure of in vitro fertilization (IVF) was described previously [31]. Briefly, oocytes were aspirated from abattoir ovaries and were cultured in TCM 199

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supplemented with 20% (v/v) estrus sheep serum (eSS), at 38.5℃, in 5% CO2 in saturated humidity air for about 22 h. Frozen-thawed sperm were incubated in synthetic oviduct fluid (SOF) supplemented with 2% eSS for 30 min for swimming up. Highly motile spermatozoa were collected and co-incubated with matured oocytes in fertilization medium for 20 to 22 h. Fertilized ova were transferred into 6

SOF supplemented with 8 mg/ml bovine serum albumin (BSA), 2% essential amino acids and 1% non-essential amino acids (SOFaa), and then cultured in this medium at 38.5℃ in 5% CO2, 7% O2 and 88% N2.

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2.4. SCNT

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SCNT was carried out as described previously [14]. Matured oocytes were

incubated with 2% hyaluronidase for 1 min to remove cumulus cells by gently

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pipetting. Denuded oocytes were stained in 10 μg/ml Hoechst 33342 for 10 min and

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then transferred to micromanipulation in medium Hepes-buffered TCM199 (H199)

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supplemented with 10% FBS and 5 μg/ml cytochalasin B. The oocyte nucleus and

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polar body were removed and a donor cell was placed into the perivitelline space of

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enucleated oocyte. Fusion was induced by applying 2.4 kV/cm 40 μs direct current twice, on a cell fusion apparatus (BLS CF-150/B, Budapest, Hungary). Fused

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embryos were activated by 5 μM ionomycin for 5 min, followed by incubation in 2

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mM 6-dimethylaminopurine (6-DMAP) for 4 h. Finally, the cloned embryos were cultured in SOFaa medium.

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2.5. Immunofluorescent staining

Cells or embryos were fixed in 4% paraformaldehyde at 4℃ overnight, and then they were washed twice in 0.05% Tween 20 in PBS before being permeated in 0.5% 7

Triton X-100 in PBS for 30 min at room temperature. Afterwards, cells or embryos were blocked at 37℃ for 2 h in 1% BSA and 0.2% Triton X-100 in PBS. If used for the detection of DNA methylation, the cells were denatured by 2 M HCl for 30 min before being blocked.

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The samples were stained with relevant primary antibodies against H3K9me3 (1:300 dilution; Upstate/Millipore, Washington, DC, USA), H3K9me2 (1:50 dilution;

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Abcam, Cambridge, UK), 5-methylcytosine (5mC) (1:400 dilution; Eurogentec, Liege,

Belgium) or 5-hydroxymethylcytosine (5hmC) (1:400 dilution; Active motif, Carlsbad,

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CA, USA) at 37℃ for 1 h or at 4℃ overnight.

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After 3 times of washes with 0.05% Tween-20 in PBS, samples were incubated in

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appropriate secondary antibodies for 1 h at 37℃. Finally, samples were incubated

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briefly with 5 μg/ml 4, 6-diamidino-2-phenylindole (DAPI) in PBS and then mounted

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onto a glass slide with a coverslip in antifading solution containing 0.25% DABCO. Observations were performed on Qlympus BX51 epifluorescence microscope

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(Olympus Corporation, Tokyo, Japan). All images were recorded with a

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high-resolution CCD camera (Olympus Corporation). Total fluorescence intensity was measured by manually outlining all nuclei with ImageJ 1.37v software (National

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Institutes of Health, Bethesda, MD, USA).

