E XP ER I ME NTAL C E LL RE S E ARCH

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Research Article

Foxa1 contributes to the repression of Nanog expression by recruiting Grg3 during the differentiation of pluripotent P19 embryonal carcinoma cells Tuanhui Chen, Sijia He, Zhen Zhang, Wei Gao, Li Yun, Yongjun Tann State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Biology, Collaborative Innovation Center for Chemistry and Molecular Medicine, Hunan University, Hunan, China

article information

abstract

Article Chronology:

Transcription factor Foxa1 plays a critical role during neural differentiation and is induced

Received 30 March 2014

immediately after retinoic acid (RA)-initiated differentiation of pluripotent P19 embryonal

Received in revised form

carcinoma cells, correlated with the downregulated expression of pluripotency-related genes

25 April 2014

such as Nanog. To study whether Foxa1 participates in the repression of pluripotency factors, we

Accepted 28 April 2014

expressed Foxa1 ectopically in P19 cells and identified that Nanog was repressed directly by Foxa1. We confirmed that Foxa1 was able to interact with Grg3, which is a transcriptional

Keywords:

corepressor that expresses in P19 cells as well as during RA-induced P19 cell differentiation.

Foxa1 transcription factor

Knockdown of Foxa1 or Grg3 delayed the downregulation of Nanog expression during

Grg3 corepressor

RA-induced P19 cell differentiation. Furthermore, we found that Foxa1 recruited Grg3 to the

Nanog

Nanog promoter 2 kb upstream region and switched the promoter to an inactive chromatin

P19 embryonal carcinoma cells

status represented by typical modifications in histone H3. Together, our results suggested

Histone modification

a critical involvement of Foxa1 in the negative regulation of Nanog expression during the differentiation of pluripotent stem cells. & 2014 Published by Elsevier Inc.

Introduction Pluripotent stem cells can self-renew indefinitely in vitro while maintaining the ability to differentiate into advanced derivatives of all three germ layers [1]. Among the well-established pluripotent cells such as embryonic stem cells (ESCs) and embryonic germ cells (EGCs), embryonal carcinoma cells (ECCs) are derived from teratocarcinomas and have been well characterized as pluripotent cells [2]. The mouse P19 EC cell line was derived

from a teratocarcinoma in C3H/He mice by grafting a seven-day old embryo to testes of an adult male mouse [3]. Like ES cells, P19 cells express pluripotent marker genes such as Oct4, Nanog, and Sox2, and possess high activity of alkaline phosphatase [4]. The teratomas formed by P19 cells in a nude mouse contain all three embryonic germ layers [4]. Particularly, P19 cells can differentiate with retinoic acid (RA) treatment in vitro to neuronal and glial cells [5,6], which have been widely used as a model for molecular analysis of neural induction and differentiation [7–11].

n

Correspondence to: State Key Laboratory of Chemo/Biosensing and Chemometrics, Department of Biomedical Engineering, College of Biology, Collaborative Innovation Center for Chemistry and Molecular Medicine, Hunan University, 1 Denggao Rd., Changsha, Hunan 410082, China. Fax: þ86 731 888 23211. E-mail addresses: [email protected] (L. Yu), [email protected] (Y. Tan). http://dx.doi.org/10.1016/j.yexcr.2014.04.020 0014-4827/& 2014 Published by Elsevier Inc.

Please cite this article as: T. Chen, et al., Foxa1 contributes to the repression of Nanog expression by recruiting Grg3 during the differentiation of pluripotent P19 embryonal carcinoma cells, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.04.020

