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. 2010 Sep;43(3):334-41.
doi: 10.1165/rcmb.2009-0149OC. Epub 2009 Oct 23.

Multiple Mechanisms Influence Regulation of the Cystic Fibrosis Transmembrane Conductance Regulator Gene Promoter

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Free PMC article

Multiple Mechanisms Influence Regulation of the Cystic Fibrosis Transmembrane Conductance Regulator Gene Promoter

Marzena A Lewandowska et al. Am J Respir Cell Mol Biol. .
Free PMC article

Abstract

The cystic fibrosis transmembrane conductance regulator (CFTR) gene is driven by a promoter that cannot alone account for the temporal and tissue-specific regulation of the gene. This has led to the search for additional regulatory elements that cooperate with the basal promoter to achieve coordinated expression. We previously identified two alternative upstream exons of the gene that were mutually exclusive of the first exon, and one of which showed temporal regulation in the human and sheep lung. We now demonstrate that this alternative splice product generates a stable protein, which initiates translation at an ATG in exon 4, and thus lacks the N terminus of CFTR. The other splice variant inhibits translation of the protein. In a search for the promoter used by the upstream exons, we identified a novel element that contributes to the activity of the basal CFTR promoter in airway epithelial cells, but does not function independently. Finally, we demonstrate that, in primary airway cells, skin fibroblasts, and both airway and intestinal cell lines, the CFTR promoter is unmethylated, irrespective of CFTR expression status. Thus, methylation is not the main cause of inactivation of CFTR transcription.

Figures

Figure 1.
Figure 1.
(A) Diagram of the constructs containing the alternative human exons −1a and 1a or sheep Ov1aS and Ov1aL exons joined to cystic fibrosis transmembrane conductance regulator (CFTR) exons 2–24. Vertical arrows denote putative alternative initiation codons (together with their location on AC000111 or AF325415, and the predicted amino acid [aa] length resulting from their usage). The ATGs in exons 3 and 4 are also shown. (B) Sequence of exons −1/1a joined to exon 2 and exon −1a joined to exon 2. Sequence of alternative exons is presented as following: exon −1a, Roman font; exon 1a, italics; exon 2, bold. The potential CTG and ATG initiation codons are marked in bold.
Figure 2.
Figure 2.
In vitro transcription and translation of constructs containing alternative upstream exons joined to CFTR exons 2–24. (A and B) In vitro transcription and translation. 35S-labeled proteins separated by SDS-PAGE and visualized by autoradiography. The products from full-length CFTR (936C) and exons 2–24 are marked by arrows (A and B, respectively). (A) Inclusion of exon −1a in the transcript generates the same products as exons 2–24 alone, whereas inclusion of exons −1a/1a together inhibits translation. (B) Mutation of the potential CTG and ATG initiation codons in exon 1a does not restore translation from more distal ATGs in exon 4. (C) In vitro transcription. RNA transcripts were generated with T7 polymerase, denatured after RNase-free DNase treatment, and separated on a formaldehyde agarose gel, poststained by ethidium bromide. Stable RNA was generated from all constructs. (D) Partial sequence of exon 1a and location of potential initiation sites. The CTG and ATG potential initiation sites are in italics, and the mutations in capitals.
Figure 3.
Figure 3.
CFTR promoter analysis. (A) Diagram of the 5′ region of the human CFTR gene (to scale). The alternative exons, −1a and 1a, and exon 1 are shown (filled boxes). Putative promoters, with their predicted scores, are shown by arrows. Numbering refers to AC000111. At the bottom are shown each of the constructs used in reporter gene assays, with the name denoting the length of promoter fragment. The star denotes the A/G mutation that disrupts the putative promoter with a score of 1. (B and C) Luciferase reporter gene assays with CFTR promoter fragment constructs. Each bar chart shows the luciferase activities for each construct relative to pGL3B245 (CFTR basal promoter construct = 1) in B, 16HBE14o and Caco2 cells. (B and C) Luciferase activities were normalized for transfection efficiency by cotransfection with pCMV/β-galactosidase. Each bar is the average of at least three transfection experiments, with each sample assayed in triplicate. Error bars represent SD. (B) Stars indicate statistical significance of a comparison between pGL3B245 values and those of the other construct (**P < 0.01). (C) The effect of the 18,841 A/G mutation in the 1,750 construct on promoter activity in 16HBE14o and Caco2 cells. Stars indicate statistical significance of the pGL3B1750 to pGL3B1750 A/G comparison in 16HBE14o cells (P < 0.01).
Figure 4.
Figure 4.
DNA methylation analyses by sodium bisulfite sequencing of the CFTR 5′ region. The figure depicts representative examples of four regions sequenced after sodium bisulfite treatment for two primary tracheal epithelial cells (pHTE) samples, 16HBE14o, and Caco2. (Top) Scaled map of the 5′ region of the CFTR gene. The bold arrow represents the ATG, and numbers in each sequenced region represent the first and last CpG dinucleotide. Small arrows represent scores for putative promoters analyzed in silico. (Bottom) Each circle represents a CpG dinucleotide. A total of 12 clones or alleles were sequenced for each region. A total of 58 CpGs in all regions were analyzed by this method. Unmethylated, open circles; methylated, filled circles. Percentages of DNA methylation are indicated on the right of the panel.

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