Cell type-specific protein-DNA interactions at the cAMP response elements of the prohormone convertase 1 promoter. Evidence for additional transactivators distinct from CREB/ATF family members.
The proximal promoter region of the neuroendocrine-specific human prohormone convertase 1 (PC1) gene contains two distinct cAMP response elements (CRE-1 and CRE-2). Both elements are essential in directing the cAMP-mediated hormonal regulation of PC1 gene transcription. In this study, we have demonstrated that CRE-1 binds several trans-acting factors. In electrophoretic mobility shift assay experiments with nuclear extracts prepared from neuroendocrine AtT-20 and beta-TC3 cells and non-neuroendocrine COS-1 cells, three specific protein-DNA complexes (I-III) were detected. Complexes II and III were shown to contain CREB-1 and ATF-1, respectively. The most slowly migrating complex I was only detected with the neuroendocrine cell lines and appeared to comprise a c-Jun-containing heterodimer. In addition, CRE-2 was shown to bind a protein that was only detected in nuclear extracts derived from the neuroendocrine cell lines. Antibody supershift experiments indicated that both the c-Jun-interacting protein in CRE-1 complex I and the CRE-2-interacting protein are distinct from known members of the basic domain, leucine zipper family of transcription factors. UV cross-linking experiments demonstrated that these potential novel proteins are approximately 100 and 60 kDa in size, respectively. Site-specific mutagenesis experiments demonstrated that the formation of both CRE-1 and CRE-2 complexes is correlated with the transcriptional activity of the proximal PC1 promoter as has been shown in transient transfections with wild-type and mutant promoter constructs. In addition, it was shown that both CREB-1 and ATF-1 transactivate the human PC1 promoter in transient transfection experiments.
Jansen
E
E
Laboratory for Molecular Oncology, Center for Human Genetics, University of Leuven and the Flanders Interuniversity Institute for Biotechnology, Herestraat 49, B-3000 Leuven, Belgium.
Ayoubi
T A
TA
Meulemans
S M
SM
Van de Ven
W J
WJ
D051697:Activating Transcription Factor 1; D051698:Activating Transcription Factor 2; D016282:Aspartic Acid Endopeptidases / Q000235:genetics*; D001665:Binding Sites; D001667:Binding, Competitive; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism*; D004268:DNA-Binding Proteins; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D011401:Promoter Regions, Genetic; D043484:Proprotein Convertases; D011487:Protein Conformation; D014157:Transcription Factors / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D014466:Ultraviolet Rays
D051697:Activating Transcription Factor 1; D051698:Activating Transcription Factor 2; D017362:Cyclic AMP Response Element-Binding Protein; D004268:DNA-Binding Proteins; D014157:Transcription Factors; D043484:Proprotein Convertases; D016282:Aspartic Acid Endopeptidases