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Keywords:

  • SOX9;
  • PIAS1;
  • SUMO-1;
  • sex determination;
  • chondrogenesis

Abstract

SOX9 is a transcription factor that harbors a HMG box and plays critical roles in developmental processes including sex determination and chondrogenesis. Currently the regulation of its molecular activity is not well characterized. In the present study, we have identified PIAS1 as a regulator for SOX9 activity. Using the GST pull-down, co-immunoprecipitation, and co-localization methods in tissue culture cells and mouse embryonic tissues, we demonstrated that SOX9 interacts with PIAS1 in vitro and in vivo. PIAS1 enhanced the SUMOylation at lysine 396 of mouse SOX9. Mutant SOX9 with a conversion of lysine 396 to arginine had a distinct nuclear localization from SOX9 with covalently attached SUMO-1. Effects of SOX9 SUMOylation on its transcriptional activity were examined by reporter assays using Vanin-1 promoter and Col11a2 enhancer constructs. The lysine 396 to arginine conversion significantly increased the reporter gene activity, while covalent attachment of SUMO-1 to SOX9 by gene fusion dramatically compromised its transcriptional activity on the reporter gene. Effects of SOX9 interaction with PIAS1 on its transcriptional activity were examined by similar reporter assays. PIAS1 was able to repress both wild type SOX9 and SUMOylation-deficient SOX9-K396R, suggesting that SOX9 SUMOylation is not absolutely required for the repression by PIAS1. However, the repression was further enhanced by exogenous SUMO-1 while SUMO-ligase-deficient PIAS1 was not able to repress SOX9 activity. Thus, PIAS1 appears to repress SOX9 activity by at least two SUMO-ligase dependent mechanisms: (1) the SUMOylation of SOX9 and (2) SUMOylation of unknown factors associated with SOX9 and/or PIAS1. Mol. Reprod. Dev. 74: 1446–1455, 2007. © 2007 Wiley-Liss, Inc.