Role of Epigenetic Regulation by the REST/Corest/HDAC Corepressor Complex of Moderate NANOG Expression in Chicken Primordial Germ Cells

STEM CELLS AND DEVELOPMENT Volume 27, Number 17, 2018 Mary Ann Liebert, Inc. DOI: 10.1089/scd.2018.0059

Role of Epigenetic Regulation by the REST/CoREST/HDAC Corepressor Complex of Moderate NANOG Expression in Chicken Primordial Germ Cells

Hyun Gyo Jung,* Young Sun Hwang,* Young Hyun Park, Ho Yeon Cho, Deivendran Rengaraj, and Jae Yong Han

Primordial germ cells (PGCs), the precursors of gametes, have regulatory mechanisms involving transcription factors and epigenetic modiﬁcations. The transcription factor NANOG is a key regulator of germ cell and embryonic stem cell characteristics. However, the epigenetic regulation of NANOG with a histone deacetylase (HDAC) complex in PGCs has not been studied. In this study, we investigated the epigenetic regulation, and in particular the histone acetylation, of NANOG in chicken PGCs. Intriguingly, although NANOG was highly activated in chicken PGCs, the upstream region of its promoter was moderately suppressed by histone deacetylation. HDAC inhibition induced histone H3 lysine 9 acetylation (H3K9ac) and derepressed NANOG expression. Furthermore, knockdown studies revealed that HDAC complex members, such as RE1-silencing transcription factor (REST) and REST corepressor 3 (RCOR3), are important epigenetic modulators of NANOG expression in chicken PGCs. We demonstrate that moderate regulation of NANOG by the REST/CoREST/HDAC complex might be crucial for maintaining the integrity of PGCs.

Keywords: chicken, primordial germ cells, NANOG, RE1-silencing transcription factor, REST corepressor, histone deacetylation

Introduction VASA and deleted in azoospermia like (DAZL), in a pre- determined manner [7,8]. In contrast with mammals, chicken rimordial germ cells (PGCs) are the precursors of PGCs migrate to the genital ridge through blood vessels after Psperm and oocytes, which are the only cells that transmit specification [9]. genetic information to the next generation. PGCs undergo In terms of epigenetic regulation, the dynamics and land- epigenetic dynamics during embryogenesis to establish cel- scape of histone modification have not been established yet in lular specification and suppress somatic lineage differentia- avian PGCs. In recent study, global enrichment of H3K9 tri- tion [1]. The epigenetic status of PGCs is regulated by DNA methylation (H3K9me3) was observed in chicken PGCs [10]. methylation and histone modifications (histone acetylation or Also, H3K9ac formed foci in euchromatin in chicken PGCs as deacetylation), which have roles in regulating the expression the activating histone acetylation marker [10], indicating this Downloaded by Seoul National Univ. Medical College from www.liebertpub.com at 11/01/18. For personal use only. of genes related to pluripotency and germness [2]. mark could play an important role in epigenetic regulation in Specifically, histone H3 lysine 9 acetylation (H3K9ac) chicken PGCs. However, gene expression regulation by his- transiently and markedly increases in PGCs to establish tone modification in chicken PGCs has been largely unknown. transcriptional activation of chromatin at the point of entry NANOG is an important transcription factor that regulates into the genital ridge and decreases during colonization [3]. pluripotency in early embryos, embryonic stem cells (ESCs), Also, maintenance of H3K9ac is involved in proper chro- and induced pluripotent stem cells (iPSCs) [11]. In mammals, mosomal segregation during the formation of gametes [4,5]. NANOG expression was also found from migrating germ cells Thus, histone acetylation is a crucial epigenetic marker of to gonadal germ cells [12,13], and shown to prevent apoptosis germ cell specification and development. of migrating germ cells [14]. Moreover, in later stage, NANOG Avian PGCs are important for biotechnology and germ cell is involved in differentiation and maturation of mouse PGCs in biology due to their unique characteristics [6]. Chicken PGCs genital ridge [15–17]. In chicken, NANOG is required for the are specified by maternally inherited germ plasm, including maintenance of pluripotency in chicken ESCs [18] and was

Department of Agricultural Biotechnology, Research Institute of Agriculture and Life Sciences, College of Agriculture and Life Sciences, Seoul National University, Seoul, Korea. *These authors contributed equally to this work.

