Young et al. 10.1073/pnas.0611419104. |
Fig. 7. ChIP studies for the 14 target genes were performed with asynchronous cells and pure mitotic cells isolated by nocodazole synchronization and mitotic shakeoff. ChIP products were analyzed by radiolabeled PCR using the primer sets targeting the promoters of the 14 Runx2 target genes. Negative control PCRs used primer sets that target the CDC6 3'-UTR and the promoter of the PHOX gene. Positive control PCRs used a primer set targeting the proximal promoter of the RUNX2 gene.
SI Materials and Methods
Cell Culture and Cell Synchronization.
Saos-2 osteosarcoma cells were maintained in McCoy's medium containing 15% FBS (Invitrogen, Carlsbad, CA) plus 2 mM L-glutamine and a penicillin/streptomycin mixture. HeLa cells were maintained in DMEM plus 2 mM L-glutamine/penicillin/streptomycin mixture. ROS cells were maintained in F12 plus 2 mM L-glutamine/penicillin/streptomycin mixture with 5% FBS. Cells were blocked in mitosis for biochemical fractionation and chromatin immunoprecipitation (ChIP) assays by adding 200 ng/ml nocodazole for 24 h followed by shakeoff of mitotic cells. For block-release studies Saos-2 cells were synchronized by the addition of 200 ng/ml nocodazole for 24 h. Cells were released by two washes in serum-free medium followed by the addition of McCoy's medium containing 15% FBS plus 2 mM L-glutamine. Cell cycle analysis was performed by propidium iodide-stained cells subjected to fluorescence-activated cell sorting (FACS; University of Massachusetts Medical Core Facility).Expression Constructs.
The following constructs have been reported previously: HA-Runx2 and Xpress-Runx2 (1). The R182Q mutant of Runx2 was generated by PCR-based site-directed mutagenesis of the HA-Runx2 construct using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). Amino acid numbering for this mutation is based on mouse GenBank accession no. D14637. The following primers were used for mutagenesis: forward, 5'-GAG ATT TGT GGG CCA GAG CGG ACG AGG-3'; and reverse, 5'-CCT CGT CCG CTC TGG CCC ACA AAT CTC-3'. Mutations were confirmed by sequencing with the primer 5'-ATG CGC CCT AAA TCA CTG AG-3'.Electrophoretic Mobility Shift Assay.
The upper strands (200 ng) of oligonucleotides including a Runx-binding sequence were labeled with 32P for 1 h at 37°C in a 50-ml volume using T4 polynucleotide kinase (New England Biolabs, Beverly, MA) as indicated by the manufacturer. The reaction was stopped by heat inactivation at 65°C for 1 h. Annealing was performed by the addition of a 2-fold excess amount of bottom strand followed by boiling for 5 min and slow cooling to room temperature. The unincorporated nucleotides were removed using a quick-spin Sephadex G-25 column (Roche Molecular Biochemicals, Indianapolis, IN) according to the manufacturer's instructions. Electrophoretic mobility shift assay (EMSA) reaction mixtures were prepared using 50 fmol of probe/50 mM KCl/12 mM Hepes, pH 7.5/1 mM EDTA/1 mM DTT/12% (vol/vol) glycerol/2 mg of poly(dI-dC)·(dI-dC)/0-4 ml of IVTT protein extract protein using HA-Runx2 (R182Q) or wild-type HA-Runx2. Aliquots were loaded onto a 4% nondenaturing polyacrylamide gel. The gels were electrophoresed for 1.5 h at 200 V, dried, and exposed to film for autoradiography.In Situ
Immunofluorescence Microscopy. Saos-2 and HeLa cells grown on gelatin-coated coverslips were processed for in situ immunofluorescence by using standard techniques. In brief, cells were rinsed twice with ice-cold PBS and fixed in 3.7% formaldehyde in PBS for 10 min on ice. After rinsing once with PBS, the cells were permeabilized in 0.1% Triton X-100 in PBS, and rinsed twice with PBSA (0.5% BSA in PBS) followed by antibody staining. Antibodies and their dilutions used are as follows: rabbit polyclonal antibodies against Runx2 (1:200, M-70; Santa Cruz Biotechnology, Santa Cruz, CA), rabbit polyclonal antibody raised against the HA epitope (1:400, M-70; Santa Cruz Biotechnology), and mouse monoclonal raised against the Xpress epitope (1:400, M-70; Invitrogen). The secondary antibodies used were either anti-mouse Alexa 594 or anti-rabbit Alexa 488 (1:800; Molecular Probes, Eugene, OR). DNA was visualized by DAPI (4',6-diamidino-2-phenylindole) staining. Immunostaining of cell preparations was recorded using an epifluorescence Zeiss Axioplan 2 (Zeiss Inc., Thorwood, NY) microscope attached to a CCD camera.siRNA Knockdown Experiments.
