E2F1 Mediates the Retinoic Acid-Induced Transcription of Tshz1 during Neuronal Differentiation in a Cell Division-Dependent Manner

Antibodies and primers.The antibodies against Tuj1 (MMS-435p-100; Covance), p27 (ab7961; Abcam), E2F1 (sc-251; Santa Cruz Biotechnology), green fluorescent protein (GFP; sc-9996; Santa Cruz Biotechnology), Flag (F3165; Sigma-Aldrich), GAPDH (glyceraldehyde-3-phosphate dehydrogenase; AM4300; Ambion), pRb (554136; BD Pharmigen,), p107 (SC-250; Santa Cruz), p130 (SC-374521; Santa Cruz), cyclin B1 (sc-254; Santa Cruz Biotechnology), and α-tubulin (ab18251; Abcam) were commercially purchased. The secondary antibody conjugated with fluorescent dye (Alexa Fluor 488; Life Technologies) was also purchased. The mouse and rabbit IgG-horseradish peroxidase (HRP) antibodies were purchased from Sigma and Millipore, respectively. For depletion of E2F1 and Tshz1, siE2F1-1 (5′ CGC UAU GAA ACC UCA CUA ATT-3′), siE2F1-2 (5′-GUG GAU UCU UCA GAG ACA UTT-3′), and siTshz1 (5′-CCC AGA UAC UCA AGU GCA UTT-3′) were used. A scrambled siRNA sequence (siCTL; 5′-UUC UCC GAA CGU GUC ACG UTT-3′) was used as a control. RNAiMAX (13778-075; Invitrogen) was used for siRNA transfection according to the manufacturer's protocol.

For the RT-PCR and qPCR analyses, we used primer sets of Tshz1 (forward, 5′ GCT GGC CCA TTT CAA AAG CTC 3′; reverse, 5′ ATC CAA TGC TAG GCT AGA CCA 3′), Hmbs (forward, 5′ ACT CTG CTT CGC TGC ATT G 3′; reverse, 5′ AGT TGC CCA TCT TTC ATC ACT G 3′), β-actin (forward, 5′ CAT CAT GAA GTG TGA CGT TG 3′; reverse, 5′ ATG ATC TTG ATC TTC ATG GT 3′), and GAPDH (forward, 5′ CTG CCT TGG AGA AGC TCA GT 3′; reverse, 5′ CAC CAA GTC GAT CAG ACC AA 3′).

For the ChIP-qPCR analysis, we used primer sets targeting the Tshz1 coding region (forward, 5′ GCT GGC CCA TTT CAA AAG CTC 3′; reverse, 5′ ATC CAA TGC TAG GCT AGA CCA 3′), the Tshz1 promoter region (forward, 5′ ACC CCA GGA TCC CTG CCC 3′; reverse, 5′ CGC TCG GGG TTG ATT CGT ACC A 3′), cdc2 (forward, 5′ ACA GAG CTC AAG AGT CAG TTG GC 3′; reverse, 5′ CGC CAA TCC GAT TGC ACG TAG A3′).

Cell culture, drug treatment, and neuronal differentiation.P19 embryonic carcinoma cells were cultured in Dulbecco's modified Eagle medium (DMEM; LM001-05; Welgene) supplemented with 10% fetal bovine serum (FBS), 5 μg/ml ANT-MPT (InvivoGen), and antibiotics (S101-01; Welgene).

For the cell cycle block experiments, the cells were first synchronized at G₁ phase using a single or double thymidine block. The single thymidine block was performed with 2 mM thymidine (T9250; Sigma) for 12 h. The double thymidine block was performed with 2 mM thymidine (T9250; Sigma) for 12 h, followed by 6 h of release into thymidine-free medium and thymine treatment for another 12 h. Upon thymidine release, the cells were treated with cell cycle-blocking drugs. For G₂ arrest, the cells were treated with RO3306 (10 μM) for 12 h.

To generate polyclonal stable cell lines, 4 × 10⁴ P19 cells were seeded on a 24-well plate. The next day, 1 μg of plasmid DNA was transfected using Lipofectamine 3000 (L3000001; Invitrogen) according to the manufacturer's instructions. One day after the transfection, the cells were transferred to a 100-mm dish and subjected to G418 selection for 1 to 2 weeks.

To differentiate P19 cells to neurons, the conventional method was performed essentially according to the previous report (39). Briefly, P19 cells (10⁵ cells/ml) were cultured in suspension in a petri dish in DMEM containing 10% FBS that was treated with 1 μM all trans-retinoic acid (R2656; Sigma) to allow for the EB formation for at least 2 days (or as otherwise indicated in the figure legend). The EBs were then trypsinized and replated into a poly-l-lysine-coated tissue culture dish and cultured in FBS-free B27-supplemented DMEM (catalog number 17504044; Gibco) for up to 4 days.