1215 1216 JUNG ET AL.

detected during early embryonic development from preovula- DMEM (Gibco) supplemented with 10% FBS. The cultured tory oocyte to intrauterine stages [19]. Also, Jak1/Stat3 sig- PGCs and DF-1 were treated with HDAC inhibitors, 0.25 mM naling supports NANOG expression and pluripotency in early valproic acid (VPA; Sigma-Aldrich), and 50 nM romidepsin chicken embryos [20]. (Selleckchem, Boston, MA), for three consecutive days. In chicken PGCs, NANOG is expressed in the germinal crescent at Hamburger and Hamilton stage 5 (HH5) to HH8 Immunostaining [21]. In addition, NANOG expression was similar to the PGC-specific genes CVH and dead end homolog 1 (DND1) VPA- and romidepsin-treated cells were harvested and wa- during PGC migration and colonization in gonads [21], in- shed twice in phosphate-buffered saline (PBS) and fixed with dicating a role for NANOG in maintaining the self-renewal 4% paraformaldehyde for 20 min. After permeabilization with ability of chicken PGCs in various stages. The recent report 0.1% Tween-20 and 1% Triton X-100, nonspecific binding was showed that NANOG solely activated enhancers upstream blocked with 1% normal goat serum. Cells were then incubated of germ cell specifiers to generate PGC-like cells from with rabbit anti-acetyl-histone H3K9 antibody (06-942; Milli- epiblast-like cells, but not in ESCs in mouse [22], indicating pore, Bedford, MA), mouse anti-acetyl-histone H3 lysine 27 that its function differs according to cell type. (H3K27ac) antibody (GTX50903; GeneTex, CA), rabbit anti- The role of histone acetylation and regulation of the trimethyl-histone H3K9 antibody (07-442; Millipore), and rabbit histone deacetylase (HDAC) complex in pluripotency and anti-trimethyl-histone H3K27 (H3K27me3) (07-449, Millipore) somatic cell reprogramming in ESCs and iPSCs has been at 4C overnight. Cells incubated in the absence of primary reported [23–25]. Despite the emerging role of NANOG in antibody were used as a negative control. After extensive germ cell development, the role of histone acetylation and washing, cells were incubated with Alexa Fluor 594-conjugated the HDAC complex in the regulation of NANOG expression goat anti-rabbit IgG H&L (ab150080; Abcam, Cambridge, in avian PGCs is unclear. United Kingdom), Alexa Fluor 488-conjugated goat anti- In this study, we demonstrated the epigenetic regulation of rabbit IgG H&L (ab150077; Abcam), and FITC-conjugated NANOG expression by H3K9ac and the HDAC corepressor goat anti-mouse IgG (sc-2010; Santa Cruz, CA) for 1 h. complex in chicken PGCs. We used an HDAC inhibitor to After washing, cells were mounted with 4¢,6-diamidino-2- induce aberrant histone acetylation and evaluated the ex- phenylindole (DAPI) and visualized using Ti-U fluorescence pression of germ cell-specific genes, including NANOG. In microscope (Nikon, Tokyo, Japan). addition, upstream regulators of NANOG, such as DNA- dependent factors and HDAC complexes, were examined to RNA isolation and reverse transcription-quantitative evaluate epigenetic regulation in chicken PGCs. This study polymerase chain reaction increases our understanding of the molecular mechanisms Total RNA was extracted using TRIzol reagent (Invitrogen, involved in the fundamental specification of germ cells. Carlsbad, CA) according to the manufacturer’s protocol. One microgram of total RNA from each sample was reverse- Materials and Methods transcribed using the Superscript III First-strand Synthesis Experimental animals and care System (Invitrogen) according to the manufacturer’s protocol. The polymerase chain reaction (PCR) mixture contained The care and experimental use of White Leghorn (WL) 2 mL of PCR buffer, 0.5 mL of 10 mM dNTP mixture, 10 pmole chickens were approved by the Institute of Laboratory Animal each of forward and reverse primers (Table 1), 1 mL of com- Resources, Seoul National University (SNU-150827-1). Chick- plementary DNA (cDNA), and 1 U of Taq DNA polymerase ens were maintained according to a standard management pro- in a 20 mL final volume. PCR amplification involved an initial gram at the University Animal Farm, Seoul National University, incubation at 95C for 10 min, followed by 40 cycles at 95C Korea. The procedures for animal management, reproduction, for 30 s, 60C for 30 s, and 72C for 30 s. PCR was terminated and embryo manipulation adhered to the standard operating with a final incubation at 72C for 5 min, and at the dissoci- protocols of our laboratory. ation temperature. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was performed using a

