Transcription factor FoxA1 plays a critical role during embryonic ... Knockdown of FoxA1 expression during P19 cell neural differentiation results in prevention.
STEM CELLS AND DEVELOPMENT Volume 19, Number 9, 2010 © Mary Ann Liebert, Inc. DOI: 10.1089/scd.2009.0386
Increased Levels of FoxA1 Transcription Factor in Pluripotent P19 Embryonal Carcinoma Cells Stimulate Neural Differentiation Yongjun Tan, Zhongqiu Xie, Miao Ding, Zhendong Wang, Qiqi Yu, Lei Meng, Hong Zhu, Xiaoqin Huang, Li Yu, Xiangxian Meng, and Yan Chen
Transcription factor FoxA1 plays a critical role during embryonic development and is activated during retinoic acid (RA)-induced neural differentiation of pluripotent P19 embryonal carcinoma cells at the early stage, which is marked by decreased expression of Nanog and increased expression of neural stem cell marker Nestin. To further understand how FoxA1 mediates neural differentiation, we have overexpressed FoxA1 through an adenovirus vector in P19 cells and identified that early neurogenesis-related sonic hedgehog (Shh) gene is activated directly by FoxA1. Knockdown of FoxA1 expression during P19 cell neural differentiation results in prevention of Shh and Nestin induction. FoxA1 binds to Shh promoter at −486 to −462 bp region and activates the promoter in cotransfection assays. Furthermore, overexpression of FoxA1 alone in P19 cells stimulates expression of Nestin and results in decreased protein levels of Nanog. During RA-induced P19 cell differentiation, elevated levels of FoxA1 increase the population of neurons, evidenced by stimulated expression of neuron-specific Neurofilament-1 and Tubulin βIII. Together, our results suggest a critical involvement of FoxA1 in stimulating neural differentiation of pluripotent stem cells at early stages.
Introduction
T
ranscription factor FoxA1 (previously known as HNF-3α) belongs to the fork head/winged-helix family of transcription factors that play important roles in cellular proliferation and differentiation during embryonic development [1–4] and also play emerging roles in cancer [5]. Expression of FoxA1 initiates during gastrulation of mouse embryogenesis in notochord, ventral floor plate of neural tube, and gut endoderm, and spreads to midbrain and spinal cord regions and to liver primordium [6–9], suggesting that FoxA1 plays a role in the early development of central nervous system and endoderm-derived organs like liver and pancreas. The range of FoxA1 expression in the adult includes tissues derived from endoderm (liver, lung, pancreas, stomach, intestine, prostate, and bladder), mesoderm (kidney, vagina and uterus, mammary glands, and seminal and coagulating glands), and neuroectoderm (brain and olfactory epithelium) [10], indicating multiple functions of FoxA1 in different adult organs. The expression pattern of FoxA1 in developing neural tube and adult brain structures
implicates its important roles in neurogenesis and brain functions. This is supported by a recent discovery in which FoxA1 was found to regulate multiple phases of midbrain dopaminergic neuron development by stimulating expression of multiple neural differentiation-related genes such as Ngn2, Nurr1, and engrailed 1, at different stages of the neuronal differentiation [11]. Mouse embryonal carcinoma (EC) cell lines are derived from teratocarcinomas and have been well characterized as pluripotent cell lines that can be maintained as undifferentiated cells and induced under controlled conditions to differentiate in vitro to any cell type of all 3 germ layers [12], providing an attractive cell model system for studying differentiation of pluripotent stem cells [13]. The P19 EC cell line was derived from a teratocarcinoma in C3H/He mice, produced by grafting an embryo at 7 days of gestation to testes of an adult male mouse [14]. The cells contain a normal karyotype, predicting the cells do not possess any gross genetic abnormalities. When injected into mouse blastocysts, P19 cells differentiate into a broad range of cell types in the
Biomedical Engineering Center and State Key Laboratory of Chemo/Biosensing and Chemometrics, Hunan University, Changsha, Hunan, China.