2.6. Western blotting

Cells were harvested by lysis buffer (Beyotime) with phenylmethanesulfonyl 8

fluoride (PMSF), then they were denatured with loading buffer at 100℃ for 10 min. Samples were loaded on 12% SDS-PAGE gel electrophoresis and transferred onto nitrocellulose membranes. Membranes were blocked by 5% skim milk at 4℃ overnight and then incubated with primary antibodies at room temperature for 2 h,

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followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies for another 2 h. The primary antibodies included H3K9me2, H3K9m3,

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H3K4me3 (Easybio, Beijing, China), H3K9Ac (Millipore)，SUV39H1 (GeneTex,

Irvine, CA, USA)，SUV39H2 (GeneTex), G9A (Cell Signaling Technology, Danvers,

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MA,USA) and β-actin (Abcam) antibodies. The membranes were treated with super

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2.7. Quantitative PCR analysis (qPCR)

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ECL Plus and then the western blotting signals were detected by exposure on films.

Total RNA was extracted from SFF cells using the TRNzol Reagent (Tiangen

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Biotech, Beijing, China) according to the manufacturer’s instruction. Reverse

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transcription was completed by using the FastQuant RT Kit (Tiangen Biotech) following the standard protocol. The cDNA was used as template for qPCR using SuperReal PreMix (Tiangen Biotech). Reaction was performed on an ABI 7500

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system (Applied Biosystems). The housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control. The primers used are presented in Table 1. The cycle threshold (CT) value of each gene was obtained from three replicates. The expression of target genes was normalized to that of GAPDH. 9

The expression levels were compared between chaetocin treatment and non-treatment groups.

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2.8. Statistical analysis

All the statistical analyses were performed using Graphpad Prime 5 Software.

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Data from cell viability assays were statistically analysed by One-way ANOVA, and

other data were analysed by Student’s two-tailed t test. Results were presented as

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means ± SEM. Differences were considered significantly at three levels (*p <0.05,

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**p <0.01, ***p <0.001).

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3. Results

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3.1. Comparison of H3K9 methylation levels between IVF and SCNT embryos

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H3K9 methylation patterns in IVF and SCNT embryos were detected and analyzed by the immunostaining method. As shown in Fig. 1, both H3K9me2 and H3K9me3 levels were significantly higher in SCNT embryos than their IVF

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counterparts at the 1-cell stage (Fig. 1A and 1B) and maintained at the 2-cell stage (Fig. 1C). No significant differences in H3K9 methylation were observed between SCNT and IVF embryos during subsequent cleavage stages (Fig. 1C and 1D).

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3.2. Effects of chaetocin on H3K9 methylation in sheep fetal fibroblast cells

To assess the effect of chaetocin on H3K9 methylation, SFF cells were cultured in medium containing various concentrations of chaetocin for 48 h. Assays by western

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blotting showed that both H3K9me2 and H3K9me3 levels were decreased by chaetocin in a concentration-dependent manner, but other histone modifications

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including H3K9 acetylation (H3K9Ac) and H3K4 trimethylation (H3K4me3), were

not influenced (Fig. 2). However, high concentrations of chaetocin appeared to have

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an adverse effect on cell viability (Fig. 3A). Further assays revealed significant

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cytotoxicity of chaetocin at 15 and 20 nM compared to 10 nM (Fig. 3B). Therefore,

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we chose the concentration of 10 nM for subsequent studies.

Immunofluorescent

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staining confirmed that 10 nM chaetocin was sufficient to induce significant

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H3K9me2/3 decline and did not affect DNA methylation or hydroxymethylation (Fig. 4A-C). These results indicated that chaetocin had a specific effect on inhibition of

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H3K9 methylation.

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3.3. Effects of chaetocin on the expression of H3K9 methyltransferases

The expression of H3K9 methyltransferases was examined in chaetocin-treated

cells. qPCR analysis showed that mRNA levels of SUV39H1 and SUV39H2 were significantly down-regulated by chaetocin, while the transcription of G9A was not altered (Fig. 5A). However, the protein levels of all these three enzymes significantly 11

declined in chaetocin-treated cells when detected by western blotting (Fig. 5B). Thus, chaetocin played a role in suppressing the expression of H3K9 methyltransferases.

