Fig. 3. MYCN and Mash1 have opposite functions in the regulation of the cyclin-D2 promoter and E-box activity. (A) CGNPs were isolated and co-electroporated with MYCN, Mash1 or empty vector as indicated, together with a luciferase reporter plasmid containing the cyclin-D2 promoter and a CMV–Renilla-luciferase plasmid as internal control. After 48 hours in the absence of Shh, luminescence was measured and plotted as the mean value of three experiments. (B) The same experiment as above was performed, using a luciferase reporter plasmid containing five repetitions of the E-box motif (CACGTG) within the pGL3 plasmid. (C) ChIP assay of the interaction of MYCN and Mash1 with the cyclin-D2 promoter. The chromatin from 1 million CGNP cells was immunoprecipitated with 1 or 3 µg of antibody. (D) Western blot analysis of CGNPs cultured in the absence of Shh and electroporated with MYCN, Mash1 or empty vector as indicated. Levels of MYCN protein, β-tubulin III, cyclin D2 and -actin were determined. Mash1 expression did not affect MYCN protein levels and was able to downregulate cyclin-D2 expression and raise β-tubulin-III levels. (E) A model for MYCN and Mash1 antagonism in cell-cycle control. In the presence of Shh, MYCN expression is upregulated. The complex formed with its partner, Max, binds to and activates the E-box sequences (CACGTG) that are present in the cyclin-D2 promoter, turning on the proliferative response. When a differentiator stimulus such as BMP2 is added, Mash1 expression increases. Mash1 then forms heterodimers with E12, displacing the MYCN-Max complex from the E-box sequences within the cyclin-D2 promoter, repressing cyclin-D2 transcription and shutting down proliferation.