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First published online 10 February 2009
doi: 10.1242/jcs.037556

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Expression of the proneural gene encoding Mash1 suppresses MYCN mitotic activity

Department of Cell Death and Proliferation, Institute for Biomedical Research of Barcelona, IIBB-CSIC-IDIBAPS, Barcelona, Spain

View larger version (45K): [in this window] [in a new window]   Fig. 1. Mash1 expression promotes cell-cycle exit and differentiation in CGNPs. (A) CGNPs were isolated and electroporated in suspension with a bicistronic GFP plasmid (pCIG) containing Mash1 or empty vector (green cells). Cells were then plated in the presence of Shh (3 µg/ml) for the indicated periods of time and BrdU staining was performed (red cells). Proliferating transfected cells are yellow. On the left, representative pictures of the experiment are shown; on the right, the percentage of BrdU-positive cells out of all transfected cells is shown. (B) The same experiment as above but cells were stained with β-tubulin III (red) as a differentiation marker. On the left, representative images at 72 hours post-electroporation are shown; on the right, the percentage of β-tubulin-III-positive cells out of all transfected cells is shown. (C) TUNEL assay to measure apoptosis. The experiment was performed as above and the percentage of apoptotic cells out of all transfected cells was determined.

View larger version (26K): [in this window] [in a new window]   Fig. 2. Mash1 opposes the proliferating activity of MYCN but not of cyclin-D2. (A) CGNPs were isolated and co-electroporated as described in the Materials and Methods with the indicated amounts of empty vector, MYCN or Mash1. Cells were then plated in the absence of Shh and, after 48 hours, [3H]-thymidine incorporation was measured. Note that Mash1 expression overcomes MYCN-induced proliferation in a dose-dependent manner. (B) The same experiment as above was performed using the cyclin-D2 expression plasmid. Note that Mash1 expression cannot suppress the proliferation induced by cyclin D2 when expressed alone or together with MYCN.

View larger version (34K): [in this window] [in a new window]   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.

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