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. 1997 Apr;17(4):2336–2346. doi: 10.1128/mcb.17.4.2336

SOX9 is a potent activator of the chondrocyte-specific enhancer of the pro alpha1(II) collagen gene.

V Lefebvre 1, W Huang 1, V R Harley 1, P N Goodfellow 1, B de Crombrugghe 1
PMCID: PMC232082  PMID: 9121483

Abstract

The identification of mutations in the SRY-related SOX9 gene in patients with campomelic dysplasia, a severe skeletal malformation syndrome, and the abundant expression of Sox9 in mouse chondroprogenitor cells and fully differentiated chondrocytes during embryonic development have suggested the hypothesis that SOX9 might play a role in chondrogenesis. Our previous experiments with the gene (Col2a1) for collagen II, an early and abundant marker of chondrocyte differentiation, identified a minimal DNA element in intron 1 which directs chondrocyte-specific expression in transgenic mice. This element is also a strong chondrocyte-specific enhancer in transient transfection experiments. We show here that Col2a1 expression is closely correlated with high levels of SOX9 RNA and protein in chondrocytes. Our experiments indicate that the minimal Col2a1 enhancer is a direct target for Sox9. Indeed, SOX9 binds to a sequence of the minimal Col2a1 enhancer that is essential for activity in chondrocytes, and SOX9 acts as a potent activator of this enhancer in cotransfection experiments in nonchondrocytic cells. Mutations in the enhancer that prevent binding of SOX9 abolish enhancer activity in chondrocytes and suppress enhancer activation by SOX9 in nonchondrocytic cells. Other SOX family members are ineffective. Expression of a truncated SOX9 protein lacking the transactivation domain but retaining DNA-binding activity interferes with enhancer activation by full-length SOX9 in fibroblasts and inhibits enhancer activity in chondrocytes. Our results strongly suggest a model whereby SOX9 is involved in the control of the cell-specific activation of COL2A1 in chondrocytes, an essential component of the differentiation program of these cells. We speculate that in campomelic dysplasia a decrease in SOX9 activity would inhibit production of collagen II, and eventually other cartilage matrix proteins, leading to major skeletal anomalies.

