E1AF is a member of the Ets transcriptional factor family, and it plays a crucial role in tumor metastasis. However, the molecular mechanisms regulating its activity are not well characterized. In this study, we show that E1AF is surnoylated at four lysine residues, both in vivo and in vitro. Replacement of these lysines by arginine enhanced the transcriptional activity of E1AF, suggesting that sumoylation negatively regulates E1AF activity. We further demonstrated that PIASy enhanced sumoylation of E1AF as a specific SUMO-E3 ligase. In addition, PIASy repressed the transcriptional activity of both the wild-type and sumoylation defective mutants. However, the C342A mutant of PIASy, which abrogates SUMO-E3 ligase activity, had a significantly decreased ability to repress E1AF activity. Taken together, our results indicate that PIASy negatively regulates E1AF-mediated transcription by both E1AF sumoylation in a dependent and independent fashion. (c) 2007 Elsevier Inc. All rights reserved.