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Molecular control of the pluripotent state resides in a core circuitry of master transcription factors [12], such as the homeodomain-containing protein Nanog [13,14]. Nanog is highly expressed in pluripotent cells and is reduced rapidly during differentiation [13]. The expression regulation of Nanog at epigenetic levels has been well documented [11,15–18]. It has been found that in pluripotent cells, the promoter sequence of Nanog is hypomethylated [15], and the highly acetylated histone H3 is found in Nanog gene locus, where Lys 4 in histone H3 (H3K4) is hypermethylated [16–18]. This epigenetic modification status keeps the high expression of Nanog in pluripotent cells. On the other hand, during the differentiation of pluripotent cells, the Nanog promoter becomes hypermethylated [11], and the deacetylation of H3 and the demethylation of H3K4 occur in Nanog gene locus area [19]. Moreover, the hypermethylation of H3K9 (Lys 9 in histone H3) and H3K27 (Lys 27 in histone H3) can be found in Nanog gene locus [16,19]. The changes of epigenetic modifications remodel the chromatin of Nanog locus and make an irreversible inactivation of Nanog during the pluripotent cell differentiation. Dnmt3a/b and HDACs are found to be responsible for the epigenetic regulation of Nanog expression [11,20]. However, the transcription factors that mediate the genomic occupancy of these epigentic modification enzymes to Nanog gene locus largely remain to be identified. Transcription factor Foxa1 belongs to the forkhead/wingedhelix (Fox) family of transcription factors that play important roles in cellular proliferation and differentiation during embryonic development [21–24]. Typically, Foxa1 protein acts as a “pioneer“ factor in the transcriptional regulation, due to its ability to bind to highly compacted chromatins [25,26] and facilitate the binding of other transcription factors such as estrogen receptor (ER) to DNA [27]. Furthermore, Foxa1 can bind to mitotic chromosomes, functioning as “bookmarks” and facilitating the timely reactivation of target genes post-mitosis [28]. In addition, it is found that Foxa1 binds more frequently to distant enhancers, rather than proximal promoters, possibly depending on the distribution of H3K4 dimethylation (H3K4me2) preferring in the distant enhancers of promoters [29]. In most studies, Foxa1 is revealed to activate the expression of its target genes. Nonetheless, accumulated data suggest that a subset of Foxa1 bindings also result in the repression of its target gene transcription [30,31]. Recently, Foxa1 is reported to interact with Grg3 [32], a transcriptional corepressor of Gro/TLE/Grg family, whose complex with specific DNA-binding proteins causes gene repression [33]. We have previously discovered that Foxal is a critical stimulator of RA-induced P19 cell neural differentiation and the expression of Foxa1 is induced immediately following the RA treatment. The upregulation of Foxal is ahead of the reduced expression of pluripotent transcription factor Nanog during P19 cell differentiation [34]. In this study, we investigated whether Foxa1 mediated the repression of Nanog transcription directly during P19 cell differentiation. We found that the ectopic expression of Foxa1 alone resulted in the decreased levels of Nanog expression in P19 cells. We determined that Foxa1 was able to interact with Grg3 and the knockdown of Foxa1 or Grg3 delayed the downregulation of Nanog expression during RA-induced P19 cell differentiation. Foxa1 recruited Grg3 to the Nanog promoter  2 kb upstream region and switched the promoter to an inactive chromatin status. Together, our results suggested a critical involvement of Foxa1 in

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the negative regulation of Nanog expression during the differentiation of pluripotent stem cells.

Materials and methods Cell culture and RA-induced P19 cell differentiation P19 EC cell line was purchased from ATCC (USA) and 293A cell line was purchased from Invitrogen (USA). P19 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) containing 7.5% calf serum, 2.5% fetal bovine serum (Gibco, USA) and 0.5% penicillin streptomycin (Gibco, USA) at 37 1C in 5% CO2. 293A cells were maintained in DMEM containing 10% fetal bovine serum. For RA-induced differentiation, P19 cell aggregates were formed by placing 3  106 cells in a 100 mm bacteriological dish (Petri dish) (Greiner, Germany) with addition of 5  10  7 M all-trans-RA (Sigma, USA).

Isolation RNA, reverse transcription, and quantitative real-time PCR (qPCR) The total RNA was isolated by Total RNA Kit (Omega, USA) according to the manufacturer's protocols. The cDNAs were synthesized with M-MLV Reverse Transcriptase (Promega, USA) from total RNA samples. qPCR was performed with SYBR Green (TOYOBO, Japan) in the realplex2 qPCR system (Eppendorf, Germany). PCR amplification was performed with following sense (S) and antisense (AS) primers: mNanog-S, 50 -CCA GGT TCC TTC CTT CTT CC-30 and mNanog-AS, 50 -GGT GAG ATG GCT CAG TGG AT30 ; mFoxa1-S, 50 -GGA TGG TTG TGT CGG CCG GG-30 and mFoxa1AS, 50 -GGA CTC AGG CCG GCC CCT AA-30 ; mGrg3-S, 50 -GAT TTT GCC TGC TGT ATT TG-30 and mGrg3-AS, 50 -TGT CCA TCT GCC TAT CAT TC-30 ; mGapdh-S, 50 -AGG TCG GTG TGA ACG GAT TTG-30 and mGapdh-AS, 50 -TGT AGA CCA TGT AGT TGA GGT CA-30 . Each sample was analyzed in triplicate with mGapdh as the internal control.

Western blot assays To measure protein levels, cell lysates were resolved by denaturing gel electrophoresis before electrotransfering to PVDF membrane (Millipore, USA). The membrane was subjected to Western blot analysis with antibodies against proteins of interest. The signals from the primary antibody were amplified by horse radish peroxidase (HRP)-conjugated anti-mouse IgG (1:10000; GE LNA931VAD, USA) or anti-rabbit IgG (1:10000; GE LNA934VAE, USA), and detected with Enhanced Chemiluminescence Plus (Beyotime, China). The following antibodies and dilutions were used: rabbit anti-Nanog (1:1000; Millipore AB9220, USA), rabbit anti-Foxa1 (1:1000; Abcam ab237438, UK), rabbit anti-Grg3 (1:1000; Santa cruz sc-9124, USA), rabbit anti-V5 (1:2000; Abcam ab15828, UK), mouse anti-β-actin (1:5000; Beyotime AA128, China).