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Selected References

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  1. Baxevanis A. D., Landsman D. The HMG-1 box protein family: classification and functional relationships. Nucleic Acids Res. 1995 May 11;23(9):1604–1613. doi: 10.1093/nar/23.9.1604. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Cancedda R., Descalzi Cancedda F., Castagnola P. Chondrocyte differentiation. Int Rev Cytol. 1995;159:265–358. doi: 10.1016/s0074-7696(08)62109-9. [DOI] [PubMed] [Google Scholar]
  3. Cheah K. S., Lau E. T., Au P. K., Tam P. P. Expression of the mouse alpha 1(II) collagen gene is not restricted to cartilage during development. Development. 1991 Apr;111(4):945–953. doi: 10.1242/dev.111.4.945. [DOI] [PubMed] [Google Scholar]
  4. Chen C. A., Okayama H. Calcium phosphate-mediated gene transfer: a highly efficient transfection system for stably transforming cells with plasmid DNA. Biotechniques. 1988 Jul-Aug;6(7):632–638. [PubMed] [Google Scholar]
  5. Connor F., Cary P. D., Read C. M., Preston N. S., Driscoll P. C., Denny P., Crane-Robinson C., Ashworth A. DNA binding and bending properties of the post-meiotically expressed Sry-related protein Sox-5. Nucleic Acids Res. 1994 Aug 25;22(16):3339–3346. doi: 10.1093/nar/22.16.3339. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Connor F., Wright E., Denny P., Koopman P., Ashworth A. The Sry-related HMG box-containing gene Sox6 is expressed in the adult testis and developing nervous system of the mouse. Nucleic Acids Res. 1995 Sep 11;23(17):3365–3372. doi: 10.1093/nar/23.17.3365. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Coustry F., Maity S. N., de Crombrugghe B. Studies on transcription activation by the multimeric CCAAT-binding factor CBF. J Biol Chem. 1995 Jan 6;270(1):468–475. doi: 10.1074/jbc.270.1.468. [DOI] [PubMed] [Google Scholar]
  8. Dailey L., Yuan H., Basilico C. Interaction between a novel F9-specific factor and octamer-binding proteins is required for cell-type-restricted activity of the fibroblast growth factor 4 enhancer. Mol Cell Biol. 1994 Dec;14(12):7758–7769. doi: 10.1128/mcb.14.12.7758. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Davis R. L., Weintraub H., Lassar A. B. Expression of a single transfected cDNA converts fibroblasts to myoblasts. Cell. 1987 Dec 24;51(6):987–1000. doi: 10.1016/0092-8674(87)90585-x. [DOI] [PubMed] [Google Scholar]
  10. Denny P., Swift S., Connor F., Ashworth A. An SRY-related gene expressed during spermatogenesis in the mouse encodes a sequence-specific DNA-binding protein. EMBO J. 1992 Oct;11(10):3705–3712. doi: 10.1002/j.1460-2075.1992.tb05455.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Ferrari S., Harley V. R., Pontiggia A., Goodfellow P. N., Lovell-Badge R., Bianchi M. E. SRY, like HMG1, recognizes sharp angles in DNA. EMBO J. 1992 Dec;11(12):4497–4506. doi: 10.1002/j.1460-2075.1992.tb05551.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Foster J. W., Dominguez-Steglich M. A., Guioli S., Kwok C., Weller P. A., Stevanović M., Weissenbach J., Mansour S., Young I. D., Goodfellow P. N. Campomelic dysplasia and autosomal sex reversal caused by mutations in an SRY-related gene. Nature. 1994 Dec 8;372(6506):525–530. doi: 10.1038/372525a0. [DOI] [PubMed] [Google Scholar]
  13. Giese K., Cox J., Grosschedl R. The HMG domain of lymphoid enhancer factor 1 bends DNA and facilitates assembly of functional nucleoprotein structures. Cell. 1992 Apr 3;69(1):185–195. doi: 10.1016/0092-8674(92)90129-z. [DOI] [PubMed] [Google Scholar]
  14. Giese K., Kingsley C., Kirshner J. R., Grosschedl R. Assembly and function of a TCR alpha enhancer complex is dependent on LEF-1-induced DNA bending and multiple protein-protein interactions. Genes Dev. 1995 Apr 15;9(8):995–1008. doi: 10.1101/gad.9.8.995. [DOI] [PubMed] [Google Scholar]
  15. Goldberg H., Helaakoski T., Garrett L. A., Karsenty G., Pellegrino A., Lozano G., Maity S., de Crombrugghe B. Tissue-specific expression of the mouse alpha 2(I) collagen promoter. Studies in transgenic mice and in tissue culture cells. J Biol Chem. 1992 Sep 25;267(27):19622–19630. [PubMed] [Google Scholar]
  16. Goodfellow P. N., Lovell-Badge R. SRY and sex determination in mammals. Annu Rev Genet. 1993;27:71–92. doi: 10.1146/annurev.ge.27.120193.000443. [DOI] [PubMed] [Google Scholar]
  17. Grosschedl R., Giese K., Pagel J. HMG domain proteins: architectural elements in the assembly of nucleoprotein structures. Trends Genet. 1994 Mar;10(3):94–100. doi: 10.1016/0168-9525(94)90232-1. [DOI] [PubMed] [Google Scholar]
  18. Harley V. R., Jackson D. I., Hextall P. J., Hawkins J. R., Berkovitz G. D., Sockanathan S., Lovell-Badge R., Goodfellow P. N. DNA binding activity of recombinant SRY from normal males and XY females. Science. 1992 Jan 24;255(5043):453–456. doi: 10.1126/science.1734522. [DOI] [PubMed] [Google Scholar]
