Transcription of Cystic Fibrosis Transmembrane Conductance Regulator Requires a CCAAT-like Element for both Basal and cAMP-mediated Regulation

Abstract

The cystic fibrosis transmembrane conductance regulator (CFTR) gene in man is controlled by a tightly regulated and weak promoter. The architecture 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 promoter through deletion and mutational analysis reveals that expression is contingent on the conservation of the inverted CCAAT sequence. Basal expression of CFTR transcription and cAMP-mediated transcriptional regulation require the presence of an imperfect and inverted CCAAT element recognized as 5 -AATTGGAAGCAAAT-3 , located between 132 and 119 nucleotides upstream of the translational start site. RNA isolated from a transfected pancreatic cell line carrying integrated wild-type and mutant CFTR-directed transgenes was used to map the 5 termini of the transgenic transcripts. Analysis of the transcript termini by ribonuclease protection analysis reflects the direct association of the conserved inverted CCAAT sequence in promoting transcript initiation. Because of the requirement for the inverted CCAAT sequence for promoting transcription of CFTR, the involvement of CCAAT-binding factors is suspected in the regulation of CFTR gene transcription. To test this, we used electrophoretic mobility shift assays to demonstrate that the majority of the binding to the inverted CCAAT element, between ؊135 and ؊116, was easily competed for by binding to cognate nucleotide sequences for CCAAT-enhancer binding protein (C/EBP). An antibody specific for the C/EBP-related protein, C/EBP␦, detected C/EBP␦ as part of a nuclear protein complex bound to the inverted CCAAT sequence of the CFTR gene. Also, the detection of specific activating transcription factor/cyclic-AMP response element binding protein antigens by antibody supershift analysis of nuclear complexes suggest that species of this family of transcription factors could be involved in the formation of complexes with C/EBP␦ within the CFTR gene inverted CCAAT-like element. These studies raise the possibility of interactions between individual 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 promoter.The gene responsible for cystic fibrosis encodes the cystic fibrosis transmembrane conductance regulator (CFTR) 1 gene product (1, 2). The expression of CFTR is predominantly confined to specific epithelial cell types and is ordinarily expressed in low levels. The low levels and cell type-specific expression of CFTR appears to be dictated, primarily, by genomic sequences 5Ј upstream of the translational start of CFTR, which correspond to functional promoter sequences (3, 4). The requirements for active and cell type-specific CFTR transcription relies presumably within a narrow band of nucleotide sequences, proximal to the m...

Transcriptional Repression of the Cystic Fibrosis Transmembrane Conductance Regulator Gene, Mediated by CCAAT Displacement Protein/cut Homolog, Is Associated with Histone Deacetylation

“…CFTR transcription appears to be influenced by additional cis-acting elements, including a site located in the first intron of the human CFTR (14). The activation of CFTR transcription by cAMP correlates with increasing sensitivity to DNase I overlapping the Y-box element (12). This may reflect the role of trans-acting factors in modifying local chromatin structure.…”

“…This may reflect the role of trans-acting factors in modifying local chromatin structure. Although it has been shown previously that individual members of bZIP families of transcription factors have the capacity to bind and activate CFTR transcription (12,13), little is known about the parameters and proteins involved in directing transcription of the CFTR in vivo.…”

“…Thus, an intact CCAAT consensus on the opposing strand of the human CFTR appears to be a requirement for accurate transcript initiation (12). Both basal and cAMP-mediated regulation of CFTR transcription have implicated the Y-box (12) in tandem with a weak CRE nucleotide consensus downstream (13). CFTR transcription appears to be influenced by additional cis-acting elements, including a site located in the first intron of the human CFTR (14).…”

“…Despite the absence of a TATA signal, the human CFTR gene can initiate RNA transcription through multiple, discrete start sites requiring conserved residues within the Y-box. Thus, an intact CCAAT consensus on the opposing strand of the human CFTR appears to be a requirement for accurate transcript initiation (12). Both basal and cAMP-mediated regulation of CFTR transcription have implicated the Y-box (12) in tandem with a weak CRE nucleotide consensus downstream (13).…”

“…The tight transcriptional control of CFTR requires a conserved inverted CCAAT (Y-box) element (12). Despite the absence of a TATA signal, the human CFTR gene can initiate RNA transcription through multiple, discrete start sites requiring conserved residues within the Y-box.…”