Downloaded by Seoul National Univ. Medical College from www.liebertpub.com at 11/01/18. For personal use only. Culture of chicken PGCs and chemical treatment StepOnePlus real-time PCR system (Applied Biosystems, Foster City, CA) with EvaGreen (Biotium, Hayward, CA). Chicken PGCs were cultured in accordance with our stan- Each test sample was assayed in triplicate. Relative quanti- dard procedure [26,27]. Briefly, PGCs from male WL embry- fication of target gene expression was performed using onic gonads at 6 days old (HH stage 28) were maintained in the following formula: 2-DDCt,whereDDCt = (Ct of the target knockout Dulbecco’s modified Eagle’s medium (DMEM; gene - (Ct of GAPDH + Ct of ACTB)/2) treatment - (Ct of the Gibco, Grand Island, NY) supplemented with 20% fetal bovine target gene - (Ct of GAPDH + Ct of ACTB)/2) control. serum (FBS; Hyclone, South Logan, UT), 2% chicken serum (Sigma-Aldrich, St. Louis, MO), 1· nucleosides (Millipore, Histone extraction and western blot analysis Billerica, MA), 2 mM L-glutamine (Gibco), 1· nonessential amino acids (Gibco), b-mercaptoethanol (Gibco), 1 mM so- Cells were harvested and washed in ice-cold PBS for two dium pyruvate (Gibco), and 1· antibiotic/antimycotic (Gibco). times. Harvested cells were resuspended in Triton extraction Human basic fibroblast growth factor (Koma Biotech, Seoul, buffer [TEB; PBS containing 0.5% Triton · 100 (v/v), 2 mM Korea) at 10 ng/mL was used for PGC self-renewal. The cul- phenylmethylsulfonyl fluoride, 0.02% (w/v) NaN3]. PBS tured PGCs were subcultured onto mitomycin-inactivated and TEB contain 5 mM sodium butyrate to preserve levels mouse embryonic fibroblasts at 5- to 6-day intervals by gentle of histone acetylation. After harvest of cell pellets, the nu- pipetting without any enzyme treatment. DF-1 was cultured in clear pellets were extracted with 0.2 N HCl for overnight at THE MODERATE EXPRESSION OF NANOG IN CHICKEN PGCS 1217

Table 1. Primers Used for Reverse Transcription-Quantitative Polymerase Chain Reaction Gene symbol Description Accession no. Primer sequence (5¢ / 3¢) NANOG Nanog homeobox NM_001146142 F: AACTCTGCGGGGCTGTCTTG R: AAAAGTGGGGCGGTGAGATG POUV/POU5F3 POU domain class 5 transcription NM_001110178 F: TGAAGGGAACGCTGGAGAGC factor 3 R: ATGTCACTGGGATGGGCAGAC VASA Chicken VASA homolog NM_204708 F: CCTTGCAGCCTTTCTTTGTC R: ACGACCAGTTCGTCCAATTC DAZL Deleted in azoospermia like NM_204218 F: CAACTATCAGGCTCCACCAC R: CTCAGACGGTTTTCAGGGTT BRACHYURY T brachyury transcription factor NM_204940 F: ACGCCATGTACTCCTTCCTG R: TGTTGGTGAGCTTGACCTTG GSC Goosecoid homeobox NM_205331 F: AACGGAGCTGCAGTTACTCAA R: TTCCATTTCTGTGCATTTTCC SOX7 SRY (sex determining region Y)- XM_420036 F: CTGGGAGAGATGGACCGTAA box 7 R: CCTTCCTCAGCAGGTCTACG GATA4 GATA-binding protein 4 NM_420041 F: ACCAGTCAGCCATCAGCCAG R: AGAGCAGGGGAGGAGGGAAA SOX3 SRY (sex determining region Y)- NM_204195 F: GACAAAACGAAAAACCGACCC box 3 R: ATTGCGGACACGAACCGAG AFP Alpha-fetoprotein XM_015276399 F: CTCCTATGCTCTCAGGAGGC R: TTCTTTGCAGCACTGTTCCC MMP7 Matrix metallopeptidase 7 NM_001006278 F: GCTGCCATTCTCCTACCTCA R: CACATCTGGGCTGTTGCATT NR6A1 Nuclear receptor subfamily 6 group XM_015279584 F: GTTTGCCAGGACTTCACAGAG A member 1 R: CGGGACATTCACCATCTTTC CXCR4 C-X-C motif chemokine receptor 4 NM_204617 F: TCTGTGGCTGACCTCCTCTT R: TCAGGCACTGTGAGAAGCAC REST RE1-silencing transcription factor XM_015276447 F: AAAGAGCAAACAAAAGGGGA R: TTGCTCGTTGGCTTCTTTT SIN3A SIN3 transcription regulator family NM_001293184 F: GGAAGAGGAGGAAGAAGAGG member A R: CTCTCCCTTGTCTCCTCTTC SIN3B SIN3 transcription regulator family XM_015300175 F: GGGGAGAAAAGAAAAGACCAA member B R: CCTCGCTTCCTTTCGTTC MTA1 Metastasis-associated 1 NM_001012953 F: AACAAGCCAAACCCCAACC R: GTTTGGTCCTGGTCTCTCTC MTA3 Metastasis-associated 1 family, XM_015283840 F: GCTCTTCCTTTCTCGCCAGT member 3 R: ACGGTCTGTAAGGGGGCTAT RCOR1 REST corepressor 1 XM_015287816 F: AACAGAGCGAAGAGGAAACC R: CTCTTTGCCGTGTTCTGC RCOR3 REST corepressor 3 NM_001079727 F: ATCGACAGGCTCGAAAGCTTGCTA R: CATTCTCAGCGTTCCAAATGCCG GAPDH Glyceraldehyde-3-phosphate NM_204305 F: GGTGGTGCTAAGCGTGTTAT dehydrogenase R: ACCTCTGTCATCTCTCCACA ACTB Actin, beta NM_205518 F: AGGAGATCACAGCCCTGGCA R: CAATGGAGGGTCCGGATTCA