  19. Harley V. R., Lovell-Badge R., Goodfellow P. N. Definition of a consensus DNA binding site for SRY. Nucleic Acids Res. 1994 Apr 25;22(8):1500–1501. doi: 10.1093/nar/22.8.1500. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. He X., Treacy M. N., Simmons D. M., Ingraham H. A., Swanson L. W., Rosenfeld M. G. Expression of a large family of POU-domain regulatory genes in mammalian brain development. Nature. 1989 Jul 6;340(6228):35–41. doi: 10.1038/340035a0. [DOI] [PubMed] [Google Scholar]
  21. Hosking B. M., Muscat G. E., Koopman P. A., Dowhan D. H., Dunn T. L. Trans-activation and DNA-binding properties of the transcription factor, Sox-18. Nucleic Acids Res. 1995 Jul 25;23(14):2626–2628. doi: 10.1093/nar/23.14.2626. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Kent J., Wheatley S. C., Andrews J. E., Sinclair A. H., Koopman P. A male-specific role for SOX9 in vertebrate sex determination. Development. 1996 Sep;122(9):2813–2822. doi: 10.1242/dev.122.9.2813. [DOI] [PubMed] [Google Scholar]
  23. Kim J. B., Spiegelman B. M. ADD1/SREBP1 promotes adipocyte differentiation and gene expression linked to fatty acid metabolism. Genes Dev. 1996 May 1;10(9):1096–1107. doi: 10.1101/gad.10.9.1096. [DOI] [PubMed] [Google Scholar]
  24. Kim J., Zwieb C., Wu C., Adhya S. Bending of DNA by gene-regulatory proteins: construction and use of a DNA bending vector. Gene. 1989 Dec 21;85(1):15–23. doi: 10.1016/0378-1119(89)90459-9. [DOI] [PubMed] [Google Scholar]
  25. Koopman P., Gubbay J., Vivian N., Goodfellow P., Lovell-Badge R. Male development of chromosomally female mice transgenic for Sry. Nature. 1991 May 9;351(6322):117–121. doi: 10.1038/351117a0. [DOI] [PubMed] [Google Scholar]
  26. Krebsbach P. H., Nakata K., Bernier S. M., Hatano O., Miyashita T., Rhodes C. S., Yamada Y. Identification of a minimum enhancer sequence for the type II collagen gene reveals several core sequence motifs in common with the link protein gene. J Biol Chem. 1996 Feb 23;271(8):4298–4303. doi: 10.1074/jbc.271.8.4298. [DOI] [PubMed] [Google Scholar]
  27. Kwok C., Weller P. A., Guioli S., Foster J. W., Mansour S., Zuffardi O., Punnett H. H., Dominguez-Steglich M. A., Brook J. D., Young I. D. Mutations in SOX9, the gene responsible for Campomelic dysplasia and autosomal sex reversal. Am J Hum Genet. 1995 Nov;57(5):1028–1036. [PMC free article] [PubMed] [Google Scholar]
  28. Laudet V., Stehelin D., Clevers H. Ancestry and diversity of the HMG box superfamily. Nucleic Acids Res. 1993 May 25;21(10):2493–2501. doi: 10.1093/nar/21.10.2493. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Lefebvre V., Garofalo S., Zhou G., Metsäranta M., Vuorio E., De Crombrugghe B. Characterization of primary cultures of chondrocytes from type II collagen/beta-galactosidase transgenic mice. Matrix Biol. 1994 Aug;14(4):329–335. doi: 10.1016/0945-053x(94)90199-6. [DOI] [PubMed] [Google Scholar]
  30. Lefebvre V., Garofalo S., de Crombrugghe B. Type X collagen gene expression in mouse chondrocytes immortalized by a temperature-sensitive simian virus 40 large tumor antigen. J Cell Biol. 1995 Jan;128(1-2):239–245. doi: 10.1083/jcb.128.1.239. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Lefebvre V., Zhou G., Mukhopadhyay K., Smith C. N., Zhang Z., Eberspaecher H., Zhou X., Sinha S., Maity S. N., de Crombrugghe B. An 18-base-pair sequence in the mouse proalpha1(II) collagen gene is sufficient for expression in cartilage and binds nuclear proteins that are selectively expressed in chondrocytes. Mol Cell Biol. 1996 Aug;16(8):4512–4523. doi: 10.1128/mcb.16.8.4512. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Leger H., Sock E., Renner K., Grummt F., Wegner M. Functional interaction between the POU domain protein Tst-1/Oct-6 and the high-mobility-group protein HMG-I/Y. Mol Cell Biol. 1995 Jul;15(7):3738–3747. doi: 10.1128/mcb.15.7.3738. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Mallein-Gerin F., Olsen B. R. Expression of simian virus 40 large T (tumor) oncogene in mouse chondrocytes induces cell proliferation without loss of the differentiated phenotype. Proc Natl Acad Sci U S A. 1993 Apr 15;90(8):3289–3293. doi: 10.1073/pnas.90.8.3289. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Mansour S., Hall C. M., Pembrey M. E., Young I. D. A clinical and genetic study of campomelic dysplasia. J Med Genet. 1995 Jun;32(6):415–420. doi: 10.1136/jmg.32.6.415. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Molkentin J. D., Black B. L., Martin J. F., Olson E. N. Cooperative activation of muscle gene expression by MEF2 and myogenic bHLH proteins. Cell. 1995 Dec 29;83(7):1125–1136. doi: 10.1016/0092-8674(95)90139-6. [DOI] [PubMed] [Google Scholar]