Chromatin immunoprecipitation assays Chromatin immunoprecipitation (ChIP) assays were performed as previously described [34]. The following antibodies were used for immunoprecipitation: rabbit anti-H3K9ac (Millipore 17-615, USA),

Please cite this article as: T. Chen, et al., Foxa1 contributes to the repression of Nanog expression by recruiting Grg3 during the differentiation of pluripotent P19 embryonal carcinoma cells, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.04.020

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rabbit anti-H3K4me2 (Millipore 07-030, USA), rabbit antiH3K27me3 (Millipore 17-622, USA), rabbit anti-IgG (Millipore PP64, USA), rabbit anti-Foxa1 (Abcam ab237438, UK), and rabbit anti-Grg3 (Santa cruz sc-9124, USA). For immunoprecipitation, 2 μg of each interested antibody was used. The ChIP DNA sample or 1% total input was used in qPCR with the following primers: mNanog upstream  2060 bp forward: 50 -GTT CCA GGA CAG CCA GAG-30 and  1879 bp backward: 50 -CAG CCT GAC TGA ACC TCT TT-30 ; mNanog upstream 177 bp forward: 50 -AGT CTG GGT CAC CTT ACA GC-30 and þ47 bp backward: 50 -ACC AAC CAA ATC AGC CTA TC-30 .

DNA methylation assays The total genomic DNA was isolated by genomic DNA Kit (Tiangen, China) according to the manufacturer's protocols. Genomic DNA (500 ng) of each sample was treated with sodium bisulfate from EZDNA methylation kit (Zymo Research, USA) according to the manufacturer's protocols. Treated DNA was subjected to nested PCR with two pairs of primers of Nanog promoter (Outside:  620 bp to þ91 bp; Inside:  510 bp to þ43 bp) as follow: Outside-S, 50 -AGG TAA TAG AGA AAA ATT TGT TTT AAA-30 and Outside-AS, 50 -AAT ACA AAA TTA TCA AAA AAT CAA AA-30 ; InsideS, 50 -GTA TTT TTG GAG GGA AGA TTT TT-30 and Inside-AS, 50 -ACA AAA AAA ACA AAA CAC CAA CC-30 . For sequencing analysis, the PCR products were cloned into T-vectors (Invitrogen, USA) and individual clones sequenced by Sangon (Shanghai) Co., Ltd, China.

Plasmid construction and siRNA treatment The cDNA of Foxa1 (Gene ID: 15375) was PCR amplified by Pfu DNA polymerase (Fermentas, Canada) from the template of mouse genome, then the cDNA was inserted into pcDNA3.1 vector (Invitrogen, USA) and the sequence of the cDNA was confirmed by Sequencing Analysis (Sangon Co., Ltd, China). The cDNA of mouse Grg3 (Gene ID: 21887) was purchased from Changsha Yingrun Biotechnology Co., Ltd., China and cloned to pcDNA3.1 vector with a V5 tag in C-terminal. The pEGFP-Foxa1 plasmid was maintained in our lab [35]. Lipofectamine 2000 (Invitrogen, USA) was used for transfection experiments following the manufacturer's instructions. For siRNA treatment, Foxa1 siRNA (sc-37931), Grg3 siRNA (sc36684), and control siRNA (sc-37007) were purchased from Santa Cruz Biotechnology, Inc. (USA). The siRNA transfection was performed according to the manufacturer's instructions.

Electrophoretic mobility shift assays (EMSA) For EMSA experiments, FAM-labeled double-strand DNA oligonucleotides were synthesized by Sangon (Shanghai) Co., Ltd, China, based on the sequence 50 -TTT TTT GTT TGT TTG TTT GGT TGG T-30 from Nanog upstream region ( 2025/ 2001 bp), which contained the Foxa1 consensus binding site (underlined). In the binding reactions, nuclear proteins (10 μg) isolated from cells were incubated with 1 pmol of the FAM-labeled probe, 2 μl of 5  binding buffer (Beyotime, China) in a total volume of 10 μl for 30 min at room temperature. The reactions were resolved in 4% native polyacrylamide gel electrophoresis in 0.5  TBE. In all of the EMSA experiments, the dose chosen for the competitive experiments was in the 100  molar excess. The unlabeled

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oligonucleotides for Foxa1 or Foxa1-mut (50 -TTT TTT GTC CAG CCA CCT GGT TGG T-30 ) were used as competitors or mutatedcompetitors. For the super-shift analysis, 1 μg of anti-Foxa1 antibody (Abcam ab237438, UK)) was added to the binding reaction.