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3.4. The development of SCNT embryos produced from chaetocin-treated cells

SFF cells treated with 10 nM chaetocin were used as donor cells for nuclear

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transfer. As shown in Fig. 6A, at the pronuclear stage, cloned embryos derived from

chaetocin-treated cells displayed significantly lower levels of H3K9me2 or H3K9me3

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than that from non-treated cells. H3K9me2 levels remained unchanged during the

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pronuclear development, but H3K9me3 levels were up-regulated when the embryos

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developed from 3 h to 16 h post activation. The lower H3K9me2 level in embryos

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from chaetocin-treated cells was maintained at the 2-cell stage, but no difference was

stages (Fig. 6B).

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found between chaetocin treatment and non-treatment groups at subsequent cleavage In addition, there were no significant differences in both cleavage

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and blastocyst development between chaetocin and control groups (Table 2).

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3.5. The development of embryos directly treated with chaetocin

To suppress the H3K9 methyltransferases in embryos, SCNT embryos after

activation were treated with 10 nM chaetocin for 24 h. Immunofluorescent staining showed that both H3K9me2 and H3K9me3 levels were significantly decreased by chaetocin (Fig. 7). However, the embryos treated with chaetocin failed to develop to 12

the blastocyst stage (Table 3). Similarly, the development of IVF embryos was also severely impaired by chaetocin treatment (Table 4).

4. Discussion

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Aberrant H3K9 methylation has been observed in SCNT embryos in several species and was thought to be one of main epigenetic marks that influence the

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development of SCNT embryos [13-15, 21]. Consistent with previous reports, in this

study we found that both H3K9me2 and H3K9me3 were hypermethylated in cloned

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ovine embryos, especially during the very early cleavage cycles. This may suggest

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that the donor somatic nucleus cannot be rapidly reprogrammed by the recipient

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cytoplasm. Recently, the transcriptome analysis of cloned mouse 2-cell embryos has

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identified many reprogramming resistant regions (termed as RRRs) [21]. These RRRs

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were characterized by significant enrichment of Suv39h1/2-deposited H3K9me3 and low DNase I accessibility, both of which are general features of heterochromatin in

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several somatic cell types analyzed. As H3K9me2/3 is closely associated with gene

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repression, removal of these somatic epigenetic marks would be beneficial to the reactivation of embryonic genes and the improvement of SCNT embryo development. To induce the reduction of H3K9 methylation, we applied chaetocin, a specific

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inhibitor of SUV39 family histone methyltransferases, to treat the cultured ovine somatic cells. We showed that chaetocin specifically reduced the levels of both H3K9me2 and H3K9me3 in cells but had no effects on other epigenetic modifications tested. These results support previous reports showing that the activity of not only 13

H3K9 trimethyltransferase SUV39H1 but also dimethyltransferase G9a could be inhibited by chaetocin [24, 25]. Notably, we found that chaetocin suppressed the expression of H3K9 methyltransferases including SUV39H1, SUV39H2 and G9A. Recent reports also described that chaetocin could induce a significant reduction of

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SUV39H1 mRNA or its protein in cultured human cancer cells [28, 29, 32]. In our study, both mRNA and protein levels of either SUV39H1 or SUV39H2 were

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decreased by chaetocin treatment. The transcription of G9A appeared not to be influenced by chaetocin, but its protein level was found to decline. These results

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suggest that in addition to suppress the enzymatic activity, chaetocin may also reduce

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the expression or protein stability of H3K9 methyltransferases in cells. However, the

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underlying mechanism in regarding to this function of chaetocin is unclear.

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Nevertheless, since chaetocin is a potent inhibitor of all of three H3K9

methylation.