Immunocytochemistry analysis.P19 cells were seeded on cover glass and were fixed with cold methanol for 10 min and washed three times with ice-cold phosphate-buffered saline (PBS). After incubation in PBST (0.1% Triton X-100 in PBS) for 10 min, the cells were blocked with blocking solution (3% bovine serum albumin in 0.3% Triton X-100 in PBS) for 30 min. Then, cells were incubated with primary antibodies at room temperature diluted in blocking solution for 1 h. Cells were washed three times with PBST and incubated with fluorescent dye-conjugated secondary antibodies diluted in blocking solution for 30 min. Afterwards, cells were washed twice with PBST, incubated with 4,6-diamidino-2-phenylindole (DAPI) solution for 3 min, and washed twice with PBST. The cover glasses were mounted on a slide glass with ProLong Gold antifade reagent (P36930; Life Technologies). Images were acquired from fluorescence microscopies equipped with digital cameras (Olympus IX51 equipped with QImaging QICAM Fast 1394 or Olympus IX81 equipped with ANDOR iXonEM+) and processed in ImagePro 5.0 (Media Cybernetics).

Neurite outgrowth assay.Typically, pictures of 30 to 50 neurons from three separate coverslips from each experiment were taken using a fluorescence microscope (Leica DMI6000B). Representative cells with strong Tuj1 immunoreactivity labeled neurite (axonal and dendritic) processes were analyzed. Neurites with lengths that were at least twice the diameter of the cell body were considered neurite-positive cells. Neurite lengths from the cell body and cell body diameter were traced and measured using NeuronJ (ImageJ) software, and the data were compiled and analyzed using Prism (GraphPad).

Immunoblot analysis.Cells were lysed on ice for 20 min with radioimmunoprecipitation assay (RIPA) lysis buffer (150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris-HCl at pH 8.0, 10 mM NaF, 1 mM Na₃VO₄, 1 mM EDTA, and 1 mM EGTA) containing a protease inhibitor cocktail (P8340; Sigma-Aldrich) and centrifuged at 12,000 rpm for 15 min at 4°C. The supernatants were mixed with 5× SDS sample buffer (250 mM Tris-HCl at pH 6.8, 10% SDS, 50% glycerol, and 0.02% bromophenol blue) and 10 mM DTT (0281-25G; Amresco). Mixtures were boiled for 7 min. The cell lysates (20 to 30 μg protein) were loaded onto an SDS-polyacrylamide gel and transferred onto nitrocellulose membranes (GE Healthcare). For the analysis of Rb family proteins, 4 to 6% SDS-polyacrylamide gel was used. The membranes were blocked with blocking solution (5% nonfat milk in 0.1% Tween 20 in Tris-buffered saline [TBS]) for 1 h at room temperature and incubated with primary antibodies diluted in blocking solution overnight at 4°C. In the case of Rb family proteins, 5% bovine serum albumin (BSA) was used as blocking solution. Then, membranes were washed for 15 min four times with TBST (0.1% Tween 20 in TBS) and incubated with HRP-conjugated secondary antibodies in blocking solution for 45 min at room temperature and washed again with TBST. To detect the signals of secondary antibodies, an enhanced chemiluminescence (ECL) solution (LF-QC0101; ABfrontier) and X-ray films (CP-BU NEW; Agfa) were used.

RT-PCR and qPCR.Total RNAs were extracted using TRIzol (Invitrogen), and reverse transcription was performed from 2 μg total RNAs using RnaUsScript RTase (LeGene) according to the manufacturer's instructions. Then, the samples were first subjected to RT-PCR using 1 μl of reverse transcription product with the 2×HOT PCR mastermix (catalog number MP00505; Doctor Protein) according to the manufacturer's instructions. Briefly, a total of 20 μl reaction mixture was prepared containing 1 μl of reverse transcription product, 5 pmol of each forward and reverse PCR primer, and the 2× Taq polymerase mixture provided by the manufacturer. Then, the mixture was subjected to incubation at 95°C for 2 min followed by 30 cycles of 95°C for 30 s, 55°C for 30 s, and 72°C for 30 s. Ten microliters of the amplified reaction mixture was electrophoresed on a 1.5% agarose gel followed by staining with ethidium bromide and imaging for PCR product. For qPCR, 1 μl of reverse transcription product, 10 pmol of each forward and reverse PCR primer, and SYBR TOPreal qPCR 2×PreMix (Enzynomics) were used. The abundance of mRNA was determined by an ABI prism 7500 system. The quantity of mRNA was calculated using the ΔΔC_T method (where C_T is threshold cycle), and beta-actin, Gapdh, and Hmbs were used as control.

