Apoptosis-antagonizing transcription factor (AATF), also termed Che-1, was identified as interacting protein of Dlk/ZIP kinase and RNA polymerase II, respectively. Che-1 has additionally been shown to bind Rb, thereby activating transcription factor E2F and promoting cell cycle progression. Moreover, AATF enhances steroid receptor-mediated transactivation in a hormone- and dose-dependent manner (Leister, P., Burgdorf, S., and Scheidtmann, K. H., (2003) Signal Transduction 3, 18-25). These data suggest that AATF exerts its functions through interaction with different transcription factors. In search of novel interaction partners of AATF, we identified the tumor susceptibility gene product TSG101, which had also been recognized as a co-regulator of nuclear hormone receptors. Interestingly, TSG101 and AATF functioned as cooperative coactivators in androgen receptor-mediated transcription. Because TSG101 was also shown to play a role in regulation of ubiquitin conjugation, we asked whether its coactivating function might be linked to ubiquitination. Indeed, TSG101 enhanced monoubiquitination of the androgen receptor in a ligand-dependent manner, and this correlated with enhanced transactivating capacity. Furthermore, a dominant-negative mutant of ubiquitin preventing polyubiquitination also stimulated androgen receptor-mediated transcription, which in this case could not be enhanced by TSG101. We propose that TSG101 activates androgen receptor-induced transcription by transient stabilization of the monoubiquitinated state, thus revealing a novel regulatory mechanism for nuclear receptors.