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methyltransferases, it would be an ideal chemical agent for down-regulation of H3K9

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Previous studies showed that down-regulation of H3K9me2 alone by

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G9A-specific inhibitor could not improve the development of cloned sheep and goat embryos [14, 16, 19]. In mice, reducing the H3K9me3 levels via overexpression of KDM4 or knockdown of the H3K9 methyltransferases, Suv39h1/2, in donor cells

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prior to nuclear transfer, greatly promotes the development of resulting SCNT embryos [21, 33, 34]. In the present study, after the nuclei of chaetocin-treated donor cells were transferred into the oocyte cytoplasm, the SCNT embryos remained genome-wide lower levels of both H3K9me2 and H3K9me3 during pronuclear 14

development. We expected that erase of the preexisting H3K9me2/3 marks in somatic cells would facilitate the epigenetic reprogramming and the development of SCNT embryos. However, we did not observe significant enhancement of in vitro development of the cloned ovine embryos from chaetocin-treated cells. It seems that

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chaetocin-induced H3K9me2/3 reduction could not completely rescue the developmental defects of SCNT embryos. Otherwise, chaetocin treatment may

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produce some unknown adverse effects that neutralize the beneficial role from

reduced H3K9 methylation. However, cell viability assays indicated no evident

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cytotoxicity of chaetocin at 10 nM that we used to treat the donor cells. Other latent

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influence of chaetocin on cells needs to be further investigated. Many studies have

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suggested that chaetocin is a potential agent for inhibiting the growth of various

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human cancer cells, such as leukemia [28-30], hepatoma [35], ovarian cancer [36] and

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lung cancer [37]. In our study on ovine somatic cells, we observed a severe cytotoxicity of chaetocin at 20 nM, a concentration far lower than that was applied for

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killing cancer cells, which generally ranges from 50 nM to 500 nM in most previous

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studies. Therefore, the toxic effects of chaetocin on normal cells would be concerned when it is used as an anti-cancer drug. Nevertheless, in our study on SCNT, chaetocin-treated cells appeared to support normal blastocyst development of SCNT

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embryos. Future work may be required to address whether in vivo development of the embryos can be improved by this strategy. We noted that H3K9me3 levels tended to increase with the pronuclear development in SCNT embryos. Previous studies on the mouse also showed that 15

reduced H3K9me3 levels in donor cells are rapidly restored after nuclear transfer [33, 34]. To inhibit the methyltransferase activity existing in oocyte cytoplasm, we attempted to directly treat the embryos with chaetocin after nuclear transfer. Treatment of the SCNT embryos with chaetocin significantly reduced the H3K9me2

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and H3K9me3 levels. However, the development of embryos was severely impaired by chaetocin treatment. The reasons for this adverse effect of chaetocin on embryos

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are unclear. Null-mutation studies on mouse demonstrate that G9a knockout or

Suv39h1/h2 double knockouts lead to embryonic lethal at post-implantation stages but

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not during preimplantation development [38, 39]. Moreover, down-regulation of

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H3K9m2/3 in SCNT embryos by overexpression of KDM4 has a beneficial role on

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the embryo development [21-23, 40, 41]. Therefore, the developmental failure of

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chaetocin-treated embryos would not be attributed to the H3K9 hypomethylation. The

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molecular mechanisms of action of chaetocin on cells have not been elucidated clearly.

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Previous studies have linked the anti-cancer effect of chaetocin with its

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inhibitory role on SUV39H1 [28, 29, 32]. However, the mechanism of cancer cell-killing effects of chaetocin may be complex. Chaetocin may target other pathways in addition to H3K9 methyltransferases. Some studies revealed that

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chaetocin can induce generation of reactive oxygen species (ROS) via competitive inhibition of the antioxidant enzyme thioredoxin reductase, and thereby produce oxidative damage to cancer cells [42, 43]. This would also be the case in embryos. Chaetocin may induce ROS production in the embryos by targeting related reductases 16

or pathways. It is well-known that increased ROS has detrimental effects on preimplantation development of mammalian embryos [44, 45]. If this is the case, supplementation of antioxidants during embryo culture would counteract the cytotoxic effects of chaetocin-induced ROS. Alternatively, as embryos appear to be more

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sensitive to chaetocin treatment than somatic cells, reducing the chaetocin concentration or optimizing the treatment duration may be conducive to establish an

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appropriate protocol for applying chaetocin to the embryos.