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1366 resulting chimeras [15]. Like embryonic stem (ES) cells, P19 cells express pluripotent marker genes such as Oct4, Nanog, and Sox2 and possess high activity of alkaline phosphatase. Teratomas formed by P19 cells in a nude mouse contain all 3 embryonic germ layers. P19 cells can differentiate in vitro into derivatives of all 3 germ layers depending on chemical treatment and growth conditions. For example, P19 cell aggregates (embryoid bodies) differentiate to cardiac and skeletal muscle when treated with dimethylsulfoxide [16,17], or neuronal and glial cells with retinoic acid (RA) treatment [16,18], similar to pluripotent ES cells. The model of RA-induced P19 cell neural differentiation has been widely used for molecular analysis of neural induction and differentiation [19–24]. As an important molecule for controlling cell growth and differentiation in both embryo and adult, RA functions by binding to ligandinducible transcription factors (nuclear receptor proteins RARs and RXRs) that activate or repress the transcription of downstream target genes [25]. The temporal patterns of gene expression during RA-induced P19 cell neural differentiation display 3 phases: the initial primary response phase (0–24 h following RA treatment), the neural differentiation phase (1–3 days following RA treatment), and the terminal differentiation phase (5–6 days following RA treatment). The expression of primary response genes is increased rapidly upon RA treatment without new protein synthesis, evidenced by in the presence of cycloheximide. Many of the primary RA response genes are transcription factors, which regulate the expression of a much larger number of other genes that display altered expression and participate in decision of neural differentiation and terminal differentiation at later time points (1–6 days) following RA treatment. FoxA1 is induced within 6 h and peaks at 1 day during RA-induced P19 cell neuronal differentiation [26], and is one of the primary targets of RA action through a RA responsive element (RARE) in its promoter [27]. Our previous data demonstrated that adenovirus-mediated increase of FoxA1 in F9 EC cells, which differentiate to visceral endoderm lineage by RA, stimulates expression of its downstream target genes that are associated with visceral endoderm differentiation [28], providing us an efficient way to study the functions of FoxA1 during pluripotent stem cell differentiation. In this study, we have investigated the possible role of FoxA1 in neural differentiation of pluripotent P19 EC cells. We have shown that in response to RA treatment, P19 cells lose their pluripotency evidenced by decreased expression of pluripotent stem cell marker Nanog [29,30], and first become neural stem-like cells, which express neural stem cell marker Nestin [31], and finally differentiate to neuronal cells. We have used the adenovirus-based FoxA1 expression vector to induce FoxA1 levels in P19 cells. The increased levels of FoxA1 stimulate the expression of Shh and Nestin, and result in decreased expression of Nanog, suggesting that FoxA1 promotes pluripotent P19 cells to become neural stem-like cells. On the other hand, knockdown of FoxA1 expression during P19 cell neural differentiation results in prevention of Shh and Nestin induction. We have further demonstrated that elevated levels of FoxA1 enhance the overall neural differentiation, evidenced by increased populations of neurons at the late time point of RA-induced P19 cell neural differentiation.
TAN ET AL.
Materials and Methods Cell culture and RA-induced neural differentiation The P19 EC cell line and mouse fibroblast BALB/3T3 cell line were purchased from ATCC. Adenovirus purification 293A cell line was purchased from Invitrogen (Carlsbad, CA). P19 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 7.5% calf serum (Gibco, Grand Island, NY), 2.5% fetal bovine serum (Gibco), and 0.5% penicillin streptomycin (Gibco) at 37°C in 5% CO2. BALB/3T3 and 293A cells were maintained in DMEM containing 10% fetal bovine serum. For neural differentiation, P19 cell aggregates were formed by placing 3 × 106 P19 cells in a 100-mm bacteriological dish (Petri dish; Falcon, Franklin Lakes, NJ) with addition of 5 × 10−7 M all-trans-RA (Sigma, St. Louis, MO) for 4 days. Subsequently, the aggregates were replated on tissue culture dishes (Corning Inc., Corning, NY) for further differentiation.
Adenovirus purifi cation and infection, and siRNA treatment The constructions of FoxA1 expression adenovirus AdFoxA1 and control virus AdLacZ or AdGFP were described previously [28]. For large-scale adenovirus purification, 20 tissue culture plates (150 × 25 mm) of 293A cells (1–2 × 107 cells/plate) were infected with adenovirus at a multiplicity of infection (m.o.i.) of 1–5 plaque-forming units (pfu)/cell. After an additional 3-day culture, the cells were harvested, resuspended in DMEM (20 mL/109 cells), and lysed by freeze-thawing (−80°C 10 min/37°C 10 min) 3 times. After 10 min centrifugation at 2,000 rpm, the supernatant of the sample was layered on a cesium chloride gradient (1.4 g/mL and 1.2 g/mL in 50 mM Tris–HCl, pH 7.8, 10 mM MgCl2) and centrifuged at 22.5K rpm for 3 h at 4°C. The viral band was removed and mixed with an equal volume of buffer (50 mM Tris–HCl, pH 7.8, 10 mM MgCl2). The sample was layered on the CsCl gradient again and centrifuged at 22.5K rpm overnight at 4°C. The viral band was removed and mixed with 4 volumes of freezing solution (0.1% BSA, 50% Glycerol, 10 mM Tris, 100 mM NaCl) and viral stocks were stored at −80°C for long-term storage. For viral infections, cultures were grown to 60%–70% confluence and washed with non-serum DMEM once, and then exposed to DMEM containing adenovirus (20 m.o.i.) for 1 h at 37°C. This medium was replaced with serum-containing DMEM and cells were cultured for additional different time courses. For siRNA treatment, mFoxA1 (HNF-3α) siRNA (sc-37931), mShh siRNA (sc-37205), and control siRNA (sc-37007) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The siRNA transfection was performed according to the manufacturer’s instructions.
