NONO Is a Negative Regulator of SOX2 Promoter

Materials and Methods

Cell lines. Human breast cancer cell lines, MCF-7 and T-47D were obtained from American Type Culture Collection (Manassas, VA, USA). These cell lines were cultured in Dulbecco's modified Eagle medium/Ham's Nutrient Mixture F-12 (DMEM/F-12) (Wako Pure Chemical, Osaka, Japan) supplemented with heat-inactivated 10% fetal bovine serum (Cambrex, East Rutherford, NJ, USA), 100 units/ml penicillin and 100 μg/ml streptomycin sulfate (both from Invitrogen, Carlsbad, CA, USA), and maintained at 37°C with 5% CO₂.

Identification of the region essential for transcriptional activity in the SOX2 promoter. To identify SOX2 promoter essential region, luciferase assay was used. The human SOX2 promoter region was amplified from genomic DNA of MCF-7 cells (5). It was amplified using KOD-Plus-Neo DNA polymerase with proof reading activity (TOYOBO, Osaka, Japan) and 0.3 μM of specific primers. The sequences of the primers used were: P789 forward: 5’-GGTACCGGCCAAAGAGCTGAGTTGG-3’, P629 forward: 5’-GGTACCAACTTCTAGTCGGGACTGTG-3’, P467 forward: 5’-GGTACCCTGGCTGTTTCCAGAAATAC-3’, P227 forward: 5’-GGTACCCTCAGTGGCTGGCAGGC-3’, P68 forward: 5’-GGTACCGCTGATTGGTCGCTAGAAAC-3’; reverse: 5’-AAGCTTGAGGCAAACTGGAATCAGGATC-3’). Thermal cycling procedure consisted of an initial denaturation for 2 min at 94°C. This was followed by 30 cycles of denaturation at 98°C for 10 s, annealing at 55-58°C for 30 s and extension at 68°C for 30 s. A-Tailing was then carried out by incubating 5.9 μl of the polymerase chain reaction (PCR) products with 1 μl of GoTaq polymerase (Promega, Madison, WI, USA), 2 μl of 5 × Go Taq reaction buffer, 0.6 μl of 2.5 mM MgCl₂, and 0.5 μl of 4 mM dATP for 30 min at 70°C. The A-tailed PCR products were then ligated into pGEM-T easy vectors (Promega), which were further digested with KpnI and HindIII (TAKARA Bio, Shiga, Japan) enzymes. Fragments obtained from the restriction digestion were inserted into pGL3-Basic luciferase vector (Promega). An electroporator was used to co-transfect 1 μg of generated vectors and 25 ng of the internal control, pRL-TK vector (Promega) into 5×10⁵ cells with or without 0.5 μg of complementary DNA (cDNA) expression vectors (CUY21SC; NepaGene, Ichikawa, Japan). Transfected cells were seeded into one well. Forty-eight hours after transfection, luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega) with a luminometer MiniLumat (Berthold, Bad Wildbad, Germany). The results were normalized by dividing the luciferase reporter activity by that of the Renilla reporter, which were then statistically compared among the groups using Mann–Whitney U-test.

