Interactions between PIAS Proteins and SOX9 Result in an Increase in the Cellular Concentrations of SOX9

Abstract

We have identified PIAS1 (protein inhibitor of activated STAT-1), -3, -xα, and -xβ as SOX9-associated polypeptides using the Gal4-based yeast two-hybrid system and a cDNA library derived from a chondrocytic cell line. These PIAS proteins were shown to interact directly with SOX9 in two-hybrid, co-immunoprecipitation, and electrophoretic mobility shift assays. SOX9 was sumoylated in cotransfection experiments with COS-7 cells using PIAS and SUMO-1 (small ubiquitin-like modifier-1) expression vectors. SOX9 was also sumoylated in vitro by PIAS proteins in the presence of SUMO-1, the SUMO-activating enzyme, and the SUMO-conjugating enzyme. In COS-7 cells, PIAS proteins stimulated the SOX9-dependent transcriptional activity of a Col2a1 promoter-enhancer reporter. This increase in reporter activity was paralleled by an increase in the cellular levels of SOX9. Cotransfection with a SUMO-expressing vector further enhanced the transcriptional activity of this SOX9-dependent Col2a1 reporter in COS-7 cells, and this additional activation was inhibited in the presence of either SUMO-1 mutants or PIAS RING domain mutants or by coexpression of a desumoylation enzyme. Immunofluorescence microscopy of SOX9-transfected COS-7 cells showed that the subnuclear distribution of SOX9 became more diffuse in the presence of PIAS1 and SUMO-1. Our results suggest that, by controlling the cellular concentrations of SOX9, PIAS proteins and sumoylation may be part of a major regulatory system of SOX9 functions. We have identified PIAS1 (protein inhibitor of activated STAT-1), -3, -xα, and -xβ as SOX9-associated polypeptides using the Gal4-based yeast two-hybrid system and a cDNA library derived from a chondrocytic cell line. These PIAS proteins were shown to interact directly with SOX9 in two-hybrid, co-immunoprecipitation, and electrophoretic mobility shift assays. SOX9 was sumoylated in cotransfection experiments with COS-7 cells using PIAS and SUMO-1 (small ubiquitin-like modifier-1) expression vectors. SOX9 was also sumoylated in vitro by PIAS proteins in the presence of SUMO-1, the SUMO-activating enzyme, and the SUMO-conjugating enzyme. In COS-7 cells, PIAS proteins stimulated the SOX9-dependent transcriptional activity of a Col2a1 promoter-enhancer reporter. This increase in reporter activity was paralleled by an increase in the cellular levels of SOX9. Cotransfection with a SUMO-expressing vector further enhanced the transcriptional activity of this SOX9-dependent Col2a1 reporter in COS-7 cells, and this additional activation was inhibited in the presence of either SUMO-1 mutants or PIAS RING domain mutants or by coexpression of a desumoylation enzyme. Immunofluorescence microscopy of SOX9-transfected COS-7 cells showed that the subnuclear distribution of SOX9 became more diffuse in the presence of PIAS1 and SUMO-1. Our results suggest that, by controlling the cellular concentrations of SOX9, PIAS proteins and sumoylation may be part of a major regulatory system of SOX9 functions. SOX9, a transcription factor of the SRY (sex-determining region, Y chromosome) family, is required for the establishment and differentiation of several cell lineages, including those of chondrocytes, Sertoli cells of male gonads, glial cells of the nervous system, and others. During chondrocyte differentiation, SOX9 is expressed abundantly in mouse chondroprogenitor cells and overtly differentiated chondrocytes (1Bi W. Deng J.M. Zhang Z. Behringer R.R. de Crombrugghe B. Nat. Genet. 1999; 22: 85-89Crossref PubMed Scopus (1444) Google Scholar) and regulates transcription of cartilage-specific extracellular matrix molecules such as collagen types II (2Lefebvre V. Huang W. Harley V.R. Goodfellow P.N. de Crombrugghe B. Mol. Cell. Biol. 1997; 17: 2336-2346Crossref PubMed Google Scholar), IX (3Zhang P. Jimenez S.A. Stokes D.G. J. Biol. Chem. 2003; 278: 117-123Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar), and XI (4Bridgewater L.C. Lefebvre V. de Crombrugghe B. J. Biol. Chem. 1998; 273: 14998-15006Abstract Full Text Full Text PDF PubMed Scopus (243) Google Scholar) and aggrecan (5Sekiya I. Tsuji K. Koopman P. Watanabe H. Yamada Y. Shinomiya K. Nifuji A. Noda M. J. Biol. Chem. 2000; 275: 10738-10744Abstract Full Text Full Text PDF PubMed Scopus (419) Google Scholar). Heterozygous mutations in the Sox9 gene cause campomelic dysplasia, a severe skeletal malformation syndrome characterized by a generalized hypoplasia of endochondral bones. SOX9 inactivation studies in mice indicate that SOX9 has an essential role in several steps of chondrogenic differentiation, including mesenchymal condensations and overt differentiation of chondrocytes (1Bi W. Deng J.M. Zhang Z. Behringer R.R. de Crombrugghe B. Nat. Genet. 