Download Supplementary Materials and Methods Plasmid vectors For the P2

Supplementary Materials and Methods
Plasmid vectors
For the P2 promoter cloning, RUNX2 genomic region spanning from -4200 to +281 of isoform I
transcription starting site was amplified by PCR and cloned into pGL3-basic vector (Promega, Milan,
Italy) upstream the luciferase gene, between KpnI and XhoI restriction sites. The short version of the P2
promoter (sP2) spanning from -103 to +281 of transcription starting site was generated by digesting the
pGL3/P2 vector with KpnI and PstI restriction endonucleases to excise the -4500/+103 fragment from the
plasmid. The plasmid was isolated by gel extraction, extremities were blunted with T4 DNA polymerase
and ligated. ENH1, ENH2 and ENH3 genomic regions and ENH3 deletion mutants were amplified by
PCR and cloned into pGL3/P2 or pGL3/sP2 vector downstream the luciferase gene in the SalI restriction
site. ENH3 point mutations were generated by site-directed mutagenesis, using primers containing the
mutated nucleotides to amplify the ENH3.C3 fragment
Chromatin Immunoprecipitation (ChIP) (additional protocol details)
Chromatin was cross-linked by incubating the cells grown on 15 cm dishes in formaldehyde 1% at room
temperature for 15 minutes. Cells were incubated for 5 minutes in 0.125 M glycine to stop the crosslinking. Cells were harvested in ice-cold PBS 1X, pelleted and resuspended in cell lysis buffer (10 mM
Tris pH 8, 85 mM Kcl, 0.5% NP40) with the addition of Complete EDTA-free Protease Inhibitors
Cocktail (Roche Diagnostics, Mannheim, Germany). After incubating on ice for 10 minutes, nuclei were
pelleted and resuspended in nuclei lysis buffer (50 mM Tris pH 8, 10 mM EDTA, 1% SDS) with the
addition of Complete EDTA-free Protease Inhibitors Cocktail. Nuclear extracts were incubated on ice for
10 minutes and then sonicated using a Sonopuls HD2070 sonicator (Bandelin, Berlin, Germany) to obtain
DNA fragments of an average length of 500-1000 bp. Chromatin was diluted 10 times in ChIP dilution
buffer (16.7 mM Tris pH 8, 167 mM NaCl, 1.2 mM EDTA, 1.1% Triton X-100, 0.01% SDS) with the
addition of Complete EDTA-free Protease Inhibitors Cocktail and incubated overnight at 4 °C with mouse
anti-PolII antibodies (ab817, Abcam, Cambridge, UK) or rabbit anti-c-JUN antibodies (H-79, sc-1694X,
Santa Cruz Biotechnology). As a control, we used normal mouse IgG (Santa Cruz Biotechnology) or
normal rabbit IgG (Santa Cruz Biotechnology) respectively. Antibody-chromatin complexes were
precipitated by incubating for 1 h at 4 °C with Protein A Sepharose CL-4B resin (GE Healthcare). The
resin bed was washed with 40 volumes of Low Salt Buffer (20 mM Tris pH 8, 150 mM NaCl, 2 mM
EDTA, 1% Triton X-100, 0.1% SDS), with 40 volumes of High Salt Buffer (20 mM Tris pH 8, 500 mM
NaCl, 2 mM EDTA, 1% Triton X-100, 0.1% SDS), with 40 volumes of LiCl Buffer (10 mM Tris pH8, 1
mM EDTA, 1% NaDeoxycholate, 1% NP40, 250 mM LiCl), and two times with 40 volumes of TE (10
mM Tris pH 8, 1 mM EDTA). Immunoprecipitated chromatin was eluted by incubating the resin with
Elution Buffer (0.1 M NaHCO3, 1% SDS) and cross-linking was reverted by incubating at 65 °C
overnight. The day after, samples were treated with Proteinase K for 1 h and DNA was purified. DNA
fragments immunoprecipitated with specific antibodies or control IgG were quantified by qPCR and
expressed as percentage of input DNA. Sequences of ChIP primers for different RUNX2 gene regions are
listed in Supplementary Table I.