Immuno-fluorescent staining Cells were fixed with 4% formaldehyde in PBS for 5 min, permeabilized with 1% Triton X-100 in PBS for 10 min, and blocked with 5% BSA in PBS for 1 h. The rabbit anti-V5 (1:200; Abcam ab15828, UK) was incubated with the cells for overnight at 4 1C and then incubated with secondary bovine anti-rabbit IgG-Texas Red (1:200; Santa Cruz sc-2787, USA). Cells were counterstained with 40 ,60 -diamidino-2-phenylindole (DAPI) and images were captured using a confocal laser scanning microscope FV500-IX70 (Olympus, Japan).

Co-immunoprecipitation For Co-immunoprecipitation (Co-IP), 293A cells were plated in 10cm dishes, transfected with pFoxa1, pGrg3-V5, or the two plasmids together. Two days later, the cells were collected and re-suspended in 500 μl of lysis buffer (150 mM NaCl, 1% NP-40, and 50 mM Tris–HCl PH7.4, protease inhibitor cocktail) and incubated for 20 min on ice. The lysates were centrifuged for 15 min at 14,000g at 4 1C, and the supernatant containing 500 μg of proteins was incubated with 2 μg anti-V5 antibody (Abcam ab15828, UK) overnight. Twenty μl of protein A/G plus-Agarose (Santa Cruz sc-2003, USA) was added to the sample and incubated for 1 h. The Agarose beads were centrifuged and washed 4 times with the lysis buffer. The washed beads were subjected to SDSPAGE followed by immunoblotting with anti-Foxa1 (1:1000; Abcam ab237438, UK) and anti-V5 (1:2000; Abcam ab15828, UK).

Luciferase assays For luciferase assays, the mouse Nanog upstream promoter regions were PCR amplified from mouse genomic DNA with the following primers: mNanog 2243 bp MluI: 50 -GTG acgcgt GGT CAA CCA GCC ACA TTA-30 and mNanog þ39 bp BglII: 50 -GCC agatct TTC CCA CAG AAA GAG CAA-30 and cloned into the corresponding MluI and BglII sites of the pGL3 basic Luciferase vector (Promega, USA). P19 cells (2  105 cells per well in a 6-well plate) were cotransfected with the luciferase reporter construct (1 μg) and pFoxa1 or pGrg3-V5. Twenty ng of pRL-CMV plasmid was used as the loading control for each transfection. The luciferase enzyme activities were measured 48 h later with the Dual-Luciferase Assay System (Promega, USA) following the manufacturer's instructions.

Statistical analysis We used Microsoft Excel Program to calculate SD and statistically significant differences between samples. The asterisks in each graph indicate statistically significant changes with P values calculated by Student T Test: nPo0.05, nnPr 0.01 and nnnPr 0.001. P values o0.05 were considered statistically significant.

Please cite this article as: T. Chen, et al., Foxa1 contributes to the repression of Nanog expression by recruiting Grg3 during the differentiation of pluripotent P19 embryonal carcinoma cells, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.04.020

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Results Nanog was silenced during RA-induced P19 cell differentiation Nanog is highly expressed in pluripotent cells and is reduced rapidly during differentiation [13]. To confirm the gene expression change of Nanog during RA-induced P19 cell differentiation, we measured the expression levels of Nanog mRNA and protein at different time points after RA treatment in P19 cell aggregates. The mRNA and protein levels of Nanog decreased dramatically when P19 cells started to differentiate as predicted (Fig. 1A and B).

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To test whether the RA treatment in P19 cells mediated epigenetic modifications in the chromatin of Nanog gene area, we analyzed the status of histone H3 modifications and DNA methylation at Nanog locus in P19 cells or P19 cells treated by RA for 2 days. Chromatin immunoprecipitation (ChIP) assays were used to measure the level changes of H3K9 acetylation, H3K4 or H3K27 methylation at  2 kb upstream region or 200 bp proximal region of Nanog promoter after RA treatment. The decreased levels of H3K9 acetylation and H3K4 methylation, and the increased levels of H3K27 methylation were found in both of the tested regions of Nanog in the RA-treated cells (Fig. 1C), suggesting that the RA treatment in P19 cell aggregates resulted in a chromatin remodeling of Nanog locus to inactivate its

