Identification of a negative Cis-regulatory element and multiple DNA binding proteins that inhibit transcription of the transforming growth factor-beta type II receptor gene.

Expression of the transforming growth factor-beta type II receptor (TGF-beta RII) is highly regulated and is a critical determinant of the cellular response to TGF-beta. Previous analysis of the promoter region for the TGF-beta RII gene introduced the possible existence of a negative regulatory element (NRE) upstream adjacent to the core promoter region (Bae et al., 1995. J. Biol. Chem. 270, 29460-29468). We have confirmed the presence of a strong NRE located between base pairs -100 and -67 relative to the transcription start site. Utilizing DNA transfection techniques and a series of synthesized oligonucleotide promoter fragments, we have shown that this NRE is active in a variety of cell lines. Electrophoretic mobility shift assays have revealed the presence of multiple DNA binding proteins specifically interacting with the NRE. At least three distinct protein complexes are variably present depending on the specific cell line examined, and mutational analysis of the NRE has identified a ten-base pair recognition sequence which is shared by all three complexes. This palindromic sequence has not been previously reported and does not share homology with any known transcription factor consensus sequences. When inserted into an E4Delta heterologous promoter construct, the NRE binding sequence failed to inhibit either basal or activated transcription of the target gene, indicating that the NRE does not act as a general repressor but may specifically operate within the context of the TGF-beta RII core promoter.