Sequence specific protein binding to and activation of the TGF-β3 promoter through a repeated TCCC motif

Abstract

We have previously characterized the TGF-β3 promoter and shown that the activity of this promoter is highly variable in different cell types. Although the promoter contains a proximal cAMP responsive element, which is critical to basal and forskolin-induced promoter activity, this element is not responsible for the variable, cell-specific regulation of the promoter. In this paper, we identify a 25 base pair sequence in the proximal region of the TGF-β3 promoter that binds a novel DNAbinding protein. This region includes the sequence TCCCTCCCTCCC, (3 × TCCC), and mutation of these TCCC repeats inhibits protein binding. Further, we show that in the cell line A375, which we have previously shown expresses high levels of TGF-β3 mRNA, this region is responsible for mediating high level TGF-β3 promoter activity. Immediately 3' to the 3 x TCCC sequence is a consensus AP-2 binding site, however, we show that this region does not bind AP-2, and AP-2 does not transactivate the TGF-β3 promoter. Therefore, we provide strong evidence that high level expression of TGF-β3 in A375 cells results from transactivation of the TGF-β3 promoter by a protein that binds to a repeated TCCC motif in the promoter and suggest that this ONA-binding protein likely also regulates aspects of developmental and tissue-specific expression of this cytokine. © 1991 Oxford University Press.