Fig. 1 – Nanog was silenced during RA-induced P19 cell differentiation. (A) and (B) Gene expression analysis of Nanog at different time points during RA-induced P19 cell differentiation of by qPCR (A) or western blotting (B). Gapdh and β-actin were used as the loading control for qPCR and western blotting respectively. (C) The histone H3 modifications of Nanog locus were changed during RA-induced P19 cell differentiation. The chromatin of P19 cells, RA-induced (2days) P19 cells was cross-linked, sonicated, and then immunoprecipitated with indicated antibodies. qPCR was used to measure Nanog promoter fragments 2060 bp to 1879 bp and 177 bp to þ47 bp. Data were represented as the percentage of input and an average of three independent experiments. (D) DNA methylation levels of Nanog promoter increased during RA-induced P19 cell differentiation. Genomic DNA was extracted from undifferentiated P19 cells or RA-induced (4 days) P19 cells. PCR products amplified from bisulfite-treated genomic DNA were cloned and sequenced to reveal the methylation status of individual CpG sites in Nanog promoter region ( 510 to þ43 bp). Filled circles indicated the methylated CpG sites. Please cite this article as: T. Chen, et al., Foxa1 contributes to the repression of Nanog expression by recruiting Grg3 during the differentiation of pluripotent P19 embryonal carcinoma cells, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.04.020

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expression. Furthermore, bisulfite sequencing analyses were used to measure the levels of DNA methylation at Nanog proximal promoter. At day 4 post RA treatment, the levels of DNA methylation at the tested Nanog promoter region increased dramatically (Fig. 1D, Fig. S1-S2), indicating a process of de novo methylation at Nanog promoter accompanying its gene silencing during P19 cell differentiation. Taken together, our data confirmed that Nanog was silenced by epigenetic changes during RA-induced differentiation of P19 EC cells, similar to the published data during the differentiation of ESCs [11].

Foxa1 and Grg3 were required for the down regulation of Nanog expression during P19 cell differentiation We discovered that Foxal was induced immediately following the RA treatment and the upregulation of Foxal was ahead of the reduced expression of Nanog during P19 cell differentiation [34]. Foxa1 was able to interact with the transcriptional corepressor Grg3 [32], providing a mechanism of Foxa1 to repress the transcription of its target genes. As the previous report, the expression of Foxa1 increased dramatically following RA treatment in P19 cells, while the Grg3 was expressed robustly in P19 cells and the RA treatment did not cause obvious changes of Grg3 expression (Fig. 2A and B). To test the hypothesis that Foxa1 might act as a negative regulator of Nanog expression by recruiting Grg3 to Nanog promoter region, we scanned  5 kb upstream region of mouse Nanog gene with the Foxa1 DNA binding consensus

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sequence and found multiple tandem Foxa1 putative binding sites at the  2020 bp to  2004 bp region of Nanog. We performed ChIP assays to test whether the bindings of Foxa1 and Grg3 existed at this region of endogenous Nanog promoter. The crosslinked chromatin fragments of P19 cells or RA-treated (2 days) P19 cells were immunoprecipitated (IP) with Foxa1 or Grg3 antibodies, and the IgG antibodies were used as the IP control. The amounts of Nanog promoter fragments associated with Foxa1 or Grg3 were measured by qPCR with primers specific to Nanog promoter region  2060 bp to  1879 bp. Compared to the P19 cell samples showing no obvious Foxa1 binding on the tested region, the RA-treated P19 cell samples showed an elevated binding activities of Foxa1 on this region of Nanog promoter (Fig. 2C). Furthermore, the differentiation of P19 cells caused a dramatic increase of Grg3 binding on this region of Nanog promoter (Fig. 2C). Grg3 could not bind to DNA by itself [33] and it was expressed in P19 EC cells. These findings suggested that the increased binding of Grg3 on Nanog promoter was probably mediated by DNA binding proteins such as Foxa1, which was able to interact with Grg3 and was induced at the early stage of P19 cell differentiation. This idea was confirmed by the ectopic expression of Foxa1 in P19 stem cells, in which both the mRNA and protein levels of Nanog were suppressed by the Foxa1 overexpression (Fig. 2D and E). We noticed that the effects of Foxa1 overexpression on Nanog mRNA levels (decreased around 8 fold in Fig. 2D) were more profound than that on Nanog protein levels (decreased around 4 fold in Fig. 2E), implicating a possible

