Daxx mediates the small ubiquitin-like modifier-dependent transcriptional repression of Smad4.
Daxx has been shown to function as an apoptosis regulator and transcriptional repressor via its interaction with various cytoplasmic and nuclear proteins. Here, we showed that Daxx interacts with Smad4 and represses its transcriptional activity via the C-terminal domain of Daxx. In vitro and in vivo interaction studies indicated that the binding of Smad4 to Daxx depends on Smad4 sumoylation. Substitution of Smad4 SUMO conjugation residue lysine 159, but not 113, to arginine not only disrupted Smad4-Daxx interaction but also relieved Daxx-elicited repression of Smad4 transcriptional activity. Furthermore, chromatin immunoprecipitation analyses revealed the recruitment of Daxx to an endogenous, Smad4-targeted promoter in a Lys(159) sumoylation-dependent manner. Finally, down-regulation of Daxx expression by RNA interference enhanced transforming growth factor beta-induced transcription of reporter and endogenous genes through a Smad4-dependent, but not K159R-Smad4-dependent, manner. Together, these results indicate that Daxx suppresses Smad4-mediated transcriptional activity by direct interaction with the sumoylated Smad4 and identify a novel role of Daxx in regulating transforming growth factor beta signaling.
Chang
Che-Chang
CC
Graduate Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan, Republic of China.
Lin
Ding-Yen
DY
Fang
Hsin-I
HI
Chen
Ruey-Hwa
RH
Shih
Hsiu-Ming
HM
D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D001120:Arginine / Q000737:chemistry; D015153:Blotting, Western; D019556:COS Cells; D002352:Carrier Proteins / Q000378:metabolism / Q000502:physiology*; D002460:Cell Line; D002843:Chromatin / Q000378:metabolism; D047369:Chromatin Immunoprecipitation; D056970:Co-Repressor Proteins; D004268:DNA-Binding Proteins / Q000378:metabolism*; D017353:Gene Deletion; D017930:Genes, Reporter; D006801:Humans; D047468:Immunoprecipitation; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism / Q000502:physiology*; D008156:Luciferases / Q000378:metabolism; D008239:Lysine / Q000737:chemistry; D008856:Microscopy, Fluorescence; D018832:Molecular Chaperones; D009154:Mutation; D009687:Nuclear Proteins / Q000378:metabolism / Q000502:physiology*; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D034622:RNA Interference; D025842:SUMO-1 Protein; D015398:Signal Transduction; D051901:Smad4 Protein; D025841:Small Ubiquitin-Related Modifier Proteins / Q000378:metabolism*; D015534:Trans-Activators / Q000378:metabolism*; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000378:metabolism*; D020798:Two-Hybrid System Techniques
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D002843:Chromatin; D056970:Co-Repressor Proteins; C106985:DAXX protein, human; D004268:DNA-Binding Proteins; D047908:Intracellular Signaling Peptides and Proteins; D018832:Molecular Chaperones; D009687:Nuclear Proteins; C497796:SMAD4 protein, human; D025842:SUMO-1 Protein; C495501:SUMO1 protein, human; D051901:Smad4 Protein; D025841:Small Ubiquitin-Related Modifier Proteins; D015534:Trans-Activators; D016212:Transforming Growth Factor beta; D001120:Arginine; D008156:Luciferases; D008239:Lysine