Downloaded by Seoul National Univ. Medical College from www.liebertpub.com at 11/01/18. For personal use only. 4C. Supernatants containing histone protein are neutralized levels were quantitated with ImageJ (National Institutes of by 2 M NaOH at 10% volume of 0.2 N HCl. Protein (*10 mg) Health, Bethesda, MD) [28]. from total cell lysate was loaded in each lane for separation on 15% SDS-polyacrylamide gels. Resolved proteins were Prediction of the RE1-silencing transcription transferred onto a Hybond 0.45 PVDF membrane (GE factor-binding site in the proximate promoter Healthcare Life Sciences, Little Chalfont, United Kingdom) of NANOG, POUV, and VASA and blocked with 3% skim milk for 1 h at room temperature (Sigma-Aldrich). The blocked membrane was incu- The region upstream of NANOG, POUV,andVASA tran- bated overnight at 4C in the presence of primary antibody scriptional start site (TSS) was predicted by MatInspector, a (dilution: 1:3,000) and then for 1 h with horseradish Genomatix program (www.genomatix.de) using TRANSFAC peroxidase-conjugated secondary antibody (31460; Ther- matrices (vertebrate matrix; core similarity 1.0 and matrix mo Fisher Scientific, Inc., Waltham, MA) at a dilution of similarity 0.8). 1:4,000. Primary antibodies were as follows: anti-histone Chromatin immunoprecipitation H3 (ab1791; Abcam) and anti-H3K9Ac antibody (06-942; Millipore). Immunoreactive proteins were visualized with Treated cells were fixed in 1% methanol-free formalde- the ECL western blot detection system (Thermo Fisher Sci- hyde (Thermo Fisher Scientific, Inc.) in DMEM for 20 min at entific, Inc.). All blots were performed in triplicate, and protein room temperature, followed by 10-min blocking in 125 mM 1218 JUNG ET AL.

Table 2. Primers Used for Chromatin Immunoprecipitation-Polymerase Chain Reaction and Chromatin Immunoprecipitation-Quantitative Polymerase Chain Reaction

Primer Description Primer sequence (5¢ / 3¢) ChIP-REST REST binding site on NANOG promoter F: GAGGCGTAGGTGGGTAGCAAGAC R: GTCAGTTGATTGGAGGGGAGTGG

glycine. Cells were rinsed two times in ice-cold PBS and buffer, 0.5 mL of 10 mM dNTP mixture, 10 pmol of each of harvested. Suspension cultured cells were fixed in the plate the forward and reverse primers (Table 2), 1 mL of ChIP and harvested by pipetting after washing with PBS. Fixed DNA, and 1 U of Taq DNA polymerase in a 20 mL final cell pellets were dissociated in radioimmunoprecipitation volume. ChIP-PCR amplification involved an initial incu- (RIPA) lysis buffer (10 mM Tris-HCl, pH 7.5, 1 mM EDTA, bation at 95C for 10 min, followed by 30 cycles at 95C 0.5 M EGTA, 1% Triton X-100, 0.1% SDS, 0.1% Na- for 30 s, 60C for 30 s, and 72C for 30 s. The reaction deoxycholate, and 140 mM NaCl; Thermo Fisher Scientific, was terminated by a final incubation at 72C for 5 min. Inc.) with protease and phosphatase inhibitors (Sigma- For chromatin immunoprecipitation-quantitative polymerase Aldrich). Chromatin was sheared to an average length of chain reaction (ChIP-qPCR), the reaction mixture, thermal 500–700 base pairs (bp) using a sonicator (Q500; Qsonica, conditions, and all other procedures were as stated above. Newtown, CT). DNA concentration and the efficiency of ChIP-qPCR was performed using a StepOnePlus real-time shearing were determined by agarose gel electrophoresis. PCR system (Applied Biosystems) with EvaGreen (Bio- An anti-H3K9Ac antibody (06-942; Millipore) was used for tium). Each test sample was assayed in triplicate. Relative immunoprecipitation. Ten micrograms of sonicated chroma- quantification of gene expression was performed using the tin samples was used. Chromatin immunoprecipitation (ChIP) following formula: ((2-DCt) · 100%), where DCt = (Ct [IP] - was performed using a One-Step ChIP Kit (ab117138; Ab- Ct [Input · DF]). The DF (default input fraction) was 1%, cam) according to the manufacturer’s instructions (ab117138; which is a dilution factor of 6.644 cycles (ie, log2 of 100). Abcam) and nonimmune IgG was used as a negative control. The bound chromatin/antibody complex was incubated in Small interfering RNA transfection in chicken PGCs reverse crosslinking buffer for 2 h at 65C, followed by treatment with proteinase K. Chicken PGCs were seeded at 2.0 · 105 in 1 mL of medium per well of a 12-well plate. Cells were transfected with Chromatin immunoprecipitation-polymerase chain 100 pmol of each HDAC small interfering RNA (siRNA) reaction and chromatin immunoprecipitation- (Table 3) using RNAiMAX (Invitrogen). Negative control quantitative polymerase chain reaction siRNA with no complementary sequence in the chicken genome was used as a control. After transfection for 48 h, For total chromatin control samples (inputs) purified be- total RNA was extracted using TRIzol reagent (Invitrogen) fore immunoprecipitation, add 10 mL of each chromatin according to the manufacturer’s protocol. The knockdown sample to 88 mL of buffer and 2.5 mL of proteinase K. For efficiency of HDAC complex siRNAs and their effects on chromatin immunoprecipitation-polymerase chain reaction the expression of pluripotency-, germ cell-, and three germ (ChIP-PCR), the reaction mixture comprised 2 mL of PCR layer-specific marker genes were assayed by RT-qPCR.

