TRANSCRIPTION OF CYSTIC-FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR REQUIRES A CCAAT-LIKE ELEMENT FOR BOTH BASAL AND CAMP-MEDIATED REGULATION

The cystic fibrosis transmembrane conductance regulator (CFTR) gene in man is controlled by a tightly regulated and weak promoter. The archi tecture of the CFTR promoter suggests regulatory characteristics that are consistent with the absence of a TATA-like sequence, including the ability to initiate RNA transcription at numerous positions. Detailed investigation of the most proximal region of the human CFTR gene prom oter through deletion and mutational analysis reveals that expression is contingent on the conservation of the inverted CCAAT sequence. Basa l expression of CFTR transcription and cAMP-mediated transcriptional r egulation require the presence of an imperfect and inverted CCAAT elem ent recognized as 5'-AATTGGAAGCAAAT-3', located between 132 and 119 nu cleotides upstream of the translational start site. RNA isolated from a transfected pancreatic cell line carrying integrated wild-type and m utant CFTR-directed transgenes was used to map the 5' termini of the t ransgenic transcripts. Analysis of the transcript termini by ribonucle ase protection analysis reflects the direct association of the conserv ed inverted CCAAT sequence in promoting transcript initiation. Because of the requirement for the inverted CCAAT sequence for promoting tran scription of CFTR, the involvement of CCAAT-binding factors is suspect ed in the regulation of CFTR gene transcription. To test this, we used electrophoretic mobility shift assays to demonstrate that the majorit y of the binding to the inverted CCAAT element, between -135 and -116, was easily competed for by binding to cognate nucleotide sequences fo r CCAAT-enhancer binding protein (C/EBP). An antibody specific for the C/EBP-related protein, C/EBP delta, detected C/EBP delta as part of a nuclear protein complex bound to the inverted CCAAT sequence of the C FTR gene. Also, the detection of specific activating transcription fac tor/cyclic-AMP response element binding protein antigens by antibody s upershift analysis of nuclear complexes suggest that species of this f amily of transcription factors could be involved in the formation of c omplexes with C/EBP delta within the CFTR gene inverted CCAAT-like ele ment. These studies raise the possibility of interactions between indi vidual members of the C/EBP and activating transcription factor/cyclic -AMP response element binding protein families potentially contribute to the tight transcriptional control rendered by the CFTR gene promote r.