of poly(dA?dT) at room temperature, followed by DNase I digestion and denaturing PAGE. Electromobility shift assays COS-1 nuclear extracts over-expressing SOX9 and RUNX2 were pre-incubated with poly(dI?dC) or poly(dG?dC) at room temperature, followed by reaction with [c-32P]ATP-labeled probes with or without the presence of nucleotide competitors, or antibodies for SOX9 (gift of Peter Koopman [64]) and OSF2/ RUNX2 (gift of Gerard Karsenty [65]), then subjected to non- denaturing PAGE at room temperature. The sequences of oligonucleotide COL2C1 and OSE2 were as previously described [10,65]. Col10Flag transgenic mice The mouse Col10a1 element A, a 640 bp-fragment located between 24.2 and 23.6 kb, was cloned at the 59 end of the pCol10Flag, previously known as FColX [18] consisting of 22070 to +7176 bp mouse Col10a1 genomic sequence, to generate pCol10Flag- E. The SOX9, TG-rich motif, and GLI binding sites in pCol10Flag-E were mutated to generate the single site mutants – respectively pCol10Flag-ED1, pCol10Flag-ED2, and pCol10Flag-ED3. All of these 3 motifs in pCol10Flag-E were mutated to generate pCol10Flag-ED4. Col10a1-Cre;Z/Sox9 compound mutants A 4.8 kb fragment of mouse genomic DNA (from 82 bp upstream of the start of transcription of Sox9 to 1119 bp downstream of the polyadenylation sequences), including the Sox9 coding region, its two introns and 1.1 kb of 39 flanking DNA, together with an IRES2-EGFP (Clontech) sequence inserted between the Sox9 stop codon and the polyadenylation site (at +3237 bp), was cloned downstream of the loxP-flanked bgeo/3xpA of the pCall2 vector (gift of Andras Nagy [66]) to create the pZ/Sox9 expression vector. pZ/Sox9 was transfected into 129/SvEv-derived L4 embryonic stem (ES) cells by electroporation and ES clones containing a single copy of the transgene were injected into blastocysts followed by crossing of chimeras with C57BL/6N mice to generate the mouse line Z/ Sox9. A mouse line carrying a single copy of pZ/Sox9 was generated which was then crossed with Col10a1-Cre mice [21] to obtain compound mutants. Chromatin immunoprecipitation CCL cell lysates or 13.5 dpc mouse limb tissue lysates were cross-linked followed by lysis and sonication to yield 200– 500 bp DNA fragments then immunoprecipitation with anti- bodies against acetylated histone H3/H4 (Ac-H3, Ac-H4) (Upstate), HDAC4 (Abcam), GLI1, GLI2, GLI3 (all from Santa Cruz), SOX9 (gift of Robin Lovell-Badge [67]), TRAP230/MED12 (gift of Robert Roeder [25]), or TRPS1 (gift of Yasuteru Muragaki [26]). The target elements in Col10a1, Col2a1, or Crygb genes were amplified by real-time or standard PCR. Gene expression analysis In-situ hybridization was performed as previously described [9]. The probes used were pRK26 for Col10a1 [68], pWF21 for the Flag sequence [18], p88 for full-length Sox9 (gift of Peter Koopman), pBS-Cbfa1-Sh for full-length Runx2 (gift of Gerard Karsenty), pWF98 for full-length Egfp, pBSII-Cre-frag for Cre (gift of Andrew Groves), and pNJ61 for Col2a1 [9]. Gene expression in cell culture was analyzed by RT-PCR. For additional details of all experiments, see the Text S1. SOX9 Is an Activator and Repressor in Chondrocytes Supporting Information Figure S1 Additional gene/protein expression data and EMSA. (A) Expression of Col2a1 and Col10a1 in CCL, MCTs, and MC3T3-E1 was analyzed by RT-PCR. Controls were no RNA (2) or RNA from mouse E17.5 embryo (+). Expression of Col10a1 was found only in MCTs. (B) Western blot analysis showed that both SOX9 and RUNX2 were highly expressed in CCL but weakly in MCTs. (C) Expression of SOX9 in nuclear (lane 3) and cytoplasmic (lane 4) fractions of pcDNA-Sox9 transfected COS-1 cells was compared with CCL (lane 1) and untransfected COS-1 (lane 2) nuclear extracts by Western blotting. (D) Expression of RUNX2 in nuclear (lane 3) and cytoplasmic (lane 4) fractions of pcDNA-Cbfa1 transfected COS-1 cells was verified by Western blotting. (E) A diagram showing the sequences of oligonucleotides which contained wild-type, mutant SOX9 and/or TG-rich motif for EMSA. Only the mutated nucleotides were shown in the sequence of competitors. (F) Intact DNA-binding property of SOX9 in expressing nuclear extract was tested by its interaction with the COL2A1 enhancer probe (COL2C1) in EMSA, in which the retarded band was challenged with SOX9 antibody (lane 4), unlabeled COL2C1 (lane 5–6), or Col10a1 element A C10C1 oligonucleotides (lane 7–8). The triangles represent increasing concentration of competitors from 106 (lane 5,7) to 1006 (lane 6,8) excess. (TIF) Figure S2 In vivo transcriptional activity of Col10a1 element A. (A) Expression pattern of Col10Flag-E in E15.5 transgenic fetus (mid-sagittal plane) examined by in-situ hybridization using digoxigenin-labeled probes. Flag expression was detected in the hypertrophic chondrocytes of cervical pedicle (a) and developing vertebrae (b). Weak expression was detected in the trabecular bone of palate (c). No expression was found in the nucleus pulposus (np) of the intervertebral disc (b), immature costal cartilage (g, cc), brain (h), hair papillae (i), lung (m), aorta (n), or myocardium (o). Expression of Col10a1 is shown for comparison (d–f, j–l, p–r). No expression was found in erythrocytes or nucleated blood cells (* in n and q). (B) In Col10Flag transgenic fetus, strong expression of Flag was found in the ossifying zone of pedicle (a, circled) and the trabecular bone of palate (b, circled). Weak signal was detected in the prehypertrophic zone of the pedicle cartilage (a, arrow). No expression was identified in tissue other than cartilage and bone. Expression of Col10a1 is shown for comparison (c–d). (TIF) Figure S3 Alignment data from SOX9-GLI site analysis. Multispecies alignment of regions spanning the linked SOX9- GLI sites are shown along with chromosomal location (Chr), identical nucleotides (.), insertion or deletion (-), and unaligned positions ( = ). (PDF) Additional details of experiments. Text S1 (DOC) Acknowledgments We thank Anna Niewiadomska for technical assistance; Peter Koopman, Gerard Karsenty, Andras Nagy, Andrew Groves, Veronique Lefebvre, Silvana Guioli, Robin Lovell-Badge, Robert Roeder, Yasuteru Muragaki, and James Kimura for generous gifts of reagents; David Smith and Pak Sham for advice on bioinformatics and statistical analyses; Fanxin Long for helpful discussion; and Chi-chung Hui, Robin Lovell-Badge, and Michael Zhang for critical reading of the manuscript. PLoS Genetics | www.plosgenetics.org 14 November 2011 | Volume 7 | Issue 11 | e1002356