Table 3. List of Small Interfering RNA Sequences for Knockdown Experiments Gene symbol Accession no. Primer sequence (5¢ / 3¢) REST XM_015276447 S: CAGAGACTGTCAGGAAGCTCAGAAA Downloaded by Seoul National Univ. Medical College from www.liebertpub.com at 11/01/18. For personal use only. AS: UUUCUGAGCUUCCUGACAGUCUCUG SIN3A NM_001293184 S: ACAUCUUCAUGAGGCUGCAUCAGAU AS: AUCUGAUGCAGCCUCAUGAAGAUGU SIN3B XM_015300175 S: CAGCGGGCUGUUGUUGCAUUACUCA AS: UGAGUAAUGCAACAACAGCCCGCUG MTA1 NM_001012953 S: CCGCCAAUGGAAAUGUGGAAGCAAA AS: UUUGCUUCCACAUUUCCAUUGGCGG MTA3 XM_015283840 S: CAUGUACCGAGUGGGAGAUUAUGUU AS: AACAUAAUCUCCCACUCGGUACAUG RCOR1 XM_015287816 S: CCGACGUCGUUUCAACAUAGAUGAA AS: UUCAUCUAUGUUGAAACGACGUCGG RCOR3 NM_001079727 S: CGGAUCUCCCUAACUUCACUCCCUU AS: AAGGGAGUGAAGUUAGGGAGAUCCG Nonspeciﬁc siRNAa S: CCUACGCCACCAAUUUCGU AS: GGAUGCGGUGGUUAAAGCA

aNonspeciﬁc siRNA has no complementary sequence in the chicken genome and was used as a control for gene silencing. siRNA, small interfering RNA. THE MODERATE EXPRESSION OF NANOG IN CHICKEN PGCS 1219

Statistical analysis H3K9ac compared with untreated control PGCs (Fig. 1A). Next, PGCs were treated with 50 nM Romidepsin in vitro, a Data are expressed as mean – standard error from three specific inhibitor of HDAC1 and 2 [30], which also resulted independent biological experiments. Student’s t-test was per- in a global increase in H3K9ac (Fig. 1A). Quantification of formed to evaluate differences between experimental groups H3K9ac after chemical treatment confirmed the significant using GraphPad Prism v.5 (GraphPad Software, La Jolla, CA). elevation of H3K9ac in VPA- and romidepsin-treated PGCs A value of P < 0.05 was considered indicative of statistical compared with control PGCs (Fig. 1B, C). However, ro- significance (***P < 0.001, **P < 0.01, and *P < 0.05). midepsin is a more effective HDAC inhibitor in PGCs compared with VPA (Fig. 1C). Therefore, only romidepsin Results was used for subsequent treatments. In addition to H3K9ac, Upregulation of H3K9ac and gene expression we examined H3K27ac, H3K9me3, and H3K27me3 in in PGCs by HDAC inhibitors romidepsin-treated PGCs (Supplementary Fig. S1; Supple- mentary Data are available online at www.liebertpub.com/ To assess the influence of HDAC on epigenetic regulation scd). The global increase in H3K27ac and decrease in and gene expression in chicken PGCs, we treated PGCs with H3K9me3 and H3K27me3 were shown in chicken PGCs by 0.25 mM VPA, an inhibitor of class I HDACs, closely as- romidepsin treatment, indicating the treatment modulated sociated with transcriptional activity [29]. Treatment of overall epigenetic marks in an active status. Next, we examined PGCs with VPA in vitro resulted in a global increase in romidepsin effects on the expression of a set of genes in PGCs. Downloaded by Seoul National Univ. Medical College from www.liebertpub.com at 11/01/18. For personal use only.