Fig. 2 – Foxa1 and Grg3 were required for the downregulation of Nanog expression during P19 cell differentiation. (A) and (B) The expression analysis of Foxa1 and Grg3 at different time points during RA-induced P19 cells differentiation by qPCR (A) or western blotting (B). (C) FoxA1 and Grg3 bound to Nanog promoter in vivo. ChIPs were performed with anti-Foxa1, anti-Grg3 or normal IgG for the chromatins from P19 cells, or RA-induced (2 days) P19 cells. qPCR was used to measure Nanog promoter fragment  2060 bp to  1879 bp. Data were represented as the percentage of input and an average of three independent experiments. (D-E) The expression of Nanog was inhibited by ectopic expression of Foxa1 in P19 cells. P19 cells were transfected with Foxa1 expression vector or a pcDNA vector and total RNA or protein extracts were prepared 2 days after transfection. qPCR was used to measure the mRNA levels of Foxa1 and Nanog (D). The protein levels of Foxa1, Nanog, and β-actin were measured by western blotting (E). (F) and (G) Knockdown of Foxa1 or Grg3 during RA-induced P19 cell differentiation prevented the downregulation of Nanog expression. P19 cells were transfected with Foxa1 siRNA, Grg3 siRNA, or control siRNA one day before RA-induced differentiation. Total RNAs or protein extracts were prepared 2 days after RA treatment. Gene expression analysis of indicated genes was performed by qPCR (F) or western blotting (G). Please cite this article as: T. Chen, et al., Foxa1 contributes to the repression of Nanog expression by recruiting Grg3 during the differentiation of pluripotent P19 embryonal carcinoma cells, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.04.020

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difference in the stability between Nanog mRNA and its protein. To further confirm that Foxa1 and Grg3 participated in the repression of Nanog during RA-induced P19 cell differentiation, we knocked down the levels of Foxa1 or Grg3 one day before the RA treatment in P19 cells. The transfection of Foxa1-specific or Grg3-specific siRNAs resulted in an effective knockdown of Foxa1 or Grg3 respectively in P19 cells (Fig. 2F and G). The knockdown of Foxa1 or Grg3 was able to prevent the downregulation of Nanog expression induced by RA-treatment in P19 cells (Fig. 2F and G). Together, these data implicated the crucial roles of Foxa1 and Grg3 in the repression of Nanog during RA-induced differentiation of pluripotent stem cells.

Foxa1 interacted with Grg3 and repressed Nanog promoter activities The Nanog promoter contained multiple tandem Foxa1 putative binding sites at the 2020 bp to 2004 bp region, where could be bound by endogenous Foxa1 and Grg3 during P19 cell differentiation (see above). To further confirm the binding of Foxa1 and Grg3 at this region of Nanog promoter, we performed Electrophoretic Mobility Shift Assays (EMSAs) with a FAM-labeled DNA probe synthesized from the mouse Nanog promoter region from  2025 bp to  2001 bp and the nuclear extracts containing Foxa1 or Grg3 proteins. We found that the probe could form a DNA/protein complex in EMSAs with Foxa1 protein, and the addition of an unlabeled probe (100  ) or Foxa1-specific antibody disturbed the formation of Foxa1/DNA complex while the addition of an unlabeled mutated probe (100  ) had no affects on the Foxa1/DNA complex formation (Fig. 3A). On the other hand, we found that Grg3 was not able to bind to the probe (Fig. S3), suggesting that Grg3 could not bind to this region of Nanog promoter by itself. We tried to perform the EMSA with nuclear extracts containing both Foxa1 and Grg3 together and found that Foxa1 was not able to recruit Grg3 onto this DNA probe (data not shown), consistent with the previous finding that Foxa1 could recruit Grg3 to chromatin but not to free DNA [32]. To confirm that Foxa1 interacted with Grg3, we performed co-immunoprecipitation (Co-IP) with the lysates from 293A cells, in which the exogenous Foxa1 and V5-tagged Grg3 (Grg3-V5) were co-expressed. With the immunoprecipitation of a V5-tag specific antibody, a strong interaction between Grg3 and Foxa1 was observed (Fig. 3B). This interaction between Grg3 and Foxa1 was further confirmed by the co-localization of these two proteins in the cell nucleus of 293A cells, in which Foxa1-GFP fusion protein and Grg3-V5 were co-expressed, through the direct GFP fluorescence and the immuno-fluorescent staining with V5 antibody (Fig. 3C). To test whether Grg3 mediated the repression of Nanog promoter bound by Foxa1, a luciferase reporter plasmid containing  2.3 kb of mouse Nanog promoter region was constructed and transfected into P19 cells with the Foxa1 expression vector, plus an increased amount of Grg3 expression vector. We found that the increased levels of Grg3 resulted in a continued decrease of the Nanog promoter activities when Foxa1 existed at the same levels (Fig. 3D) but the increased levels of Grg3 alone did not affect the activities of this promoter in P19 stem cells (Fig. S4). These results suggested that Foxa1 directly bound to Nanog promoter and the interaction between Foxa1 and Grg3 mediated the repression of Nanog promoter by Foxa1.

