The elucidation of molecular switches involved in deciding basic cell fate is critical to our understanding of normal development and cancer. Although c-Myc has long been implicated as a principal component in such decisions only recently the significance of the role of Mad1 in the switch from proliferation to differentiation has been recognized. Together, Myc, Max, Mad, Mxi1, and mSin3 comprise a transcription factor superfamily in which the central protein is Max. In fact, all the family members (except mSin3) must dimerize with Max through their respective HLH and LZ domains to bind DNA cooperatively. In contrast to the transactivating property of Myc/Max heterodimers, Max heterodimerization with Mad or Mxi1, followed by the tethering of mSin3 and associated co-repressor proteins to target genes, results in suppression of transcription. It has been suggested that the heterocomplex switch from Myc/Max to Mad/Max or Mxi1/Max, in turn, drives a transcriptional switch from activation to repression of genes involved in the maintenance of the undifferentiated state. Our research is focused on experiments designed to investigate the mechanism(s) by which Myc functions during proliferation and differentiation. Our results obtained through studies of stable transfectants generated from murine erythroleukemia (MEL) and human colon carcinoma cells are consistent with the existing model for activation and suppression by Myc/Max and Mad/Max. We have demonstrated that c-Myc expression is necessary for proliferation, while inactivation of the oncoprotein by a dominant negative mutant of Max results in growth retardation, accumulation of cells at the G0/G1 phase of the cell cycle and spontaneous as well as accelerated inducer-mediated differentiation. We have also shown that Mad antagonizes c-Myc function during the transition from proliferation to differentiation through an active repression mechanism. While MEL cells overexpressing Myc fail to differentiate upon chemical induction, high levels of ectopic Mad overcome the block and differentiation proceeds normally. Most recently we have identified and characterized new functional motifs within the carboxy terminus (CT) of Mad1. Removal of the last 18 amino acids of the protein (region V) abolishes its growth inhibitory function and the ability to reverse a Myc imposed differentiation block. Furthermore, deletion of region V renders a protein that binds DNA weakly and no longer represses Myc-induced transcriptional activation. In contrast, deletion of the preceding 24 amino acids (region IV) together with region V restores DNA binding and transcriptional repression suggesting a functional interplay between the two regions. In addition CKII phosphorylation in region IV appears to mediate this interplay. A better understanding of the ability of Mad1 to antagonize Myc and restore a differentiation switch may lead to novel therapeutic approaches to reverse Myc-derived malignant phenotypes. In addition to transactivation, c-Myc controls gene expression also by repression. The C/EBP-alpha gene, which encodes a transcription factor essential for adipocyte differentiation is down regulated by c-Myc in a fashion independent of Myc-specific DNA binding sites. This repression is mediated via an initiator element located in the promoter of the C/EBP-alpha gene. In colon carcinoma cells inactivation of c-Myc causes cessation of cell growth, spontaneous differentiation and accumulation of fat. Concomitantly, the expression of p42-C/EBP-alpha and Mad1, two differentiation associated genes is induced, while no apparent changes in expression of PPAR family members is detected. We found direct correlation between the induced expression of p42-C/EBP-alpha and fat synthesis. We are examining whether c-Myc plays a role in the maintenance of the malignant phenotype of colon cancer cells by repressing p42-C/EBP-alpha expression. Our goal is to understand the molecular mechanism(s) underlying this differentiation process. Activation of transcription factors (p42 C/EBP-alpha, PPAR-gamma and others) in colon cancer cells is a potential new approach for therapeutic intervention. To gain further insight into the mechanisms by which Myc activates or suppresses gene expression, RNAs derived from proliferating, differentiating and c-Myc differentiation blocked MEL cells were hybridized to a large array of cDNAs. Genes associated with a given cellular state were identified by comparing the various hybridization patterns. Potential new Myc targets were selected for further analysis.