FIG. 1. Effect of HDAC inhibitors on H3K9ac and expression of NANOG in chicken PGCs. (A) Cultured PGCs were treated with 0.25 mM VPA or 50 nM romidepsin for 3 days, and subjected to immunocytochemical analysis of H3K9ac levels. Scale bar, 100 mm. (B) Western blot analysis of H3K9ac after chemical treatment. Histone H3 was used as control. (C) Relative quantification of H3K9ac to H3 in B. (D) Expression of NANOG in PGCs treated with romidepsin by RT- qPCR. RT-qPCR was conducted in triplicate, and the relative gene expression in treatments was normalized to expression of GAPDH and ACTB. Error bars represent SE of biological triplicate analyses. Significant differences between groups are indicated as ***P < 0.001 and *P < 0.05. H3K9ac, histone H3 lysine 9 acetylation; HDAC, histone deacetylase; PGCs, primordial germ cells; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; SE, standard error; VPA, valproic acid. Color images available online at www.liebertpub.com/scd FIG. 2. Effect of romidepsin on acetylated H3K9 levels at the REST-binding site in the NANOG promoter. (A) The REST- binding site in the proximal NANOG promoter. Arrows indicate primer-binding site. (B) Agarose gel electrophoresis of ChIP- PCR after treatment with HDAC inhibitor. %Input = 100-fold diluted DNA before selection; IgG = control for nonspecific antibody binding to DNA. (C) ChIP-qPCR amplification of the proximal REST-binding site (188 bp from the TSS) after treatment with HDAC inhibitor. Untreated PGCs were used as the control. Error bars represent SE of biological triplicate analyses. Significant differences between groups are indicated as *P < 0.05. bp, base pairs; ChIP-PCR, chromatin immunoprecipitation-polymerase chain reaction; ChIP-qPCR, chromatin immunoprecipitation-quantitative polymerase chain reaction; H3K9ac, histone H3 lysine 9 acetylation; HDAC, histone deacetylase; IgG, immunoglobulin G; PGCs, primordial germ cells; RE1-silencing transcription factor; SE, standard error; TSS, transcriptional start site. Color images available online at www.liebertpub.com/scd Downloaded by Seoul National Univ. Medical College from www.liebertpub.com at 11/01/18. For personal use only.

FIG. 3. REST regulates H3K9ac to repress NANOG expression in chicken PGCs. (A) Expression of REST in DF-1 cells and chicken PGCs by RT-qPCR. (B) Effect of siRNA-mediated knockdown of REST determined by RT-qPCR. (C) RT- qPCR analysis of POUV, NANOG, VASA, and DAZL expression in PGCs after knockdown of REST. RT-qPCR was conducted in triplicate, and the relative gene expression in treatments was normalized to expression of GAPDH and ACTB. (D, E) Suppression of REST increased H3K9ac levels at the REST-binding site in the proximal NANOG promoter. %Input = 100-fold diluted DNA before selection; IgG = control for nonspecific antibody binding to DNA. Nonspecific siRNA-treated PGCs were used as the control. Error bars represent SE of biological triplicate analyses. Significant differences between groups are indicated as ***P < 0.001, **P < 0.01, and *P < 0.05. siRNA, small interfering RNA; REST, RE1-silencing transcription factor; H3K9ac, histone H3 lysine 9 acetylation; PGCs, primordial germ cells; IgG, immunoglobulin G; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; SE, standard error.