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The effects of elevated Foxa1 levels on the epigenetic modifications of Nanog promoter Grg proteins have been found to inhibit the transcriptional machinery by recruiting HDACs or PRC2 proteins to the locus of certain genes [36–38] and Foxa1 is also able to interact with HDACs when it acts as a repressor for its target genes [31]. It was worthy to test whether elevating Foxa1 levels would mediate the histone modifications of Nanog gene locus to repress its transcription in P19 cells. We also intended to test whether Foxa1 would affect the DNA methylation status of Nanog promoter. We transfected Foxa1 expression vector to P19 cells and measured these epigenetic modification changes after Foxa1 overexpression. First, we performed ChIP assays to show that elevated Foxa1 levels resulted in the increased binding of endogenous Grg3 on Nanog promoter region  2060 bp to 1879 bp (Fig. 4A), further confirming that Foxa1 recruited Grg3 to this region. Consequently, the increased binding events resulted in the changes of histone H3 modification, such as H3K9 deacetylation, H3K4 demethylation, and H3K27 methylation at this Nanog promoter region (Fig. 4B). On the other hand, the DNA hypomethylation status of Nanog promoter in P19 stem cells showed no obvious change post 4 days of Foxa1 overexpression (Fig. S5 and S6). Taken together, these data suggested that Foxa1 mediated the repression of Nanog transcription probably through altering the histone modifications at Nanog locus by recruiting Grg3 in P19 cells (Fig. 4C).

Discussion Pluripotent stem cells can be propagated in culture in an undifferentiated state and are tightly regulated by the core transcription factors such as Oct4, Sox2, and Nanog [39]. The differentiation of stem cells can be view as a process in which epigenetic changes result in alteration in gene expression of the cells after they become more specialized. For example, when treated with RA, epigenetic changes including DNA methylation and histone modifications are induced in stem cells and these epigenetic changes can create heritable changes in gene expression [40]. In this study, we have observed that pluripotencyrelated Nanog is silenced through DNA methylation and histone modifications during RA-induced P19 cell differentiation. We have found that these epigenetic changes of Nanog are partially mediated by the recruitment of Grg3 to Nanog promoter through the binding of Foxa1 at this promoter region (Fig. 4C). RA-induced differentiation initiates the Foxa1 expression, which is correlated with the decreased expression of many of pluripotency-related genes, in P19 cells. As an important molecule for controlling cell growth and differentiation in both embryo and adult, RA functions by binding to ligand-inducible transcription factors (nuclear receptor proteins RARs and RXRs) that regulate the transcription of RA primary response genes [41]. It occurs rapidly within minutes to a few hours after RA addition. As one of primary targets of RA action, Foxa1 expression is stimulated within 6 h and peaks at 1 day during RA-induced P19 cell differentiation [42], through a RA responsive element (RARE) on its promoter [43]. Our previous data have demonstrated that Foxa1 expression is essential for the neural differentiation of RA-induced P19 cells and ectopic expression of Foxa1 alone promotes P19 cells to become neural stem-like cells [34].

Please cite this article as: T. Chen, et al., Foxa1 contributes to the repression of Nanog expression by recruiting Grg3 during the differentiation of pluripotent P19 embryonal carcinoma cells, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.04.020

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Fig. 3 – Foxa1 interacted with Grg3 and repressed Nanog promoter activities. (A) FoxA1 bound to Nanog promoter region 2025 bp to 2001 bp. Nuclear extracts were prepared from Foxa1 expression vector-transfected P19 cells (2 days) and used for EMSAs with a FAM-labeled DNA probe synthesized from Nanog promoter sequence  2025 bp to  2001 bp. The unlabeled probe (100  ), the unlabeled mutated probe (100  ), or 1 μg of Foxa1 antibody was added to the reaction to show specificity of Foxa1/DNA complex formation. (B-C) Foxa1 interacted with Grg3 in vivo. 293A cells were transfected with Foxa1 expression vector, Grg3-V5 expression vector, or both. Two days after transcfection, the cells were harvested and lysed for immunoprecipitation with anti-V5 antibody. Western blotting was performed with anti-Foxa1 and anti-V5 antibodies. One percent of input of lysates (500 μg) was also loaded on the gel (B). 293A cells were transfected with Foxa1-GFP expression vector and Grg3-V5 expression vector. Two days after transfection, the cells were immunostained with anti-V5 antibody and secondary anti-rabbit IgG-Texas Red. Pictures were captured using confocal microscope (C). (D) Grg3 mediated the repression of Nanog promoter bound by Foxa1. The 2.3 kb Nanog promoter-luciferase reporter plasmid (1 μg) and loading control pRL-CMV luciferase reporter plasmid (20 ng) were cotransfected into P19 cells with Foxa1 expression vector (0.2 μg) and Grg3-V5 expression vector (increasing amounts: 0, 0.1, 0.2, 0.5 μg). Protein lysates were prepared at 48 h after transfection and used for the measurement of dual Luciferase activities. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Interestingly, ectopic expression of Foxa1 alone in P19 cells also decreases the expression levels of Nanog, suggesting a mechanism of Foxa1-mediated transcriptional repression. Foxa1 is able to scan chromatin and bind to its recognition motif in condensed DNA structures [26,44]. Of note, the epigenetic marker H3K4me2, which correlates with active enhancers in promoters [45], is thought to facilitate Foxa1 binding [29].

