Characterization of the cAMP response element of the cystic fibrosis transmembrane conductance regulator gene promoter.

The Journal of Biological Chemistry
R P Matthews, G S McKnight

Abstract

A dominant negative inhibitor of the cAMP-dependent protein kinase has been shown to inhibit the basal expression of the cystic fibrosis transmembrane conductance regulator (CFTR) gene in the human colon carcinoma cell line, T84. A functional cAMP response element (CRE) was localized at -48 in the CFTR promoter, and we have analyzed the interactions of this regulatory region with transcription factors. An adjacent inverted CCAAT element (Y box) at position -60 was also investigated. Mutation of the CRE or the Y box decreases the activity of the promoter in transient transfections of T84 or JEG-3 cells. Electrophoretic mobility shift assays demonstrate that CRE-binding protein (CREB) binds to the CFTR CRE with high affinity and independently of the adjacent Y box and that the CFTR CRE binds CREB and activating transcription factor-1 in nuclear extracts of T84 and CaLu-3 cells. In transient transfections of JEG-3 cells, activation of the CFTR promoter is blocked by a dominant negative CREB mutant. The CFTR CRE will also drive cAMP-mediated expression when placed upstream of a heterologous basal promoter. These results demonstrate that CFTR is a bona fide CRE-dependent gene, and we suggest that CFTR expression levels in vivo may b...Continue Reading

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