Electromobility Shift Assay (EMSA) (additional protocol details)
To prepare nuclear extracts for EMSA, cells grown on 15 cm dishes were scraped in ice-cold PBS 1X and
pelleted. Volume of the pellet was estimated and cells were resuspended in 10 volumes of cell lysis buffer
(10 mM Hepes pH 8, 10 mM NaCl, 5 mM MgCl2, 0.5 mM DTT) with the addition of Complete EDTAfree Protease Inhibitors Cocktail and PhosSTOP Phosphatase Inhibitors Cocktail (Roche Diagnostics).
After incubating on ice for 30 minutes, nuclei were pelleted and 1 volume of nuclei lysis buffer was
added (10 mM Hepes pH 8, 300 mM Kcl, 5 mM MgCl2, 0.5 mM DTT, 20% glycerol, Complete EDTAfree Protease Inhibitors Cocktail, PhosSTOP Phosphatase Inhibitors Cocktail). Nuclear extracts were
incubated for 30 min on a rotating wheel at 4°C and then centrifuged to remove insoluble pellet. Nuclear
extracts were quantified and 5 μg were used for each EMSA reaction.
S3A probe corresponding to ENH3 fragment C3A was obtained by Integrated DNA Technologies
(Leuven, Belgium), as well as S3A mutants. The wild-type probe was radiolabeled with [γ-32P]-ATP
(Perkin Elmer, Waltham, MA, USA) using T4 polynucleotide kinase (New England Biolabs, Ipswich,
MA, USA) and purified on a Chromaspin-30 column (Clontech Laboratories, Mountain View, CA, USA).
Radiolabeled probe was incubated with or without nuclear extract in presence of 1 μg of dI-dC (SigmaAldrich, Milan, Italy) as aspecific competitor in EMSA binding buffer (20 mM Hepes pH 8, 20 mM KCl,
2.5% Glycerol). Increasing amounts of wild-type or mutated cold competitors were added in competition
experiments. A 4-fold, a 20-fold and a 100-fold excess of cold competitors were used for TPC1 nuclear
extracts. A 4-fold, a 12-fold and a 36-fold of cold competitors were used for BCPAP nuclear extracts. For
supershift experiments we used rabbit anti-c-JUN antibodies (H-79, sc-1694X, Santa Cruz
Biotechnology) or normal rabbit IgG (Santa Cruz Biotechnology) as a control. Binding reactions were
incubated 30 min on ice and then loaded on a non-denaturant polyacrylamide gel. Gels were dried and
exposed to autoradiography film.
DNA pulldown (additional protocol details)
Nuclear extracts have been prepared as described in EMSA protocol. For each reaction 50 ul of
Streptavidin conjugated Dynabeads M-280 (Life Technologies) have been washed once in Wash Buffer
(1 M NaCl, 0.02% Triton X-100, 1 mM EDTA, 10 mM Tris pH 7.5), once in PBS 1x and incubated with
20 pmol of biotinylated wild type or Mut1+2 S3A probe in 150 ul PBS 1x for 1 hour at 4 °C on a rotating
wheel. The beads have been washed as before and incubated with 1 mg of nuclear extract and 50 ug of
Salmon Sperm DNA was added as non-specific competitor. For each reaction final volume and KCl
concentration have been adjusted to 800 ul and 300 mM respectively. After 90' incubation at 4 °C on a
rotating wheel, the beads have been washed with NETN (20 mM Tris pH 7.5, 150 mM NaCl, 1 mM
EDTA, 0.5% NP40) buffer two times and with PBS 1x once. The beads have been resuspended in gel
loading buffer and bound proteins have been resolved on a SDS-PAGE gel. Detection of c-JUN has been
performed by Western blot.