Isolation of RNA, RNase protection assays, and reverse transcription polymerase chain reaction mRNA or total RNA isolated from cell cultures were routinely used for RNase protection assays (RPA) or reverse transcription polymerase chain reaction (RT-PCR), respectively. mRNA was isolated by using the Fast Track mRNA isolation kit (Invitrogen, Carlsbad, CA) and total RNA was prepared
FOXA1 STIMULATES NEURAL DIFFERENTIATION OF PLURIPOTENT CELLS by using RNAprep Pure Cell/Bacteria Kits (Tiangen Biotech, China), following the manufacturer’s instructions. For RPA, antisense RNA probes were generated by transcribing specific cDNA fragments cloned in Bluescript KSII vector using T7 RNA polymerase as described previously [32]. The following cDNAs were transcribed: rat FoxA1 (cDNA 1,388 bp–1,581 bp), mouse Shh (cDNA 604 bp–855 bp), mouse Neurofilament-1 (NF-1 cDNA 783 bp–1,083 bp), mouse glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Ambion, Foster City, CA). Hybridization reactions were carried out using the RPA II kit distributed by Ambion (Foster City, CA). For each reaction, 5 μg of oligo(dT)-selected mRNA was hybridized in hybridization buffer (80% deionized formamide, 100 mM sodium citrate, pH 6.4, 300 mM sodium acetate, pH 6.4, 1 mM EDTA) with 1 × 105 cpm of antisense RNA probe for 18 h at 45°C, following the recommendations of the kit producer. After RNase treatment, the resistant RNAs were analyzed on 5% polyacrylamide/8 M urea gels. Autoradiography of gels was performed with Kodak XAR film and Fisher Biotech L-Plus screens at −80°C for 1–4 days. For RT-PCR, the cDNAs were synthesized using RevertAid™ First Strand cDNA Synthesis Kits (Fermentas, Vilnius, Lithuania) with total RNA as templates. PCR amplification was performed with Taq DNA polymerase (Promega, Madison, WI) with following sense (S) and antisense (AS) primers, annealing temperature (Ta) and number of PCR cycles (N): mNestin-S, 5′-TCG ATG ACC TGG AGG GAC AAC-3′ and mNestin-AS, 5′-AAA TGC CTT GGG TCC TCT AGC C-3′ (Ta: 62.6°C, N: 27); mShh-S, 5′-CAA TCT GCA ACG GAA GCG AG-3′ and mShh-AS, 5′-GTG CGC TTT CCC ATC AGT TCC-3′ (Ta: 64°C, N: 35); mTubulin βIII-S, 5′-GAT GAT GAC GAG GAA TCG GAA G-3′ and mTubulin βIII-AS, 5′-AGA GGT GGC TAA AAT GGG GAG G-3′ (Ta: 58.2°C, N: 28); mGFAP-S, 5′-CAA GAG ACA GAG GAG TGG TAT CGG-3′ and mGFAP-AS, 5′-AGG AAT GGT GAT GCG GTT TTC-3′ (Ta: 59.6°C, N: 35); mFoxA1-S, 5′-AGA CAT TCA AGC GCA GCT ACC-3′ and mFoxA1-AS, 5′-GGG TCC TTG CGA CTT TCT G-3′ (Ta: 57.5°C, N: 30); rFoxA1-S, 5′-TAC GCT CCG TCC AAT CTG GG-3′ and rFoxA1-AS, 5′-TGA GTG GCG AAT GGA GTT CTG-3′ (Ta: 63.6°C, N: 30); and mCyclophilin-S 5′-GGC AAA TGC TGG ACC AAA CAC-3′ and mCyclophilin-AS 5′-TTC CTG GAC CCA AAA CGC TC-3′ (Ta: 57.5°C, N: 22).