Biotin–streptavidin pulldown assay. To isolate SOX2 promoter activity regulatory factors, biotin–streptavidin pulldown assay was performed. Nuclear fractions (100 μl) were prepared from 2×10⁶ MCF-7 cells using NE-PER Nuclear and Cytoplasmic Extraction Reagent following the manufacturer's instructions (Thermo Fisher Scientific, Pittsburg, PA, USA). Subsequently, 100 μl of the nuclear fraction was incubated with 17 μl of Streptavidin Mag Sepharose (GE Healthcare Bioscience, Pittsburgh, PA, USA) at 4°C for 2 h in 1 ml of binding buffer [20% glycerol, 20 mM Tris pH 7.5, 100 mM KCl, 1 mM dithiothreitol (DTT), 20 μg/ml bovine serum albumin (BSA), 2 mM EDTA] on a shaking incubator. After incubation, the mixtures were centrifuged at 5,000 × g for 1 min at 4°C. Supernatants were transferred to a fresh tube as the nuclear fractions were removed by avidin binding protein. Mag Sepharose-bound complex was washed twice with 100 μl of binding buffer without BSA and washed buffers were stored at −80°C. Human SOX2 promoter-derived P227 and P68 regions were then amplified from MCF-7 genomic DNA using KOD Plus-Neo with biotin-labeled primers for P227 (forward, 5’-biotin-CTCAGTGGCTGGCAGGCTGG-3’), P68 (forward: 5’-biotin-GCTGATTGGTCGCTAGAAACC-3’), and reverse: 5’-GAGGCAAACTGGAATCAGGATC-3’. The nuclear fractions bound to avidin binding protein were incubated with 50 μl of the biotin-labeled PCR products at 22°C for 4 h. The biotin-labeled DNA-nuclear protein complex was incubated with 17 μl of Streptavidin Mag Sepharose at 4°C for 1 hour and washed twice before it was re-suspended in Laemmli sample buffer with 5% (v/v) β-mercaptoethanol. To break the streptavidin–biotin interaction, 20 μl of the re-suspended protein complex was heated at 98°C before it was separated on Mini-PROTEAN® TGX™ Precast Gels (BIO-RAD, Hercules, CA, USA). Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels were then stained using SilverQuest™ Silver Staining Kit (Invitrogen), and specific bands were analyzed by liquid chromatography–mass spectrometry/mass spectrometry (LC-MS/MS) analysis.

In-gel digestion. To purify SOX2 promoter-binding proteins from SDS-PAGE, in-gel digestion was performed. The SDS-PAGE gel was rinsed twice with 15 mM potassium ferricyanide and 50 mM sodium thiosulfate solution for 15 min to destain it. The gel was then rinsed twice with ultrapure water and the sample in the gel piece was reduced twice in a solution containing 50% acetonitrile (ACN), 50 mM ammonium bicarbonate, and 5 mM DTT for 10 min. The gel piece was dehydrated in 100% ACN twice for 30 min each, and then rehydrated with an in-gel digestion reagent containing 10 μg/ml Sequencing-Grade Modified Trypsin (Promega) in 30% ACN, 50 mM ammonium bicarbonate, and 5 mM DTT. This procedure for in-gel digestion was performed overnight at 30°C. After digestion, the samples were lyophilized overnight using Labconco Lyph-Lock 1L Model 77400 (Labconco, Kansas, MO, USA). Lyophilized samples were then dissolved in 0.1% formic acid.

LC-MS/MS analysis. To identify SOX2 promoter-binding proteins, LC-MS/MS analysis was performed. Twenty-five microliters of each sample were separated on a Zorbax 300SB-C18 column (75 μm × 150 mm; Agilent Technologies, Palo Alto, CA, USA). The capillary pump of Agilent 1100 LC/MSD Trap XCT (Agilent Technologies) was operated under the following conditions: solvent A: 0.1% formic acid, solvent B: ACN in 0.1% formic acid; column flow: 0.3 μl/min, primary flow: 300 μl/min; gradient: 0-5 min 2% solvent B, 60 min 60% solvent B; stop time: 60 min. Protein identification was performed in the Agilent Spectrum Mill MS proteomics workbench against the Swiss-Prot protein database search engine (http://kr.expasy.org/sprot/) and the MASCOT MS/MS Ions Search engine (http://www.matrixscience.com/search_form_select.html). The criteria for positive identification of proteins were set as follows: filter by protein score >10.0, and filter peptide by score >8, % scored peak intensity.

