Myc genes are key regulators of cell proliferation, and their deregulation contributes to the genesis of most human tumours. Recently, a wealth of data has shed new light on the biochemical functions of Myc proteins and on the mechanisms through which they function in cellular transformation.

The Myc oncoprotein forms a binary activating complex with its partner protein, Max, and a ternary repressive complex that, in addition to Max, contains the zinc finger protein Miz1. Here we show that the E3 ubiquitin ligase HectH9 ubiquitinates Myc in vivo and in vitro, forming a lysine 63-linked polyubiquitin chain. Miz1 inhibits this ubiquitination. HectH9-mediated ubiquitination of Myc is required for transactivation of multiple target genes, recruitment of the coactivator p300, and induction of cell proliferation by Myc. HectH9 is overexpressed in multiple human tumors and is essential for proliferation of a subset of tumor cells. Our results suggest that site-specific ubiquitination regulates the switch between an activating and a repressive state of the Myc protein, and they suggest a strategy to interfere with Myc function in vivo.

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Miz1 is a member of the POZ domain/zinc finger transcription factor family. In vivo, Miz1 forms a complex with the Myc oncoprotein and recruits Myc to core promoter elements. The MYC (or c-MYC) gene and two of its relatives, MYCL and MYCN, are causally involved in the genesis of a wide variety of human tumors (32). c-MYC encodes a transcription factor (Myc) that can both activate and repress transcription. Myc activates transcription as part of a heterodimeric complex with the Max protein (1, 23). The complex binds to specific sequences, termed E-boxes, and recruits both the Gcn5 and Tip60 histone acetylase complexes to E-box elements through interaction with the TRRAP protein (8,16,17,25,26). In addition, TRRAP-independent mechanisms of transcriptional activation have been demonstrated (30). These may involve interactions of Myc with the P-TEFb complex, which regulates transcriptional elongation (14). Both directed searches and a number of array analyses have identified a large number of genes that are activated by Myc in vivo (9,27,31,45).Similarly, a large number of genes have been identified that are repressed upon activation of Myc. However, the mechanisms of transcriptional repression by Myc have remained more elusive. For a number of genes, repression of Myc has been mapped to the core promoter, suggesting that Myc affects proteins that regulate transcription at or close to the start site of transcription (24). One suggestion has been that Myc directs the synthesis of a transcriptional repressor protein and thereby indirectly represses transcription, but such a repressor has not yet been identified. A second suggestion has been that MycMax complexes directly bind to the start site of one repressed gene, p27kip1 (49), but direct binding of Myc-Max complexes to start sites of other repressed genes has not been found. A third suggestion, therefore, has been that Myc is recruited to core promoters through protein-protein interactions with other transcription factors. A number of candidate interaction partners have been identified, including TFII-I (35), YY-1 (38), Smad2 (15), Sp1 (18), and Miz1 (34).Recently, evidence has accumulated that three genes, p15Ink4b (37, 40), p21Cip1 (20,36,43,48), and Mad4 (22), are repressed by Myc through interaction with Miz1. Miz1 is a transcription factor with 13 zinc fingers and a POZ/BTB domain at its amino terminus (4). Free Miz1 binds to the core promoter of all three genes and activates transcription. Upon binding to Myc, transcriptional activation by Miz1 is abolished, and the Myc-Miz1 complex acts as a transcriptional repressor; this is in part due to competition between p300 and Myc for binding to Miz1 (40). Array analyses and chromatin immunoprecipitation (ChIp) experiments suggest that several other genes that are repressed by Myc, including p57Kip2 (12) and C/EBP␣ (24), are directly repressed by Myc through interaction with Miz1 (V. Beuger and M. Eilers, unpublished observations).These findings suggest that interaction with Miz1 plays a central role in transcriptio...