Luciferase assay.A promoter reporter construct containing the indicated regions relative to the transcription start site of the Tshz1 promoter was cloned from P19 cells' genomic DNA and inserted upstream of the firefly luciferase reporter in the pGL3-Basic vector (Promega). To generate a stable cell line of luciferase reporter, a neomycin (kanamycin)-expressing sequence was inserted into the firefly luciferase reporter vector. Twenty-four hours after transfection, P19 cells were subjected to G418 selection for 1 week. Then, polyclonal cell lines were subjected to luciferase assays using a microplate luminometer (DE/LB 96V; Berthold Technologies). For construction of Tshz1 mutants, site-directed mutagenesis was performed. One day after seeding 2 × 10⁴ stable cells in a 24-well plate, cells were treated with the indicated drugs with or without RA for 24 h. Then, cells were lysed and subjected to luciferase assay. After cell lysis, the luciferase assay was performed within 2 h. Luciferase outputs were normalized by total protein amount measured via the Bradford assay. All values were calculated as relative to pGL3 vector.

EMSA.The oligonucleotides were first biotinylated and annealed, using the biotin 3′ end DNA labeling kit (89818; Pierce). Nuclear extracts were prepared from P19 cells using nuclear extraction buffer. The binding reactions were performed on ice for 20 min with nuclear extract and 20 fmol of biotin-labeled DNA in a final volume of 20 μl, containing 2 μl of 10× binding buffer (100 mM Tris, 500 mM KCl, 10 mM DTT; pH 7.5) and 1 μl of poly(dI-dC). For supershift assays, 1 to 3 μg of E2F1 antibody (Santa Cruz Biotechnology) was added to the reaction mixture described above, followed by incubation on ice for 15 min prior to the addition of the labeled oligonucleotide probe. A double-stranded mutated oligonucleotide and unlabeled double-stranded oligonucleotides were used as competitors (400 fmol) to examine the specificity of DNA binding. For all the supershift assays, DTT-free buffers were used. The resultant DNA-protein complex was resolved from free oligonucleotide in a 6% native polyacrylamide gel in Tris borate-EDTA buffer and transferred to nylon membranes (Bio-Rad). The membranes were cross-linked by UV radiation using Gel-doc (Bio-Rad). Biotin signal detection was performed using the chemiluminescent nucleic acid detection module (89880; Thermo Fisher Scientific) in accordance with the manufacturer's instructions.

Chromatin immunoprecipitation assay.P19 cells (or HeLa cells) were cross-linked with 1% formaldehyde for 10 min at room temperature. After glycine quenching to a final concentration of 0.2 M for 10 min at room temperature, the cell pellets were lysed in buffer containing 50 mM Tris-HCl (pH 8.1), 10 mM EDTA, and 1% SDS, supplemented with complete protease inhibitor cocktail (Roche), and sonicated. Chromatin extracts containing DNA fragments with an average of 250 bp were then diluted 10 times with dilution buffer containing 1% Triton X-100, 2 mM EDTA, 150 mM NaCl, and 20 mM Tris-HCl (pH 8.1) with complete protease inhibitor cocktail, precleared with protein A/G-Sepharose (17-0780-01; GE Healthcare Life Sciences) and subjected to immunoprecipitations overnight at 4°C by incubating with 3 μg of E2F1 antibody (sc-251; Santa Cruz Biotechnology) and mouse IgG as a negative control. Immunocomplexes were captured by incubating 45 μl of protein A/G-Sepharose for 2 h at 4°C. Beads were washed with low-salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl [pH 8.1], 150 mM NaCl), high-salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl [pH 8.1], 500 mM NaCl), buffer III (0.25 M LiCl, 1% NP-40, 1% deoxycholate, 10 mM Tris-HCl [pH 8.1], 1 mM EDTA), and Tris-EDTA (TE) buffer (10 mM Tris-HCl [pH 8.0], 0.5 M EDTA) and eluted in elution buffer (1% SDS, 0.1 M NaHCO₃). The supernatant was incubated overnight at 65°C to reverse-cross-link and then digested with RNase A for 2 h at 37°C and proteinase K for 2 h at 55°C. ChIP samples as well as 1% of DNA extract from soluble chromatin that was used as input DNA were then purified for qPCR analysis using the primers indicated. Enrichment relative to input values were calculated using the percent input method.

FACS analysis.Samples were collected over indicated time points and fixed in 70% ethanol overnight. For cell cycle analysis, fixed cells were treated with RNase for 20 min before addition of 5 μg/ml propidium iodide (PI) and analyzed by fluorescence-activated cell sorter (FACS; BD Biosciences FACSCalibur).

Statistical analysis.All experiments were performed independently at least three times. Values are expressed as means ± standard deviations (SD). P values were analyzed using the two-tailed unpaired t test and one-way analysis of variance (ANOVA). P values of <0.05 were considered statistically significant.