Quantitative reverse transcription-PCR (qRT-PCR). To analyze SOX2 mRNA expression, qRT-PCR was performed on ABI Prism 7900HT (Applied Biosystems, Grand Island, NY, USA) using 2×QuantiFast SYBR Green PCR Master Mix (Qiagen, Valencia, CA, USA) with 0.5 μM primers. Specific primer sets used were as follows: human SOX2 forward: 5’-TGGACAGTTACGCGCACAT-3’, reverse: 5’-CGAGTAGGACATGCTGTAGGT-3’; and β-Actin forward: 5’-TTGCCGACAGGATGCAGAA-3’ and reverse: 5’-GCCGATCCACACGGAGTACT-3’. The gene expression of SOX2 and the endogenous control, β-Actin were analyzed in three independent qRT-PCR assays. The thermal cycling procedure included an initial denaturation step for 5 min at 95°C, followed by 40 cycles of 95°C for 10 s and 60°C for 30 s. Relative expression of the target gene was analyzed by the ΔΔCt method. Ratios of mRNA levels were expressed relative to those of the control group.

RNA repression by small-interfering RNA (siRNA). To repress NONO mRNA expression, NONO-specific siRNA was treated to MCF-7 or T-47D cells. BLOCK-iT RNAi Designer (Invitrogen) was used to design siRNAs with the following sequences were as follows: NONO sense, 5’-GCAUUCCUGAAGUCUCUAATT-3’; antisense, 5’-UUAGAGACUUCAGGAAUGCTT-3’. AllStars Negative Control siRNA (Qiagen) was used as a negative control. Trypsinized MCF-7 cells (1×10⁶ cells) were re-suspended in 100 μl of Opti-Minimal Essential Medium (Invitrogen) followed by the addition of the siRNA such that the final concentration was 4 μM. Transfections were performed in an electroporation cuvette using a CUY21SC electroporator (NepaGene). The transfected cells were transferred to DMEM/F-12 containing 10% fetal bovine serum, incubated for 48 h in a humidified incubator maintained at 37°C and 5% CO₂. After 48 h, the cells were harvested for RNA or protein preparation.

Chromatin immunoprecipitation (ChIP) assay. To confirm NONO binding on SOX2 promoter, ChiP assay was performed using anti-NONO antibody and qPCR for SOX2 promoter region. Cells were seeded in 10-cm dishes and were harvested when it reached 80% confluency. Cross-linking was performed by incubating the cells in 500 μl of 1% formaldehyde prepared in phosphate-buffered saline (PBS) for 20 min at 20°C. After incubation, the cells were washed twice in ice-cold PBS, and lysed in 100 μl of ChIP lysis buffer (0.5% SDS, 50 mM Tris-HCl pH 8.0, 150 mM NaCl, 10 mM EDTA) for 30 min at 20°C. The cells lysates were then sonicated for 30×30 s at the maximum power setting on a Bioruptor Sonicator (Diagenode, Denville, NJ, USA) to generate DNA fragments of 150-500 bp in length. Sonicated samples were then centrifuged at 19,000 × g for 10 min at 4°C, and the supernatant was pre-cleared by incubating in 50% protein G agarose slurry (GE Healthcare) prepared in BSA/PBS for 2 h at 4°C. Pre-cleared samples were then incubated at 4°C overnight with 10 μg of normal mouse IgG (sc-2025, Santa Cruz Biotechnology, Dallas, TX, USA), anti-POL2 antibody (F-12, Santa Cruz Biotechnology), anti-NONO antibody (clone 3/p54nrb (RUO), BD Bioscience, Franklin Lakes, NJ, USA). The following day, 20 μl of protein G agarose was added and incubated with rotation for 2 h at 4°C, followed by a series of washes: twice with IP buffer (20 mM Tris-HCl pH 8.0, 150 mM NaCl, 2 mM EDTA, and 1% Triton X-100), twice with high-salt buffer (20 mM Tris-HCl pH 8.0, 500 mM NaCl, 2 mM EDTA, and 1% Triton X-100), once with LiCl buffer (250 mM LiCl, 20 mM Tris-HCl pH 8.0, 1 mM EDTA, 1% Triton X-100, 0.1% NP40, and 0.5% sodium deoxycholate), and twice with 10 mM Tris -1 mM EDTA (pH 8.0) buffer. Samples were eluted from the beads with 0.1 M Na₂CO₃ and 1% SDS by heating the mixture at 67°C for 30 min with intermittent vortexing at 10-min intervals. Samples were reverse-crosslinked overnight at 65°C. Immunoprecipitated DNA and input DNA were incubated with RNase A and Proteinase K (Sigma, St Louis, MO, USA) at 45 °C for 2 h. DNA was purified using QIAquick PCR Purification Kit (Qiagen) and then analyzed using qPCR. Primer sequences were as follows: SOX2 promoter −47 to −123, forward: 5’-CCTTTCATGCAAAACCCG-3’, reverse: 5’-GGGTTTCTAGCGACCAATCA-3’, and SOX2 promoter −69 to +58, forward: 5’-CGCTGATTGGTCGCTAGAAAC-3’, reverse: 5’-CCCCTTTTGCAAACACTCTC-3’. qPCR was performed on an ABI Prism 7900HT using previously described protocol for qRT-PCR. The relative gene expression was analyzed using ΔΔCt method and compared with normal mouse IgG.