  36. Morais da Silva S., Hacker A., Harley V., Goodfellow P., Swain A., Lovell-Badge R. Sox9 expression during gonadal development implies a conserved role for the gene in testis differentiation in mammals and birds. Nat Genet. 1996 Sep;14(1):62–68. doi: 10.1038/ng0996-62. [DOI] [PubMed] [Google Scholar]
  37. Mukhopadhyay K., Lefebvre V., Zhou G., Garofalo S., Kimura J. H., de Crombrugghe B. Use of a new rat chondrosarcoma cell line to delineate a 119-base pair chondrocyte-specific enhancer element and to define active promoter segments in the mouse pro-alpha 1(II) collagen gene. J Biol Chem. 1995 Nov 17;270(46):27711–27719. doi: 10.1074/jbc.270.46.27711. [DOI] [PubMed] [Google Scholar]
  38. Südbeck P., Schmitz M. L., Baeuerle P. A., Scherer G. Sex reversal by loss of the C-terminal transactivation domain of human SOX9. Nat Genet. 1996 Jun;13(2):230–232. doi: 10.1038/ng0696-230. [DOI] [PubMed] [Google Scholar]
  39. Tanaka M., Herr W. Differential transcriptional activation by Oct-1 and Oct-2: interdependent activation domains induce Oct-2 phosphorylation. Cell. 1990 Feb 9;60(3):375–386. doi: 10.1016/0092-8674(90)90589-7. [DOI] [PubMed] [Google Scholar]
  40. Thompson J. F., Landy A. Empirical estimation of protein-induced DNA bending angles: applications to lambda site-specific recombination complexes. Nucleic Acids Res. 1988 Oct 25;16(20):9687–9705. doi: 10.1093/nar/16.20.9687. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Tjian R., Maniatis T. Transcriptional activation: a complex puzzle with few easy pieces. Cell. 1994 Apr 8;77(1):5–8. doi: 10.1016/0092-8674(94)90227-5. [DOI] [PubMed] [Google Scholar]
  42. Tontonoz P., Hu E., Spiegelman B. M. Stimulation of adipogenesis in fibroblasts by PPAR gamma 2, a lipid-activated transcription factor. Cell. 1994 Dec 30;79(7):1147–1156. doi: 10.1016/0092-8674(94)90006-x. [DOI] [PubMed] [Google Scholar]
  43. Wagner T., Wirth J., Meyer J., Zabel B., Held M., Zimmer J., Pasantes J., Bricarelli F. D., Keutel J., Hustert E. Autosomal sex reversal and campomelic dysplasia are caused by mutations in and around the SRY-related gene SOX9. Cell. 1994 Dec 16;79(6):1111–1120. doi: 10.1016/0092-8674(94)90041-8. [DOI] [PubMed] [Google Scholar]
  44. Wirth J., Wagner T., Meyer J., Pfeiffer R. A., Tietze H. U., Schempp W., Scherer G. Translocation breakpoints in three patients with campomelic dysplasia and autosomal sex reversal map more than 130 kb from SOX9. Hum Genet. 1996 Feb;97(2):186–193. doi: 10.1007/BF02265263. [DOI] [PubMed] [Google Scholar]
  45. Wotton D., Lake R. A., Farr C. J., Owen M. J. The high mobility group transcription factor, SOX4, transactivates the human CD2 enhancer. J Biol Chem. 1995 Mar 31;270(13):7515–7522. doi: 10.1074/jbc.270.13.7515. [DOI] [PubMed] [Google Scholar]
  46. Wright E. M., Snopek B., Koopman P. Seven new members of the Sox gene family expressed during mouse development. Nucleic Acids Res. 1993 Feb 11;21(3):744–744. doi: 10.1093/nar/21.3.744. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Wright E., Hargrave M. R., Christiansen J., Cooper L., Kun J., Evans T., Gangadharan U., Greenfield A., Koopman P. The Sry-related gene Sox9 is expressed during chondrogenesis in mouse embryos. Nat Genet. 1995 Jan;9(1):15–20. doi: 10.1038/ng0195-15. [DOI] [PubMed] [Google Scholar]
  48. Yuan H., Corbi N., Basilico C., Dailey L. Developmental-specific activity of the FGF-4 enhancer requires the synergistic action of Sox2 and Oct-3. Genes Dev. 1995 Nov 1;9(21):2635–2645. doi: 10.1101/gad.9.21.2635. [DOI] [PubMed] [Google Scholar]
  49. Zhou G., Garofalo S., Mukhopadhyay K., Lefebvre V., Smith C. N., Eberspaecher H., de Crombrugghe B. A 182 bp fragment of the mouse pro alpha 1(II) collagen gene is sufficient to direct chondrocyte expression in transgenic mice. J Cell Sci. 1995 Dec;108(Pt 12):3677–3684. doi: 10.1242/jcs.108.12.3677. [DOI] [PubMed] [Google Scholar]
  50. Zwilling S., König H., Wirth T. High mobility group protein 2 functionally interacts with the POU domains of octamer transcription factors. EMBO J. 1995 Mar 15;14(6):1198–1208. doi: 10.1002/j.1460-2075.1995.tb07103.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. van de Wetering M., Clevers H. Sequence-specific interaction of the HMG box proteins TCF-1 and SRY occurs within the minor groove of a Watson-Crick double helix. EMBO J. 1992 Aug;11(8):3039–3044. doi: 10.1002/j.1460-2075.1992.tb05374.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. van de Wetering M., Oosterwegel M., van Norren K., Clevers H. Sox-4, an Sry-like HMG box protein, is a transcriptional activator in lymphocytes. EMBO J. 1993 Oct;12(10):3847–3854. doi: 10.1002/j.1460-2075.1993.tb06063.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

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