1220 THE MODERATE EXPRESSION OF NANOG IN CHICKEN PGCS 1221

NANOG expression was significantly upregulated following was efficiently knocked down using REST-targeting siRNA. romidepsin treatment (Fig. 1D). Furthermore, expression of the Following successful knockdown of REST (Fig.3B),we pluripotency and germness-related genes, POUV and VASA, quantified the expression of pluripotency- and germness- was significantly upregulated following romidepsin treatment related genes by RT-qPCR. NANOG and VASA expression (Supplementary Fig. S2A), indicating that active genes in was significantly upregulated; the increase in NANOG ex- chicken PGCs, NANOG, POUV, and VASA, were further in- pression was considerable. In contrast, POUV and DAZL duced by HDAC inhibitor. Therefore, expression of these expression was unchanged (Fig. 3C). REST-binding sites genes may be suppressed by an HDAC complex in chicken were also found in the proximal POUV and VASA promoters PGCs. In contrast, the expression of three germ layer markers, (Supplementary Fig. S4). Otherwise, REST knockdown in- BRACHYURY, GSC, SOX7, GATA4, and SOX3, which are not creased VASA expression but not POUV expression (Fig. 3C), constitutively expressed in chicken PGCs, was unchanged even indicating REST to be involved in transcriptional regulation following romidepsin treatment (SupplementaryFig. S2B). We of VASA but not POUV. ChIP-PCR and ChIP-qPCR showed found that the germ cell tumor markers, such as AFP, MMP7, that suppression of REST increased H3K9ac at the REST- and NR6A1, were upregulated in romidepsin-treated PGCs binding site in the proximate NANOG promoter (Fig. 3D, E). (Supplementary Fig. S2C). While romidepsin-treated PGCs These results indicate that the REST repressor regulates showed downregulated expression of migration-related gene, the level of H3K9ac to control NANOG transcription in CXCR4 (Supplementary Fig. S2D). In chicken DF-1 cells, only chicken PGCs. NANOG was expressed after romidepsin treatment than that of POUV, DAZL,andVASA (Supplementary Fig. S3). The CoREST complex member RCOR3 mediates epigenetic regulation of NANOG expression HDAC inhibitor increases acetylated H3K9 levels Next, we elucidated the HDAC complex that mediates the at RE1-silencing transcription factor-binding sites change in H3K9ac in PGCs. We first determined the expression in the NANOG promoter of candidate members of the HDAC complex, including To identify the transcriptional repressor involved in metastasis-associated 1 (MTA1), MTA3, REST corepressor 1 modulation of NANOG expression by romidepsin, we pre- (RCOR1), RCOR3, SIN3 transcription regulator family dicted the regions affected by HDAC inhibition in the pu- member A (SIN3A), and SIN3B, in chicken PGCs and DF-1 tative promoter of NANOG.UpstreamofNANOG, we found cells. Reverse transcription-polymerase chain reaction (RT- a binding site of the transcriptional repressor, RE1-silencing PCR) showed that all HDAC complex members, except for transcription factor (REST), 188 bp from the TSS using SIN3A, were expressed in PGCs and DF-1 cells (Fig. 4A). MatInspector (Fig. 2A). Next, to examine the effect of the Second, the HDAC complex members MTA1, MTA3, RCOR1, HDAC inhibitor on the REST-binding site, we performed RCOR3,andSIN3B were knocked down in PGCs using spe- ChIP analysis on this site of the NANOG promoter (188 bp cific siRNAs. After successful knockdown of HDAC complex from TSS). ChIP-PCR and ChIP-qPCR analyses showed members in PGCs (Fig. 4B), we examined the expression of that romidepsin treatment resulted in a significant increase POUV, NANOG, VASA,andDAZL using RT-qPCR. Among of H3K9ac in the region nearby the REST-binding site the five members of the HDAC complex, only knockdown of in the NANOG promoter (Fig. 2B, C). Therefore, H3K9ac RCOR3 showed significant upregulation of NANOG expres- level and the expression of NANOG could be regulated by sion (Fig. 4C). In addition, repression of RCOR3 by siRNA an HDAC complex possibly via the REST-binding site in enriched the acetylated H3K9 residue on the REST site in the chicken PGCs. NANOG promoter (Fig. 4D, E). These results suggest that RCOR3, together with REST, deacetylates the region upstream REST represses NANOG expression via H3K9ac of the NANOG promoter, which represses NANOG expression in chicken PGCs in chicken PGCs.

We next performed an expression and knockdown anal- Discussion ysis of REST in chicken PGCs. RT-qPCR showed that REST Downloaded by Seoul National Univ. Medical College from www.liebertpub.com at 11/01/18. For personal use only. expression was significantly higher in PGCs than DF-1 cells NANOG is a notable homeodomain transcription factor (Fig. 3A). Subsequently, we conducted a loss-of-function involved in maintaining and establishing pluripotency in study of REST on gene expression in PGCs. REST expression mammals [11,31,32]. During somatic cell reprogramming ‰ FIG. 4. The CoREST complex member RCOR3 modulates epigenetic regulation of NANOG expression. (A) Expression of HDAC complexes in chicken PGCs and DF-1 cells by RT-PCR. (B) Effect of siRNA-mediated knockdown of HDAC complex members by RT-qPCR. (C) RT-qPCR analysis of POUV, NANOG, VASA, and DAZL expression in PGCs after knockdown of HDAC complex members. RT-qPCR was conducted in triplicate, and the relative gene expression in treatments was normalized to expression of GAPDH and ACTB. (D, E) Suppression of RCOR3 resulted in an increased level of H3K9ac at the REST-binding site in the proximal NANOG promoter. %Input = 100-fold diluted DNA before selection; IgG = control for nonspecific antibody binding to DNA. Nonspecific siRNA-treated PGCs were used as the control. Error bars represent SE of biological triplicate analyses. Significant differences between groups are indicated as ***P < 0.001, **P < 0.01, and *P < 0.05. RCOR3, REST corepressor 3; HDAC, histone deacetylase; REST, RE1-silencing transcription factor; H3K9ac, histone H3 lysine 9 acetylation; PGCs, primordial germ cells; RT-PCR, reverse transcription-polymerase chain reaction; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; IgG, immunoglobulin G; siRNA, small interfering RNA; SE, standard error. Downloaded by Seoul National Univ. Medical College from www.liebertpub.com at 11/01/18. For personal use only.