Western blotting. To analyze protein expression level of NONO or SOX2, western blotting was performed. pcDNA3-NONO-1xFLAG (16, 17) or pCMV-3XFLAG (Mock, provided by Dr. Koji Nakagawa, Health Science University of Hokkaido School of Nursing & Social Services) were transfected into the cells by electroporation. Cells (1×10⁶) were lysed using 100 μl of RIPA buffer (25 mM Tris-HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, and 0.1% SDS). Samples containing 20 μg of protein extracts were separated on a 14% polyacrylamide gels and transferred onto PVDF membranes (Immobilon P; Millipore, Billerica, MA, USA). Antibodies against SOX2 (D6D9, Cell Signaling Technology, Danvers, MA, USA), NONO (clone 3/p54nrb (RUO), FLAG (M2: Sigma), β-Actin (clone C4: Millipore), anti-mouse IgG secondary antibodies, horseradish peroxidase (HRP)-linked F(ab’)2 fragment sheep or anti-rabbit IgG, and HRP-linked F(ab’)2 fragment donkey (GE Healthcare Bioscience) were used. Each protein band was detected using Western Chemiluminescent HRP Substrate (Immobilon, Millipore) and visualized by LAS1000mini (FUJIFILM Corp, Tokyo, Japan).

Immunocytochemistry. To analyze protein expression and localization of NONO or -SOX2, immunohistochemistry was performed using anti-NONO antibody or SOX2 antibodies. MCF-7 cells were seeded on glass-based 24-well plates and fixed in 4% paraformaldehyde and 0.1% Triton X-100 at 20 °C for 30 min after they reached 80% confluence. They were washed twice with 1×PBS for 5 min each and incubated with two primary antibodies, anti-SOX2 (D6D9) and anti-NONO (clone 3/p54nrb; RUO). The cells were then incubated with biotinylated sheep anti-rabbit IgG or Alexa 568 conjugated goat anti-mouse IgG (Invitrogen), and avidin-conjugated Alexa 488 (Invitrogen). All the antibodies were diluted in 1× PBS pIus 3% BSA. 4,6-Diamidino-2-phenylindole (0.1 μg/ml) was used for nuclear staining in order to confirm cellular localization of NONO and SOX2.