Saos-2 cells at 30-50% confluence were transfected using Oligofectamine (Invitrogen) with siRNA duplexes specific for human Runx2 obtained from Qiagen, Inc. (Valencia, CA). For gene profiling and histone modification studies, oligonucleotides were used at 50 and 25 nM, respectively. The siRNA duplexes were r(GGUUCAACGAUCUGAGAUU)d(TT). The cells were also transfected with control siRNA duplexes specific for GFP or nonsilencing siRNA (Qiagen, Inc.) using the same concentrations and vehicle alone as a control. Opti-MEM (a reduced serum medium from Invitrogen) was used to dilute the siRNA duplexes and Oligofectamine and for transfection. After treating the cells with siRNA for 4 h, the cells were supplemented with McCoy's containing 45% FBS for a final concentration of 15% in the medium. The gene-profiling siRNA experiment was carried out for 72 h, at which time the cells were harvested for total protein and RNA to analyze the knockdown effect of Runx2 siRNA on endogenous Runx2. For histone modification studies, cells were treated with siRNA for 48 h, incubated for an additional 24 h in the presence of the microtubule-destabilizing agent nocodazole (100 ng/ml; Sigma-Aldrich, St. Louis, MO), followed by shakeoff to obtain mitotic cells; parallel plates were incubated with siRNA oligonucleotides for 72 h without nocodazole treatment to obtain asynchronous cells. To obtain cells in early G1 for microarray profiling experiments, mitotic cells were pelleted and washed twice in DMEM, refed with growth medium, and replated.Gene Expression Profiling.
Focused expression profiling was performed using the osteogenic and cell cycle-focused cDNA arrays according to the manufacturer's GEArray instructions (SuperArray Bioscience Corporation, Frederick, MD). Briefly, total RNA was isolated from Saos-2 cells at the indicated time points for cell synchronization experiments and at 72 h for the indicated treatments for siRNA experiments using TRIzol reagent (Invitrogen). cDNA was generated from purified RNA using a reverse transcription reaction (Invitrogen) with primers provided with GEArray kits and [a-32P]dCTP. Radioactive reverse-transcription cDNA products were directly hybridized to cDNA arrays for 16 h at 60°C, washed once with 1% SDS and 2´ sodium chloride/sodium citrate (SSC; pH 7.0) with rotation at 20 rpm at 60°C for 15 min, and once with 0.5% SDS and 0.1´ SSC (pH 7.0) with rotation at 20 rpm at 60°C for 15 min. Arrays were exposed to BioMax film (Eastman Kodak, Rochester, NY) for 48 h and digitized for quantitation. Digital images of gene arrays were quantified by using ImageQuant TL software (GE Healthcare, Piscataway, NJ). Signals were background-corrected and normalized to the average of four cyclophilin A cDNA spots. Exploratory analysis of gene expression patterns was performed by hierarchical cluster analysis of row-wise standardized data using dCHIP software (2).Affymetrix microarrays (Hu-U133Plus2 chips) were performed by using RNA samples isolated from mitotically synchronized cells that were pretreated with Runx2 siRNA and nonsilencing RNAs. The processing of RNA samples and hybridization were essentially performed as described previously (3, 4).
ChIP Assays.
ChIP assays were performed essentially as described (5). Briefly, asynchronously growing or mitotic cells were cross-linked in DMEM with 1% formaldehyde for 10 min, and the reaction was quenched by the addition of glycine at a final concentration of 250 mM for 10 min. Cells were scraped, pelleted, and washed twice with PBS. Cell pellets were resuspended in 2.5 ml of lysis buffer [150 mM NaCl/50 mM Tris·HCl, pH 8.0/1% Nonidet P-40/25 mM MG-132/1´ Complete protease inhibitor mixture (Roche)]. After 10 min on ice, cells were sonicated to a DNA fragment size of 500-1,000 bp as determined by agarose gel electrophoresis with ethidium bromide staining. Cell debris was precleared by centrifugation at 14,000 rpm ´ g for 20 min. Supernatant-containing protein-DNA complexes were aliquoted into three tubes (1 ml per antibody and 500 ml for input DNA) and incubated for 16 h with 3 mg of rabbit polyclonal antibody directed against Runx2 (M-70; Santa Cruz Biotechnology) and 3 mg of normal rabbit IgG (Santa Cruz Biotechnology) or 4 ml of rabbit polyclonal antibodies directed against hyperacetylated histone H4 or dimethyl K4-histone H3 (06-946 and 07-030, respectively; Upstate Biotechnology, Lake Placid, NY) followed by 1 h with 50 ml of protein A/G-conjugated agarose beads. Protein A/G-bead complexes were washed with the following buffers: low salt (20 mM Tris·Cl, pH 8.1/150 mM NaCl/1% Triton X-100/2 mM EDT/1´ Complete protease inhibitor), high salt (20 mM Tris·Cl, pH 8.1/500 mM NaCl/1% Triton X-100/2 mM EDTA), LiCl (10 mM Tris·Cl, pH 8.1/250 mM LiCl/1% deoxycholate/1% Nonidet P-40/1 mM EDTA), and twice in TE (10 mM Tris·Cl, pH 8.1/1 mM EDTA). Protein-DNA complexes were eluted in 1% SDS/100 mM NaHCO3. Cross-links were reversed by incubation for 16 h in elution buffer and 300 mM sodium acetate (pH 5.2). DNA was extracted, purified, precipitated, and resuspended in TE for quantitative PCR. ChIP enrichment was determined as a quantitative measure reflecting the percentage of input. Runx2 target gene ChIP data were normalized to the nonspecific PHOX gene.Western Blot Analysis.