Western blotting, immunostaining, and fl ow cytometry To measure protein levels, cell lysates were resolved by denaturing gel electrophoresis before electrotransfer to Protran nitrocellulose membrane. The membrane was subjected to western blot analysis with antibodies against proteins of interest as described previously [33]. The signals from the primary antibody were amplified by HRP-conjugated anti-mouse or anti-rabbit IgG (Bio-Rad, Hercules, CA), and detected with Enhanced Chemiluminescence Plus (ECLplus; Amersham Pharmacia Biotech, Piscataway, NJ). The following antibodies and dilutions were used for western blotting: rabbit anti-FoxA1 (1:2,000; abcam ab23738), rabbit anti-Nanog (1:2,500; Chemicon AB9220), rabbit anti-Nestin (1:2500; Chemicon AB5922), mouse anti-Tubulin βIII (1:1,000; Chemicon MAB1637), mouse anti-GFAP (1:500; Beyotime
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AG259), rabbit anti-Shh (1:200; Santa Cruz SC-9024), and mouse anti-β-Actin (1:20,000; Sigma AC-15). For immunostaining, cells at the certain time point were seeded on an 8-well chamber slide (Nalge Nunc International, Naperville, IL). After 24 h, cells were subjected to immunostaining. In brief, cells were fixed with 10% formalin (Sigma, St. Louis, MO), permeabilized with 0.2% Triton X-100, and blocked with PBS containing 1% bovine serum albumin. The mouse anti-Tubulin βIII (1:100; Chemicon MAB1637) was incubated with the cells for overnight at 4°C and then incubated with secondary chicken anti-mouse IgG-Texas Red (1:100; Santa Cruz). Cells were counterstained with 4′,6′-diamidino-2-phenylindole (DAPI) and images were captured using a TE2000 microscope (Nikon, Tokyo, Japan). For flow cytometry, cells at the certain time point were dissociated with 0.025% trypsin and 107 cells were incubated with 10 μL of antibody against protein of interest in 100 μL buffer (0.5% BSA, 2 mM EDTA, 1× PBS) for 10 min at 4°C. The cells were washed by adding 2 mL buffer and centrifuged 10 min (300g) and resuspended in 500 μL buffer. Samples were analyzed for flow cytometry on FACSCalibur (BD Biosciences, Mountain View, CA) with Prominin-1-PE antibody (Miltenyi Biotec 130-092-334).
Chromatin immunoprecipitation assays, electrophoretic mobility shift assays, and cotransfection assays Chromatin immunoprecipitation (ChIP) assays were performed following published methods with additional modifications [34]. In brief, sample cells were cross-linked in situ by addition of 37% formaldehyde (Fisher Scientific, Hampton, NH) to a final concentration of 1% (w/v) and incubated at 25°C for 15 min with gentle swirling. The cross-linking reaction was stopped by the addition of 2.5 M glycine to a final concentration of 0.125 M followed by an additional 5 min of gentle swirling. Cells were washed once with 4°C sterile PBS and collected by 2,000 rpm centrifugation for 10 min. The cell pellet was then resuspended in a 2× pellet volume of SDS lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris, pH 8.1) and placed on ice for 10 min. The resulting extract was sonicated on ice using a Scientz-IID Sonicator (Scientz, China) fitted with a 3-mm stepped micro-tip for total 8 min (3-s sonication plus 8-s pulse) at a power setting of 30%. At this stage, the processing of all experimental samples and total input was carried out according to the Upstate Cell ChIP Assay Protocol (catalog # 17-295). For immunoprecipitation, 2 μg of rabbit anti-FoxA1 (abcam ab23738) or rabbit control IgG anti-cdc25B (Santa Cruz SC-326) was added to 400 μL precleared and clarified samples, which were incubated at 4°C with rotation for 12–16 h and washed according to Upstate ChIP Assay Protocol. Cross-links were reversed on all samples, including 20% input, by addition of 100 μL TE (1 mM EDTA, 10 mM Tris–HCl, pH 7.4) containing 10 μg of RNaseA, Proteinase K (10 μg), and NaCl (4 μL of 5 M solution). Samples were digested for 16 h at 65°C. DNA was extracted from the digested samples using PCR purification columns following manufacturer’s instructions (Tiangen Biotech, China) to final volume 50 μL. Then 2.5 μL of the ChIP DNA sample or 5% total input was used in PCR with the following primers: mShh promoter −496 bp forward: 5′-GGG GAT GGG GTG TAA ATA AGG C-3′ and mShh
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System (Promega) was used to measure Luciferase enzyme activity following the manufacturer’s instructions.
Statistical analysis We used Microsoft Excel Program to calculate SD and statistically significant differences between samples with Student’s t-test. The asterisks in each graph indicate statistically significant changes with P values calculated by Student’s t-test: *P < 0.05, **P ≤ 0.01 and ***P ≤ 0.001. P values