1999; 22: 85-89Crossref PubMed Scopus (1444) Google Scholar, 6Akiyama H. Chaboissier M.C. Martin J.F. Schedl A. de Crombrugghe B. Genes Dev. 2002; 16: 2813-2828Crossref PubMed Scopus (1419) Google Scholar). In the absence of SOX9, no chondrocyte marker genes are expressed (1Bi W. Deng J.M. Zhang Z. Behringer R.R. de Crombrugghe B. Nat. Genet. 1999; 22: 85-89Crossref PubMed Scopus (1444) Google Scholar), but the precise mechanism of transcriptional activation by SOX9 of cartilage-specific genes is only poorly understood. The cellular concentration of SOX9 must be tightly regulated in vivo given that campomelic dysplasia is due to haploinsufficiency of SOX9 and because a modest increase in SOX9 expression results in dwarfism in mice (7Akiyama H. Lyons J.P. Mori-Akiyama Y. Yang X. Zhang R. Zhang Z. Deng J.M. Taketo M.M. Nakamura T. Behringer R.R. McCrea P.D. de Crombrugghe B. Genes Dev. 2004; 18: 1072-1087Crossref PubMed Scopus (656) Google Scholar). Members of the SOX9 family of transcription factors contain a high mobility group (HMG) 3The abbreviations used are: HMG, high mobility group; E3, SUMO-protein isopeptide ligase; E1, SUMO-activating enzyme; E2, SUMO-conjugating enzyme; HA, hemagglutinin; GST, glutathione S-transferase; siRNAs, small interfering RNAs; GFP, green fluorescent protein. box DNA-binding domain that is at least 50% identical to an equivalent domain in the sex-determining factor SRY (8Koopman P. CMLS Cell. Mol. Life Sci. 1999; 55: 839-856PubMed Google Scholar). SOX9 also contains a potent transcriptional activation domain located at its C-terminal end. Previous experiments have shown that two other members of the SOX family, L-SOX5 and SOX6, cooperate with SOX9 to activate the Col2a1 and aggrecan genes (9Lefebvre V. Li P. de Crombrugghe B. EMBO J. 1998; 17: 5718-5733Crossref PubMed Scopus (682) Google Scholar, 10Lefebvre V. Behringer R.R. de Crombrugghe B. Osteoarthritis Cartilage. 2001; 9: S69-S75Abstract Full Text PDF PubMed Scopus (312) Google Scholar). In SOX5 and SOX6 double mutants, overt differentiation of chondrocytes is severely inhibited (11Smits P. Li P. Mandel J. Zhang Z. Deng J.M. Behringer R.R. de Crombrugghe B. Lefebvre V. Dev. Cell. 2001; 1: 277-290Abstract Full Text Full Text PDF PubMed Scopus (489) Google Scholar). Several SOX9-interacting transcription proteins have been identified previously. These include a long form of c-Maf (Lc-Maf), which cooperates with SOX9 to activate a Col2a1 enhancer and increases expression of endogenous Col2a1, a SOX9 target gene (12Huang W. Lu N. Eberspaecher H. de Crombrugghe B. J. Biol. Chem. 2002; 277: 50668-50675Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar); TRAP230, a component of the Mediator complex (13Zhou R. Bonneaud N. Yuan C.X. de Santa Barbara P. Boizet B. Schomber T. Scherer G. Roeder R.G. Poulat F. Berta P. Nucleic Acids Res. 2002; 30: 3245-3252Crossref PubMed Scopus (76) Google Scholar); the ubiquitously expressed coactivator p300/CBP (cAMP-responsive element-binding protein-binding protein) (14Tsuda M. Takahashi S. Takahashi Y. Asahara H. J. Biol. Chem. 2003; 278: 27224-27229Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar); and PGC-1α, a transcription factor involved in gluconeogenesis and thermogenesis that enhances the activity of SOX9 in chondrocyte precursors and chondrocytes (15Kawakami Y. Tsuda M. Takahashi S. Taniguchi N. Esteban C.R. Zemmyo M. Furumatsu T. Lotz M. Belmonte J.C. Asahara H. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: PubMed Scopus Google Scholar). further the regulatory mechanism of SOX9-dependent transcription and to additional SOX9-interacting a yeast two-hybrid was using a cDNA library from the chondrocytic cell PIAS1 (protein inhibitor of activated is and of and were identified as SOX9-interacting PIAS1 and were identified in a for proteins that interact with and and to the transcriptional activity of polypeptides J.M. J.M. M. Mol. 2004; 18: PubMed Scopus Google Scholar). PIAS proteins have also been to interact with several other proteins N. S. Mol. 2000; PubMed Scopus Google Scholar, H. N. U. N. M. 2000; PubMed Google Scholar). was characterized as an the transcriptional activity of the PIAS proteins have been to as of the and other H. 2002; PubMed Scopus Google Scholar, A. T. T. H. K. F. H. A. Res. 2000; 278: PubMed Scopus Google Scholar). with the H. F. N. Lyons R. Dev. 1997; PubMed Scopus Google Scholar); PIAS1 also with S. Proc. Natl. Acad. Sci. U. S. A. 2002; PubMed Scopus Google Scholar, T. T. H. Mol. Cell. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). PIAS proteins have been shown to as (small ubiquitin-like isopeptide T. T. H. Mol. Cell. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, N. U. Mol. Cell. Biol. 2002; 22: PubMed Scopus Google Scholar, T. H. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar). The system of proteins to the system Mol. Cell. 2005; 18: Full Text Full Text PDF PubMed Scopus Google Scholar, T. R. G. N. H. Res. 1999; PubMed Scopus Google Scholar). is activated in an by a SUMO-activating of an is to a SUMO-conjugating and to the group of in of be with only and this the presence of proteins to be and to target studies that sumoylation by PIAS family members is to target transcription factors such as and S. Proc. Natl. Acad. Sci. U. S. A. 2002; PubMed Scopus Google Scholar) and W. M. A. R. J. 2004; PubMed Scopus Google Scholar, Z. Zhang Y. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar). PIAS proteins a RING which is required for SOX9 has sumoylation and In this that PIAS proteins directly interact with SOX9 and SOX9 sumoylation in vitro and in Our results indicate that, in COS-7 cells, and the sumoylation of SOX9 SOX9 and may The cellular levels of SOX9 to an increase in the SOX9-dependent transcription of a Col2a1 reporter. We that of SOX9 is part of a system that the cellular concentrations of SOX9. cDNA and and of SOX9 were as by and the which contains the DNA-binding domain of a cDNA library from the chondrocytic cell was in the of the which the activation of cDNA with an of of were of the cDNA library was yeast cells that been with a SOX9 or and and and and of were and was for further and the of the vector PIAS1 was in the yeast with the vector either or The vector was also with the of SOX9 as a double were in and of cells were and cellular proteins were by and in yeast and the PIAS1 and either or SOX9, the activity of the reporter gene was to and from the activity of the vector with the of SOX9 SOX9 expression vector was by SOX9 to a at its was the vector (2Lefebvre V. Huang W. Harley V.R. Goodfellow P.N. de Crombrugghe B. Mol. Cell. Biol. 1997; 17: 2336-2346Crossref PubMed Google Scholar). SOX9 mutants were in the vector with of for the and for the and and for the and of or or or and or was T. H. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar). of and of of SOX9 an was to the Sox9 gene by and the which a and an and cells for expression of The expressed was with and SUMO-1 were expressed in and as T. R. G. N. H. Res. 1999; PubMed Scopus Google Scholar). and PIAS proteins were expressed in cells and with as T. T. H. Mol. Cell. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, T. H. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar, T. R. G. N. H. Res. 1999; PubMed Scopus Google Scholar). SOX9 was also in for of The SOX9 cDNA a was by the which a and The expressed SOX9 was with and by were with of SOX9 was and was with a and cells, cells, and cells were in with experiments were using experiments were with small interfering for SUMO-1, and by SUMO-1, and from also the The of the of the were as X. M. