Abstract Cyclic AMP-dependent transcription factor-3 (ATF3), a stress sensor/mediator, plays an essential role in cells to maintain homeostasis and has diverse functions in cellular death and survival signal pathways. ATF3 binds to the consensus ATF/cAMP response element (CRE) to regulate its downstream target genes, such as CCNE2 (Cyclin E2), CD82 (KAI1), DDIT3 (GADD153), LDLR (LDL receptor), SNAI1, TP53, and TWIST1. A previous report has shown that the activities of ATF3 are modulated by ubiquitination; however, whether it is modified by small ubiquitin-related modifier (SUMO), an important modification in regulating a wide range of cellular processes, remains unknown. The aim of this study was to investigate whether ATF3 is post-translationally modified by SUMO proteins and also to determine the role of SUMOylation of ATF3 on MC2R, NR5A1, STAR, and TP53 gene activities. Here we demonstrate that ATF3 is clearly defined as a SUMO target protein both in vitro SUMOylation assay using recombinant proteins and at the cellular levels. Furthermore, ATF3 interacted with UBE2I (Ubc9), the only SUMO E2 enzyme found so far. In addition, PIAS3β (a SUMO E3 ligase) enhanced and SENP2 and SENP7 (two SUMOylation proteases) decreased SUMOylation of ATF3, respectively. In addition, we found that ATF3 is selectively SUMOylated at lysine residue 42 but the SUMOylation/deSUMOylation does not alter subcellular localization of ATF3. We next characterized the functional role of ATF3 SUMOylation on MC2R, NR5A1, STAR, and TP53 gene expression. We found that SUMOylation of ATF3 is required for full repression of NR5A1 and TP53 genes. While SUMOylation of ATF3 is required for full activation of MC2R gene activity, SUMOylation of ATF3 further enhances repression of STAR gene activity. Overall, we provide the first evidence that ATF3 is post-translationally modified by SUMO to regulate MC2R, NR5A1, STAR, and TP53 gene activities. Citation Format: Chiung-Min Wang, Victoria Brennan, Ninoska Gutierrez, Xirui Wang, Lizhong Wang, Wei-Hsiung Yang. SUMOylation of ATF3 alters MC2R, NR5A1, STAR, and TP53 gene activities. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 763A. doi:10.1158/1538-7445.AM2013-763A