FIG. 2.

Myc activates Odc transcription by relieving Mnt-mediated repression. (A) Odc expression in BALb/c-3T3 cells is serum dependent. Fibroblasts were cultured in the presence (lane 1) or absence of serum for 1, 2, and 3 days (lanes 2 to 4). After 3 days of starvation, fresh medium containing serum was added for 3 h (lane 5). Northern blot analysis was performed using a probe directed against Odc. 18S rRNA served as loading control. (B) Mnt-Max complexes occupy the Odc E-boxes in quiescent BALB/c-3T3 fibroblasts and are displaced by c-Myc-Max complexes in serum-stimulated cells. ChIP assays of serum-starved (2 days) or restimulated (for 3 h) BALB/c-3T3 cells were performed using antibodies directed against Max, c-Myc, or Mnt. The primers used for the PCR encompass the intronic E-boxes of the Odc gene. (C) Mnt represses the Odc promoter, and Mnt repression is relieved by Myc. BALB/c-3T3 cells infected with a Myc-ER-expressing retrovirus (MSCV-Myc-ER-IRES-GFP) were cotransfected with Odc promoter-luciferase constructs (containing intact [Odc-Luc] or mutated [Odc-luc5A] E-boxes) together with a Renilla luciferase control plasmid (pRL-SV40 [Promega]) and, where indicated, a Mnt expression vector (pRc-CMV-HA-Mnt). Transfections were balanced for DNA and promoter content by using the empty pRc vector plasmid. Myc-ER was activated by adding 4-HT for 24 h. Luciferase activity was determined using a luminometer. Results shown are the mean of triplicate assays and are representative of three independent experiments.