Western blot analysis was performed as described previously (6). Briefly, amounts of total cellular protein were resolved in 8 or 10% SDS/PAGE and transferred to polyvinylidene difluoride membranes (Immobilon-P; Millipore Corp., Bedford, MA). Blots were incubated with a 1:2,000 dilution of each primary antibody for 1 h. Mouse monoclonal antibodies specific for lamin B1 (1:2,000; Zymed Laboratories, Inc., San Francisco, CA) and Runx2 (7) were used. In addition, rabbit polyclonal antibodies against histone H4 (1:1,000) and phosphoserine-10 histone H3 were obtained from Upstate Biotechnology. Membranes were then incubated with horseradish peroxidase-conjugated secondary antibody, and protein bands were visualized by a chemiluminescence detection kit (Perkin-Elmer Life Sciences, Boston, MA).RNA Analysis.
Total RNA was isolated from cells at the indicated time points using TRIzol reagent (Invitrogen). Total RNA was purified using the DNA-free RNA kit (Zymo Research Corp., Orange CA). cDNA was generated from purified RNA using a reverse-transcription reaction with random hexamer primers (Invitrogen). cDNA was then subjected to a real-time PCR using SYBR chemistry (Applied Biosystems, Inc., Foster City, CA). All primers for gene validation studies span exons that are contained in all known transcripts.Statistical Analysis.
t tests were performed to assess the significance of observed changes in gene expression associated with siRNA knockdown of Runx2 protein and to determine the significance of observed binding by Runx2 to the promoters of target genes in Fig. 3. ANOVA was carried out to assess the significance of Runx2 knockdown on histone modifications at target gene promoters in Fig. 5. Data were obtained from duplicate ChIP assays using hyperacetylated histone H4 and dimethylated K4-histone H3 antibodies from asynchronous and mitotic cells. qPCR measurements were made in duplicate for each of 14 target genes and expressed as percentage input chromatin. A mixed-model analysis was performed on log-transformed ChIP data using SAS/Analyst (SAS Institute Inc., Cary, NC) with genes (8), antibodies (9), and cell cycle stage (9) incorporated as fixed effects and qPCR well position as a random effect. Separate mixed models that were grouped by cell cycle stage were also analyzed. Multiple comparisons using Tukey HSD correction established P values for pairwise comparisons of significant effects. To identify differentially expressed genes in our microarray study in Fig. 6, we used an empirical Bayes linear modeling approach that was implemented with the Bioconductor package LIMMA in R statistical computing environment. We constructed a design matrix that incorporates an intercept, effects for each of four time points, and the siRNA treatment. The analysis was performed on transformed data (i.e., log + 1). We identified 500 genes that were significantly altered on the siRNA treatment effect. R-scripts and microarray data are provided in the SI Data Sets. Gene annotation enrichment analysis was performed to elucidate the biological processes and pathways associated with each gene cluster. Runx2-responsive genes from both cluster 1 and cluster 2 were subjected to functional annotation clustering using DAVID 2006 found at http://david.abcc.ncifcrf.gov (10, 11). Top annotation clusters are shown in bar plots where the value of the abscissa reflects the annotation enrichment score. This enrichment score is computed as the negative log of the geometric mean of the P values from Fischer exact tests performed on the underlying annotation gene sets. Default gene sets were analyzed and include GO ontology, Biocarta and KEGG Pathways, and Interpro and PIR superfamily names. Group names on the ordinate were defined based on interpretation of the underlying annotations. Complete DAVID output is provided in SI Data Sets 1 and 2.1. Zaidi SK, Javed A, Choi J-Y, van Wijnen AJ, Stein JL, Lian JB, Stein GS (2001) J Cell Sci 114:3093-3102.
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