REGULATION OF THE TRANSFORMING GROWTH-FACTOR-BETA-2 GENE PROMOTER IN EMBRYONAL CARCINOMA-CELLS AND THEIR DIFFERENTIATED CELLS - DIFFERENTIAL UTILIZATION OF TRANSCRIPTION FACTORS

Previous studies demonstrated that differentiation of embryonal carcin oma (EC) cells increases the expression of the TGF-beta 2 gene and ide ntified a CRE/ATF-like motif in the TGF-beta 2 promoter that is necess ary for its activity. This suggested that differentiation may increase the transcription of this gene by differential binding of transcripti on factors to the CRE/ATF-like motif. To test this possibility, we per formed gel mobility shift analysis using double-stranded oligodeoxynuc leotides containing the TGF-beta 2 CRE/ATF-like motif and nuclear extr acts prepared from F9 EC cells and F9-differentiated cells. We determi ned that the DNA/protein complexes formed by the EC nuclear extracts, but not the complexes formed by differentiated cell nuclear extracts, are recognized and supershifted by an ATF-1 specific antibody. This ob servation is consistent with our Western immunoblot analysis that dete cts ATF-1 in the EC cells, but not in their differentiated counterpart s. In addition, we provide evidence that protein phosphorylation influ ences the formation of complexes between F9 nuclear proteins and the C RE/ATF-like motif. Together, our studies identify a likely role for th e CRE/ATF-like motif in the regulation of TGF-beta 2 and suggest that this site binds one set of nuclear proteins in EC cells, where the gen e is not expressed, and a different set of nuclear proteins in the dif ferentiated cells, where the gene is expressed. (C) 1995 Wiley-Liss, I nc.