Deregulated expression of c-myc can induce cell proliferation in established cell lines and in primary mouse embryonic fibroblasts (MEFs), through a combination of both transcriptional activation and repression by Myc. Here we show that a Myc-associated transcription factor, Miz-1, arrests cells in G1 phase and inhibits cyclin D-associated kinase activity. Miz-1 upregulates expression of the cyclin-dependent kinases (CDK) inhibitor p15INK4b by binding to the initiator element of the p15INK4b promoter. Myc and Max form a complex with Miz-1 at the p15 initiator and inhibit transcriptional activation by Miz-1. Expression of Myc in primary cells inhibits the accumulation of p15INK4b that is associated with cellular senescence; conversely, deletion of c-myc in an established cell line activates p15INK4b expression. Alleles of c-myc that are unable to bind to Miz-1 fail to inhibit accumulation of p15INK4b messenger RNA in primary cells and are, as a consequence, deficient in immortalization.

The c‐Myc protein activates transcription as part of a heteromeric complex with Max. However, Myc‐transformed cells are characterized by loss of expression of several genes, suggesting that Myc may also repress gene expression. Two‐hybrid cloning identifies a novel POZ domain Zn finger protein (Miz‐1; Myc‐interacting Zn finger protein‐1) that specifically interacts with Myc, but not with Max or USF. Miz‐1 binds to start sites of the adenovirus major late and cyclin D1 promoters and activates transcription from both promoters. Miz‐1 has a potent growth arrest function. Binding of Myc to Miz‐1 requires the helix–loop–helix domain of Myc and a short amphipathic helix located in the carboxy‐terminus of Miz‐1. Expression of Myc inhibits transactivation, overcomes Miz‐1‐induced growth arrest and renders Miz‐1 insoluble in vivo. These processes depend on Myc and Miz‐1 association and on the integrity of the POZ domain of Miz‐1, suggesting that Myc binding activates a latent inhibitory function of this domain. Fusion of a nuclear localization signal induces efficient nuclear transport of Miz‐1 and impairs the ability of Myc to overcome transcriptional activation and growth arrest by Miz‐1. Our data suggest a model for how gene repression by Myc may occur in vivo.

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...

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Transcriptional activation studies involving the human oncoprotein and transcription factor Myc and its helix-loop-helix partner protein Max in mammalian cells are critical due to the presence of endogenous Myc and Max proteins. Here we show that co-expression of the human c-myc and max genes from 2micro circle derived high copy number vectors in yeast cells stimulate the transcriptional activation of a LacZ reporter gene fused to the yeast cytochrome-c1 oxidase minimal promoter containing the adenovirus major late promoter element (AMLPE). The exchange of the single Myc binding site in the AMLPE by the two E-box DNA motifs (CACGTG) present in the Myc responsive element of a human Myc target gene (ornithine decarboxylase) in front of a promoter-reporter gene cassette results in a two-fold enhanced beta-galactosidase expression. Low expression of max and high level expression of c-myc at the same time led to a further enhancement of transcriptional activation from this promoter-reporter gene cassette.