Generation of POU binding element mutated promoter vector. To analyze the effect of mutation of POU binding element in luciferase vector, plasmid vectors were constructed. P227-pGEM T vector was used as a template for PCR. The DNA fragment was amplified by KOD-Plus-Neo using 0.3 μM of specific primers (P227 forward: 5’-GGTACCCTCAGTGGCTGGCAGGC-3’, POU-M reverse: 5’-GGGTGGGTACAGCCCTGGGGCGGGGCCT-3’, POU-M forward: 5’-AGGCCCCGCCCCAGGGCTGTACCCACCC-3’, reverse 5’-AAGCTTGAGGCAAACTGGAATCAGGATC-3’) Mutated POU binding element is underlined in the above primer sequences. PCR products were extracted from agarose gel using QIAEXII (Qiagen), which served as templates for the second round of PCR using P227 forward and reverse primers. Thermal cycling procedure involved an initial denaturation step for 2 min at 94°C, followed by 20 cycles of 98°C for 10 s, 58°C 30 s, and 68°C for 30 s. A-tailing of the PCR products was carried out, after which, they were ligated into pGEM-T vector. The pGEM-T was then digested with restriction enzymes, Kpn I and Hind III. The fragment obtained was ligated to pGL3-Basic luciferase vector. The reporter vector with mutation in the POU binding element was designated as P227-M.

Generation of NONO deletion mutants. To confirm SOX2 binding elements in NONO protein, plasmid vectors were constructed. pcDNA3-NONO-1xFLAG was used as a template for PCR. DNA fragment was amplified by KOD-Plus-Neo using 0.3 μM of specific primers (ΔN forward: 5’-AGCCGTCTTTTTGTGGGAAATC-3’, reverse: 5’-CATGGTTCTATCTCCTTCGAAG-3’, ΔR forward: 5’-TTAGATGATGAAGAGGGACT T-3’, reverse: 5’-TCGTTGGGTGAAGGTCTTC-3’, ΔCC forward: 5’-ACCTTCCCTGATGCGAGAG-3’, reverse: 5’-ATATTCATACTCAAAGGAGC-3’, ΔC forward: 5’-TATGACCCAGCCTTTCTTGTAC-3’, reverse: 5’-TCCCTTGAATCCTTCCTGCTG-3’). Thermal cycling included an initial denaturation step for 2 min at 94°C, followed by 30 cycles at 98°C for 10 s, 58°C for 30 s, and 68°C for 3 min. PCR amplicons were digested with Dpn I. Digested PCR products were incubated with T4 polynucleotide kinase (New England Biolabs, Ipswich, MA, USA) for 30 min at 37°C, followed by overnight incubation with T4 DNA ligase (Promega) at 16°C. NONO coding regions of constructed vectors were sequenced, and expression of mutated NONO was confirmed by western blotting using FLAG antibody (M2: Sigma).

Flow cytometric (FACS) analysis. To confirm the SOX2-positive cell population in NONO siRNA-treated MCF-7 cells, FACS analysis was performed using antibody to SOX2. MCF-7 cells were fixed with 4% formaldehyde for 10 min at 37°C and permeabilized in ice-cold 100% methanol for 30 min. The cells were then treated with 3% BSA for 1 h and incubated with anti-SOX2 (D6D9) for 1 h at 20 °C. After washing with PBS, the cells were incubated with CF488A-conjugated goat anti-rabbit IgG antibody (Biotium, Hayward, CA, USA) for 1 h at 20°C. Immunostained cells were analyzed by FACSAria III flow cytometer I (BD Biosciences, San Jose, CA, USA).

Sphere formation. To analyze cancer stemness in cells with repression of NONO expression, sphere formation assay was used. Control and NONO-specific siRNA transfected MCF-7 cells were seeded at 3,000/well on ultra-low-attachment 96-well plate (Sumitomo Bakelite Co., LTD, Tokyo, Japan) using FACSAria III and cultured in a sphere-formation medium (DMEM/F-12 supplemented with 20 ng/ml of epidermal growth factor, 20 ng/ml of basic fibroblast growth factor, 100 units/ml penicillin, and 100 μg/ml of streptomycin sulfate) for 7-10 days. Spheres formed were detected and counted under microscopic observation (IX73; Olympus, Tokyo, Japan).

Statistical analysis. Statistical analysis was performed to analyze whether or not there were any significant differences in the experimental results. Experiments were performed in triplicate or quadruplicate and conducted in a minimum of three independent trials. Data are presented as means±standard deviations. Two-tailed t-tests were conducted in which the acceptable level of significance was p<0.05.