In this study, we attempted to explore the possibility to modulate H3K9

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methylation in SCNT embryos by using chaetocin. As a potential agent for cancer

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therapy, the cytotoxicity of chaetocin has been tested in many cancer cell types.

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However, its adverse effects on reproduction have not been documented. In this

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regard, our study for the first time suggests a reproductive toxicity of chaetocin, in

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terms of its negative influence on embryo development. It should be noted that, in addition to the potential applications as a therapeutic drug, chaetocin may also exist in

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the food with fungal contaminations[46]. Therefore, the toxicity of chaetocin may be

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concerned in the case of its uptake from medicine or food, and such hazard is required to be further investigated. In conclusion, we demonstrate that treatment with a single chemical, chaetocin,

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can achieve simultaneous down-regulation of H3K9 di- and trimethylation in ovine somatic cells. After nuclear transfer, chaetocin-treated cells can support normal preimplantation development of the resulting cloned embryos. However, chaetocin has a detrimental effect on embryonic development when directly applied to embryos. 17

Our study may be informative for better exploring the use of chaetocin in future applications.

Acknowledgments

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This work was supported by the National Natural Science Foundation of China (Grant No. 31172208), 948 Project from Ministry of Agriculture of People’s Republic

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of China (Grant No. 2016-X36) and China Agriculture Research System (Grant No.

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CARS-39-04).

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Figure Legends Figure 1. Comparison of H3K9me2 and H3K9me3 levels between ovine IVF and SCNT embryos. (A, B) Immunofluorescence detection of H3K9me3 or H3K9me2 in

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embryos at the pronuclear stage. (C) Immunofluorescence detection of H3K9me2 (green) and H3K9me3 (red) in 2-, 4-, and 8-cell stage embryos derived from IVF and

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SCNT groups. (D) Quantification analysis of immunofluorescence intensities. The nuclei (blue) were labeled with DAPI. The arrows point to the pronuclei of embryos.

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fertilization. SCNT, somatic cell nuclear transfer.

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Scale bar = 50 μm. Data are presented as means ± SEM. ***p < 0.001. IVF, in vitro

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Figure 2. Western blotting analysis of H3K9me3, H3K9me2, H3K9 acetylation

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(H3K9Ac) and H3K4me3 proteins in sheep fetal fibroblast cells treated with different

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concentrations (0, 5, 10, 15, 20 nM) of chaetocin for 48 h. DMSO represents the cells cultured in medium containing 0.01% DMSO and served as a control to the chaetocin

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treatment. β-actin was used as loading controls.

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Figure 3. Effects of chaetocin treatment on cell viability. (A) Growth status of sheep fetal fibroblast cells treated with different concentrations (0, 5, 10, 15, 20 nM) of chaetocin for 48 h. Scale bar = 100 μm. (B) Cell viability was examined with WST-8

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assay. Each treatment group was normalized to the non-treatment group (0 nM) and values are presented as means ± SEM. *p＜0.05, ***p＜0.001. Figure 4. Immunostaining detection of H3K9 methylation and DNA methylation in sheep fetal fibroblast cells treated with chaetocin. (A, B) Immunostaining and 26

quantification analysis of H3K9me2 and H3K9me3 levels. (C) Immunostaining and quantification

analysis

of

DNA

5-methylcytosine

(5mC)

and

5-hydroxymethylcytosine (5hmC) levels. The cells were treated with 10 nM chaetocin for 48 h. The untreated cells were used as controls. Scale bar = 50 μm. Data are

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presented as means ± SEM. ***p < 0.001. Figure 5. Effects of chaetocin on the expression of H3K9 methyltransferases. The

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cells were treated with 10 nM chaetocin for 48 h. The untreated cells were used as

controls. (A) The mRNA levels of SUV39H1, SUV39H2 and G9A were analyzed by

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qRT-PCR. Data are presented as means ± SEM. N = 3; **p < 0.01. (B) The protein

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levels of SUV39H1, SUV39H2 or G9A were analyzed by western blotting. β-actin

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served as loading controls.

