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 RII receptor (TGF-be ta RII) is highly regulated and is a critical determinant of the cellular r esponse to TGF-beta. Previous analysis of the promoter region for the TGF-b eta RII gene introduced the possible existence of a negative regulatory ele ment (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 stro ng NRE located between base pairs -100 and -67 relative to the transcriptio n start site. Utilizing DNA transfection techniques and a series of synthes ized oligonucleotide promoter fragments, we have shown that this NRE is act ive in a variety of cell lines. Electrophoretic mobility shift assays have revealed the presence of multiple DNA binding proteins specifically interac ting with the NRE. At least three distinct protein complexes are variably p resent depending on the specific cell line examined, and mutational analysi s of the NRE has identified a ten-base pair recognition sequence which is s hared by all three complexes. This palindromic sequence has not been previo usly reported and does not share homology with any known transcription fact or consensus sequences. When inserted into an E4 Delta heterologous promote r construct, the NRE binding sequence failed to inhibit either basal or act ivated transcription of the target gene, indicating that the NRE does not a ct as a general repressor but may specifically operate within the context o f the TGF-beta RII core promoter. (C) 2001 Elsevier Science B.V. All rights reserved.