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar); SUMO-1, and of was with to the activity of the Col2a1 reporter and that of the were were in and and and were as PubMed Scopus Google Scholar). and and cells were with and in and the were at and concentration was the cell were in and the were with and for at The were with and the proteins were from by in and were to and the were with a and with a was by enhanced and to In in vitro sumoylation was in a of of or of of SUMO-1, and or of for at the was by and with The were also with and sumoylated proteins were with and SOX9 were and in vitro in of and in the presence of using the of the were with and directly by The of the were used for with were in of and for at The was with and SOX9 proteins were by with and by to the box was with and The contains the for including SOX9, and were at the of for with were as V. K. G. S. Eberspaecher H. de Crombrugghe B. N. Y. Acad. Sci. PubMed Scopus Google Scholar). with proteins were with of and of and those with cell were with of and of Immunofluorescence and with expression were with for at The cells were with and for at and with for at The of the were as and the cells were with and The of the were as and fluorescent PIAS1 directly was were with a PIAS with and were identified as proteins in a yeast two-hybrid of a cDNA and PIAS1 were expressed in showed expression in and but in and The presence of PIAS1 and proteins in chondrocytes was also by using in several cells, including cells mouse chondrocytes, and cells This results by and The indicate that PIAS1 and were expressed in chondrocytic cells, as as in COS-7 cells the SOX9 and PIAS used several to the PIAS1 in SOX9, SOX9 or or were expressed in yeast cells as DNA-binding domain proteins PIAS1 was as a domain protein. PIAS1 and SOX9 was by the activity of the reporter to PIAS1 was by SOX9 that the that or SOX9 and were by in vitro and with and with PIAS1 was by to These experiments the two-hybrid in showed the with PIAS1 in the yeast two-hybrid the in vitro was with The results of the suggest that the C-terminal domain of SOX9 which contains of the sumoylation is for with of SOX9 that contain two other sumoylation also to We also the SOX9 and PIAS1 by of the proteins in COS-7 that PIAS1 was with in the presence and absence of SUMO-1. other members of the PIAS family, PIAS1 has been to as a SOX9 contains an box domain that directly to a in the of In electrophoretic mobility shift using a an box of PIAS1 to SOX9 a complex and increases the of complex The presence of SOX9 and in the was by of or to a PIAS1 to the but enhanced the of SOX9. These experiments a SOX9 and -xα, and SOX9-dependent of a a of cotransfection experiments with COS-7 cells, PIAS proteins the activity of a SOX9-dependent Col2a1 reporter by This increase was In the absence of SOX9, PIAS proteins the activity of the reporter that cotransfection of SOX9 and PIAS the levels of SOX9 that the SOX9 and PIAS proteins to of the SOX9 the increase in the activity of the SOX9-dependent reporter in COS-7 cells is due in part to of SOX9, of SOX9 to also have a the of SOX9 by PIAS is due to to an inhibitor of further increase the levels of SOX9. shown H. T. Yang X. R. L.C. M. Mori-Akiyama Y. de Crombrugghe B. Biol. 2005; PubMed Scopus Google Scholar), the levels of SOX9 in the absence of This increase was by of an inhibitor of In the presence of further increase the enhanced levels of SOX9, and These results suggest that the SOX9 and PIAS the of SOX9 by the Members of the PIAS as for of proteins to increases the by and the of a SOX9 is and proteins in COS-7 In the absence of PIAS the of SOX9 from cells as a The mobility of a at was with a of sumoylation of SOX9 by endogenous of PIAS1 or a of the and the of additional that may of several SUMO-1 molecules and SOX9 PIAS proteins that were identified yeast two-hybrid showed the of SUMO-1 to SOX9, the cell were with and the was by using to SUMO-1 or to SOX9 This that the at was SOX9. PIAS proteins with mutations in the RING domain have a SUMO-1 also the of RING domain mutations in PIAS proteins sumoylation of SOX9. that coexpression of or with SOX9 and SUMO-1 SUMO-1 to SOX9 with PIAS cotransfection with SUMO-1 and the RING domain mutants of PIAS1 and the of the of sumoylated SOX9 In the of a SUMO-1 to the of the and the of SOX9 of SOX9 at the sumoylation in SOX9, SOX9 mutants mutations and in were with PIAS1 and SUMO-1. the of SOX9 were to SUMO-1, that is the sumoylation of SOX9 sumoylation was with the a role for in sumoylation We also further the of the desumoylation in COS-7 Cotransfection of and SUMO-1 with PIAS1 and SOX9 the of the of sumoylated SOX9, a no sumoylation of SOX9 PIAS proteins with an RING a that has the to be and a in SOX9. SOX9 by -xα, and In in vitro sumoylation experiments using SOX9 as a to and are to SUMO-1 to SOX9. -xα, or or SOX9 was expressed in a vector as a in by or and used for sumoylation of SOX9 in the presence of and In the absence of