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Figure 6. H3K9 methylation patterns of SCNT embryos produced from

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chaetocin-treated cells. SCNT embryos were produced from cells treated with chaetocin (10 nM) or from non-treated cells (control). (A) Immunofluorescence

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detection of H3K9me2 and H3K9me3 in SCNT embryos at 3 and 16 h after activation.

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The arrows point to the pronuclei of embryos. (B) Immunofluorescence detection of H3K9me2 and H3K9me3 in 2-, 4-, and 8-cell SCNT embryos. Scale bar = 50 μm. Data are presented as means ± SEM. *p < 0.05.

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Figure 7. H3K9 methylation patterns of chaetocin-treated SCNT embryos. After activation, the SCNT embryos were treated with 10 nM chaetocin for 24 h and were immunostained at the 2-cell stage. SCNT embryos without any chaetocin treatment were used as control. (A) Immunofluorescence staining of the embryos. (B) 27

Quantification analysis of immunofluorescence intensities. Scale bar = 50 μm. Data

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are presented as means ± SEM. ***p < 0.001.

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Figures:

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Tables: Table 1. Primers for quantitative PCR

119

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SUV39H2

Sizes(bp) 133

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Sequences (5’ to 3’) Forward: GTGTCCGCACGCTGGAGAAG Reverse: AGGTCGAAGAGGTAGGTGGC Forward: GTACCCCCATTTACGAGTGC Reverse: GCCACAGCCATTGCTAGTTCG Forward: CATCCCTTGTGTCAATGGTG Reverse: CAGTTGGAGCTGGAGCAGTC Forward: TGATGACATCAAGAAGGTGGTG Reverse: TCCTTGGAGGCCATGTAGGCCAT

Genes SUV39H1

G9A

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GAPDH

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153 240

Table 2. Preimplantation development of SCNT embryos derived from chaetocin-treated cells.

No. of reconstructed No. of cleaved embryos (%, means ± SEM) 120 115

No. of blastocysts (%, means ± SEM)

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Donor cells treated with chaetocin (nM) 0 10

96 (77.6±9.6) 106 (92.3±2.4)

8 (9.8±4.1) 11 (10.6±3.7)

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Cleavage percentage: No. of cleaved/No. of reconstructed embryos. Blastocyst

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percentage: No. of blastocysts/No. of cleaved.

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Table 3. Preimplantation development of SCNT embryos treated with chaetocin for 24 h after activation

0 10

No. of cleaved (%, means ± SEM) 74 (89.3±2.9) 60 (79.2±5.4)

No. of blastocysts (%, means ± SEM) 6 (7.6±3.1)a 0b

within a row represent significant difference between groups (P <0.05)

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a, b

No. of reconstructed embryos 83 77

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Chaetocin (nM)

Cleavage percentage: No. of cleaved/No. of reconstructed embryos. Blastocyst

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percentage: No. of blastocysts/No. of cleaved

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Table 4.

Preimplantation development of IVF embryos treated with chaetocin

for 24 h after fertilization

Chaetocin (nM)

a, b

No. of cleaved (%,means ± SEM) 119(69.4±3.2) 119(68.1±8.3)

No. of blastocysts (%,means ± SEM) 24(20.4±1.6) a 0b

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0 10

No. of embryos cultured 175 184

within a row represent significant difference between groups (P <0.05)

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Cleavage percentage: No. of cleaved/No. of embryos cultured. Blastocyst percentage:

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No. of blastocysts/No. of cleaved

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