Regulation of the transforming growth factor‐β2 gene promoter in embryonal carcinoma cells and their differentiated cells: Differential utilization of transcription factors

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Abstract
Previous studies demonstrated that differentiation of embryonal carcinoma (EC) cells increases the expression of the TGF‐β2 gene and identified a CRE/ATF‐like motif in the TGF‐β2 promoter that is necessary for its activity. This suggested that differentiation may increase the transcription of this gene by differential binding of transcription factors to the CRE/ATF‐like motif. To test this possibility, we performed gel mobility shift analysis using double‐stranded oligodeoxynucleotides containing the TGF‐β2 CRE/ATF‐like motif and nuclear extracts prepared from F9 EC cells and F9‐differentiated cells. We determined 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 observation is consistent with our Western immunoblot analysis that detects ATF‐1 in the EC cells, but not in their differentiated counterparts. In addition, we provide evidence that protein phosphorylation influences the formation of complexes between F9 nuclear proteins and the CRE/ATF‐like motif. Together, our studies identify a likely role for the CRE/ATF‐like motif in the regulation of TGF‐β2 and suggest that this site binds one set of nuclear proteins in EC cells, where the gene is not expressed, and a different set of nuclear proteins in the differentiated cells, where the gene is expressed.