PIAS E1, E2, or SUMO-1 was from the no sumoylation of SOX9 was In the absence of PIAS a sumoylated SOX9 was with no SOX9 was sumoylated with of -xα, or enhanced sumoylation of SOX9 in a the results of SOX9 sumoylation by PIAS proteins in SUMO-1 the and in the of a SOX9-dependent Col2a1 of a SUMO-1 expression vector in cotransfection experiments with COS-7 cells with SOX9 and PIAS proteins a further increase in the activity of the Col2a1 reporter This increase the of activation of the reporter by PIAS proteins and SOX9 was inhibited by a SUMO-1 to to the in cells with of and in which the RING been and no increase in transcription of the Col2a1 reporter the increase in reporter activity with the PIAS mutants SUMO-1 was the as that with the PIAS proteins This that the activity of PIAS proteins is required to increase the activity of the SOX9-dependent reporter. This was further by the of an expression vector for the desumoylation expression inhibited the increase in reporter activity due to a to reporter activity experiments that, in COS-7 cells, the increase in SOX9-dependent reporter activity by PIAS proteins was due to sumoylation of SOX9 by endogenous that the of SOX9 with PIAS1 SUMO-1 transcriptional activation of the Col2a1 reporter SOX9 to with a Col2a1 reporter in which the was or in which the enhancer was coexpression of SOX9 PIAS1 and SUMO-1 to activate the Col2a1 reporter. This also showed that of SUMO-1 further the cellular levels of SOX9. This increase in the levels of SOX9 was in with the increase in activity of the Col2a1 reporter. In the of COS-7 cells in which SOX9 was with PIAS or RING domain mutants, the levels of SOX9 were that the activity of PIAS is required for the of SOX9. further the that PIAS proteins the activity of the SOX9-dependent Col2a1 reporter in the absence of were used to sumoylation and Cotransfection of to which levels in COS-7 cells the increase in reporter activity due to SUMO-1 but the increase in reporter activity due to with to SUMO-1 or the increase in reporter activity due to PIAS1 was results were of to SUMO-1 and The of to SUMO-1, and showed results that PIAS1 the SOX9-dependent reporter of SOX9 in the of a Col2a1 mutants mutations of sumoylation and sumoylation In of COS-7 cells, SOX9 stimulated the SOX9-dependent reporter more SOX9 the levels of SOX9 were those of SOX9 This the that SOX9 may have a transcriptional activity with SOX9. Cotransfection of SOX9 with PIAS further the activity of the SOX9-dependent reporter In the levels of SOX9 in with the increase in SOX9 levels by These experiments that the sumoylation of SOX9 were for the enhanced of SOX9 by The for the transcriptional activity of SOX9 is but the results indicate that the transcriptional activity of SOX9 of SOX9. SOX9 proteins have been shown to to subnuclear as A. R. J.M. P. A. Mol. Cell. Biol. 2001; PubMed Scopus Google Scholar, S. S. S. A. 2000; PubMed Google Scholar). These which contain and other are with the matrix M. Biol. Cell. PubMed Google Scholar). the of PIAS1 and sumoylation the subnuclear of SOX9, a of COS-7 SOX9 and PIAS1 showed a SOX9 was more the PIAS1 and SOX9 were proteins were at least a more diffuse of PIAS1 and SUMO-1 in and In the SUMO-1 to the of SOX9, and SUMO-1 showed a distribution of proteins in a diffuse which was with the SUMO-1 the that the distribution be due to by the expression the were the been the subnuclear of SOX9 coexpression of PIAS1 and SUMO-1 were to that shown in the that the of SOX9 distribution was a of the distribution of endogenous SOX9 was in the chondrocytic cell which contains endogenous SOX9 and PIAS1 Immunofluorescence of cells a but more distribution of endogenous SOX9 SOX9 mutants sumoylation the diffuse but a more subnuclear distribution with PIAS1 and SUMO-1 coexpression of the desumoylation SOX9 in a more subnuclear distribution in the presence of PIAS1 and SUMO-1, coexpression of a this The proteins -xα, and were identified in a yeast two-hybrid as SOX9-interacting Several additional for a SOX9 and two-hybrid in yeast showed that the C-terminal transcriptional activation domain of SOX9 was a major but that other also in The were further by in vitro by of SOX9 and PIAS1 cotransfection of COS-7 cells, by in the of cells as by and by electrophoretic mobility shift the of a complex SOX9 and Our experiments also that, in cells and in PIAS proteins enhanced the sumoylation of SOX9, as as shown for other target proteins T. T. H. Mol. Cell. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, T. H. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar). sumoylation of SOX9 a of SOX9 to an increase of This was either a to be to or a PIAS in activity was SOX9 contains sumoylation but is the of the in SOX9 inhibited sumoylation of SOX9. In of COS-7 cells, PIAS proteins the SOX9-dependent activity of a Col2a1 reporter in which a enhancer was This increase in reporter activity was paralleled by an increase in the cellular concentration of SOX9, the that PIAS polypeptides SOX9 These experiments were to those used for the that SOX9 and PIAS are part of the We that experiments that a SOX9 and PIAS polypeptides in COS-7 We an the levels of SOX9 and the increase in reporter activity by the PIAS This increase in reporter gene activity and the increase in the levels of SOX9 in the absence of with a to be to or with a PIAS in the increase in SOX9-dependent reporter activity is that the increase in SOX9-dependent reporter activity was due to sumoylation of SOX9 by endogenous because this enhanced SOX9-dependent reporter activity in presence of the desumoylation The increase in reporter activity further enhanced by cotransfection with a SUMO-expressing in the SOX9 sumoylation which the sumoylation of SOX9, the activity of the SOX9-dependent and a the major of SOX9 an increase in the activity of the SOX9-dependent reporter to that by the with the is also a major of as the and a modest increase in the of SOX9 H. T. Yang X. R. L.C. M. Mori-Akiyama Y. de Crombrugghe B. Biol. 2005; PubMed Scopus Google Scholar). The increase in SOX9-dependent reporter activity by the with mutations in the sumoylation to be due to a transcriptional activity of SOX9 and be the of of this with or with that the increase in activity of the SOX9-dependent which was with the that sumoylation of SOX9, be as that sumoylation the transcriptional activity of SOX9 given that other experiments that the PIAS polypeptides and sumoylation the levels of SOX9 and the activity of a SOX9-dependent reporter. of SOX9 at least in and in presence of an inhibitor of the levels of SOX9 In the inhibitor further increase the cellular concentration of SOX9 in cells with This that PIAS proteins the of SOX9. in the presence of the inhibitor the cellular concentrations of SOX9 This was inhibited in the presence of the that PIAS proteins SOX9 from the of and SOX9 in a to of COS-7 cells, SOX9 showed a distribution in the Cotransfection with PIAS1 showed of SOX9 and with a more diffuse subnuclear distribution of SOX9. Cotransfection of SOX9, and SUMO-1 of proteins in a diffuse This diffuse of proteins the of SUMO-1 to be and sumoylation in SOX9. We that the of SOX9 with PIAS and the of SOX9 the This to a diffuse distribution of SOX9 with the activity of a SOX9-dependent reporter and with the cellular concentrations of SOX9. be that the in the subnuclear distribution of SOX9 was the in the distribution of with from a diffuse to a This was with of the activity of a reporter in to the increase in the activity of a SOX9-dependent for the role of PIAS in its with SOX9 and for the role of sumoylation of SOX9. We that SOX9, the and sumoylated SOX9 are in with sumoylated SOX9 be the Our that, PIAS with SOX9, SOX9, its and the This in an increase in the cellular concentration of SOX9 and of SOX9. of SOX9 also its subnuclear We that PIAS and the system have an role in controlling the cellular concentration of SOX9. such an SOX9 concentration is for the of SOX9. 50% in the cellular concentration of SOX9 as in with campomelic dysplasia, a due to mutations of SOX9, has severe for and for In a modest increase in SOX9 of in chondrocytes chondrocyte and dwarfism (7Akiyama H. Lyons J.P. Mori-Akiyama Y. Yang X. Zhang R. Zhang Z. Deng J.M. Taketo M.M. Nakamura T. Behringer R.R. McCrea P.D. de Crombrugghe B. Genes Dev. 2004; 18: 1072-1087Crossref PubMed Scopus (656) Google Scholar). In to the PIAS polypeptides and as as the system, several other are to in an concentration of SOX9. PIAS and the also have a role in the subnuclear distribution of SOX9 to its in controlling of cell differentiation and cell We are to and for for with and and for We and for and members of the de Crombrugghe for We also for the SUMO-1, and and for the