1222 THE MODERATE EXPRESSION OF NANOG IN CHICKEN PGCS 1223

FIG. 5. Epigenetic regulation of NANOG expression in chicken PGCs. Epigenetic regulation of HDAC complex controls NANOG expression in chicken PGCs. HDAC inhibitor represses deacetylation activity of the HDAC, resulting in upregulation of NANOG. The transcription factor REST binds to the upstream region of the NANOG promoter and represses NANOG expression with RCOR3 and HDAC. REST/CoREST/HDAC complex maintains moderate NANOG expression in chicken PGCs. HDAC, histone deacetylase; REST, RE1-silencing transcription factor; RCOR3, REST corepressor 3; PGCs, primordial germ cells. Color images available online at www.liebertpub.com/scd

and self-renewal in ESCs, NANOG cooperates with the mors [39]. Therefore, gene expression in PGCs may need to SIN3A/HDAC corepressor complex to activate pluripotency be regulated to the proper level. genes and repress reprogramming barrier genes [25]. In REST is a zinc finger transcription factor that represses terms of germ cell specification from the epiblast in vitro, the expression of multiple genes during embryogenesis, and NANOG activates the expressionofgermcell-specific regulates neuronal gene expression and neural stem cell transcription factors such as PRDM1 and PRDM14 as (NSC) fate in vertebrates [40–42]. In mouse, Rest has dif- an enhancer in mouse [22], suggesting a crucial role for ferential regulatory roles in ESCs and NSCs [43]. Even, Rest NANOG in germ cells. In chicken, NANOG is expressed in shared common targets with pluripotency-related factors, in- both ESCs and PGCs [18,21]; however, epigenetic regula- cluding Nanog [43], indicating the possible interaction be- tion of NANOG expression by the HDAC complex in PGCs tween REST and NANOG in other cell types. In the case of has not been investigated. chicken PGCs, to moderately regulate gene expression, Generally, acetylation of histone tails, including H3K9ac, especially NANOG, repressors such as REST seem to promote activates gene expression. In mouse, H3K9ac was increasing histone deacetylation of gene promoters. According to our sharply in PGCs at embryonic day (E) 10.5 when entering results, REST is involved in repression of NANOG expres- into genital ridges, but decreasing after settlement [3]. In sion and reduction of H3K9ac in promoter. migrating chicken PGCs, H3K9ac was restricted in foci of Furthermore, among the members of HDAC corepressor loose chromatin structures, which could be involved in germ complexes, such as CoREST [23], MTA [24], and SIN3 [25], cell-specific gene expression [10]. Treatment with an HDAC RCOR3 is a major upstream regulator of histone acetylation in inhibitor resulted in global elevation of the H3K9ac level. NANOG promoter in chicken PGCs. Mouse ESCs showed Indeed, treatment with the HDAC1/2-specific inhibitor ro- varying numbers of recruited corepressors with Rest in target midepsin resulted in a greater elevation of the H3K9ac level, sites, indicating different interactions among their complexes implying that HDAC1 or 2 could be the active deacetylase in diverse regions [44]. In terms of early neuronal differenti- in chicken PGCs. Surprisingly, although NANOG, as well as ation in mouse ESCs, the REST-CoREST complex modulates POUV and VASA, is expressed at high levels in chicken gene expression [45]. In this study, our results demonstrated PGCs, the expression was increased following the increase that the REST/CoREST/HDAC complex modulates the in H3K9ac or indirect effects induced by treatment with H3K9ac level in upstream of NANOG to regulate its moderate HDAC inhibitor. NANOG expression was also increased in expression in chicken PGCs. romidepsin-treated chicken DF-1 cells, suggesting that the In conclusion, we report that the CoREST/HDAC complex transcription of NANOG is epigenetically regulated in with REST, which binds to the NANOG promoter, functions chicken, regardless of cell type. In contrast, the expression as a transcriptional corepressor to regulate NANOG ex-

Downloaded by Seoul National Univ. Medical College from www.liebertpub.com at 11/01/18. For personal use only. levels of differentiation markers were unaffected. In mam- pression in chicken PGCs (Fig. 5). The CoREST/REST/ malian ESCs, treatment with low concentrations of HDAC HDAC repressor complex in PGCs is involved in maintaining inhibitors results in enrichment of H3K9ac to promote self- moderate expression of NANOG, which is a critical tran- renewal and pluripotency [33,34]. However, PGCs, unlike scription factor for germ cell characteristics. Therefore, ESCs, generate sperm and oocytes and transmit genetic in- regulation of NANOG expression by the REST/CoREST/ formation to the next generation; therefore, gene expression HDAC complex could be involved in maintaining the PGC in PGCs should be tightly regulated. In mammals, over- integrity. expression of NANOG causes germ cell tumors [35,36]; in contrast, suppression of NANOG induces apoptosis of germ Acknowledgments cells [14]. Correspondingly, germ cell tumor markers, such as AFP, MMP7, and NR6A1 [37], could be upregulated by This work was supported by the National Research NANOG overexpression due to romidepsin treatment in Foundation of Korea (NRF) grant funded by the Korea chicken PGCs. Also, we found that the migration-related government (MSIP) (no. NRF-2015R1A3A2033826). This gene in chicken, CXCR4 [38], was downregulated, indicat- research was also supported by the International Research & ing the possibility of aberrant migration of those treated Development Program of the National Research Foundation PGCs. The misallocated cells that could not undergo apo- of Korea (NRF) funded by the Ministry of Science, ICT and ptosis in extragonadal sites could give rise to germ cell tu- Future Planning of Korea (NRF-2016K1A3A1A21005676). 1224 JUNG ET AL.

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