Consistently, Foxa1 binding occurs primarily in intergenic enhancers in a given cell type [29]. In most studies, Foxa1 has been revealed to activate the expression of its target genes. However, in a recent study, a significant fraction of Foxa1-bound sites has been identified to have a relatively closed chromatin conformation, linked to the epigenetic signature toward repressive histone markers [30]. Gro/TLE/Grg proteins, which inhibit the transcriptional machinery

Please cite this article as: T. Chen, et al., Foxa1 contributes to the repression of Nanog expression by recruiting Grg3 during the differentiation of pluripotent P19 embryonal carcinoma cells, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.04.020

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Fig. 4 – The overexpression of Foxa1 alone in P19 cells altered the histone modifications of Nanog locus. (A) The ectopic expression of Foxa1 recruited Grg3 to Nanog promoter. P19 cells were transfected with pcDNA or Foxa1 expression vector. Two days after transfection, the cross-linked chromatin fragments of the cells were immunoprecipitated with anti-Foxa1 or anti-Grg3 antibodies. qPCR was used to measure Nanog promoter fragments  2060 bp to 1879 bp. Data were represented as the percentage of input and an average of three independent experiments. (B) The overexpression of Foxa1 alone resulted in the changes of histone H3 modification of Nanog promoter in P19 cells. ChIP assays were performed to measure the levels of H3K9ac, H3K4me2, and H3K27me3 around Nanog promoter region 2060 bp to  1879 bp in Foxa1-overexpressed P19 cells. (C) Model depicting the repression of Nanog transcription by Foxa1 altering the histone modifications at Nanog locus during RA-induced P19 cell differentiation. Foxa1 was stimulated immediately after RA induction and then bound to Nanog promoter around 2 kb region. Subsequently Foxa1 recruited Grg3 to this region. Grg3 was able to interact with HDACs and PRC2 proteins, which caused the histone modification changes at Nanog locus.

by recruiting HDACs or PRC2 proteins to the locus of certain genes [36–38], interact with Foxa1 and switch Foxa1 from an activator to a repressor of transcription [32]. In this study, we confirmed that Foxa1 interacted with Grg3 and recruited Grg3 to Nanog promoter regions to cause histone modification changes and the following repression of Nanog during P19 cell differentiation. After Foxa1 was stimulated by RA treatment, it bound to its binding sites in  2 kb Nanog promoter region and subsequently recruited Grg3 to this region. With the abilities of interacting with HDACs and PRC2 proteins, Grg3 recruited these histone modification enzymes to this region and elicited the histone modification changes at Nanog locus. The repression of Nanog is also mediated by DNA hypermethylation of its promoter during pluripotent stem cell differentiation [11]. During RA-induced P19 cell differentiation, both DNA methylation and repressive histone modifications around Nanog locus have happened but ectopic expression of Foxa1 in P19 cells does not affect the epigenetic signature of DNA methylation status

at Nanog locus. It is proposed that Nanog promoter undergoes de novo methylation by Dnmt3a/b during stem cell differentiation [11] but the transcription factors mediating the recruitment of Dnmt3a/b to Nanog promoter are waiting to be identified. Therefore, how to achieve the specific methylation in Nanog promoter during stem cell differentiation needs to be further explored.

Conflict of interest statement The authors declare no conflict of interest.

Acknowledgments This work was supported by Natural Science Foundation of China [grant number 81171949, 31161160558 to Y.T.]; and the Ministry of Science and Technology of China [grant number 2010DFB30300].

Please cite this article as: T. Chen, et al., Foxa1 contributes to the repression of Nanog expression by recruiting Grg3 during the differentiation of pluripotent P19 embryonal carcinoma cells, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.04.020

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Appendix A.

Supporting information

Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.yexcr.2014.04.020.

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Please cite this article as: T. Chen, et al., Foxa1 contributes to the repression of Nanog expression by recruiting Grg3 during the differentiation of pluripotent P19 embryonal carcinoma cells, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.04.020

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Foxa1 contributes to the repression of Nanog expression by recruiting Grg3 during the differentiation of pluripotent P19 embryonal carcinoma cells.

Transcription factor Foxa1 plays a critical role during neural differentiation and is induced immediately after retinoic acid (RA)-initiated different...
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