Abstract
Expression of the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) is tightly regulated. Using a panel of cell lines expressing different levels of CFTR mRNA, we investigated the mechanisms mediating control of CFTR transcription. In highly expressing cells, multiple sites of transcription initiation can be identified between positions -95 and +50 of the CFTR gene, and an alternatively initiated splice variant transcript is also present. Nonepithelial cell lines expressing very low levels of CFTR utilize a start site at -32. Promoter sequence elements from -83 to +111 are at least partially responsible for dictating CFTR transcriptional tissue-specificity, while multiple elements located farther 5' augment promoter strength. Analysis of the chromatin structure and methylation status of the CFTR promoter region reveals cell line differences which correlate with expression levels, suggesting that the physical context of the CFTR gene in vivo may contribute significantly to appropriate regulation of CFTR transcription. Taken together, these findings indicate that cellular control of CFTR gene expression is likely to be a complex function of several overlapping regulatory pathways.
Highlights
Expression of the gene encoding the cystic fibrosis tional cloning of the gene responsible for cystic fibrosis (6, 7 )
Using a panel of cell lines expressing regulator, or CFTR)’ provides the opportunity to study how different levels of CFTR mRNA, we investigated the transcriptional regulation of the CFTR gene relates to cystic mechanisms mediating control of CFTR transcription
The steady state abunrevealscellinedifferences which correlate with dance of CFTR mRNA varieswidely in different tissues and expression levels, suggestingthat the physical context cultured cell lines and can be of the CFTR gene in vivo may contribute significantly modulatedincultured cells by forskolin andCAMP [13], to appropriate regulation of CFTR transcription. phorbol esters [14, 15], protein kinaseC stimulants [16], and
Summary
CFPAC was converted to cDNA with Moloney murine leukemia virus reverse transcriptase (Bethesda Research Laboratories) and random. Labeled RNA was ethanol precipitated and the 483 nucleotide anti- Methylation Assays-20 pg of genomic DNA isolated from a panel sense transcript was gel purified on a denaturing 6%polyacrylamide of cell lines was digested overnight at 37 “C with 7.5 units of EcoRI gel. Hybridization and washing conditions assayed in the reaction and the probe consisted of sequences sub- were based upon standard protocols provided by the Nytran memcloned into pBluescript I1 KS via RNA PCR, using the primers 5‘- brane manufacturer, except that thenonspecific blocking DNA in the AGACATGGTCTGGATAGTTGGTGG-3‘ and 5”CAATGTCAAG- hybridization solution was sonicated human placental DNA (Sigma), GACTGGCAAGAGTG-3’, based upon published information [22]. 3.7LUC with XhoI (a unique site in the polylinker) and unique sites ently identified by primer extension and S1 nuclease protecwithin the CFTR genomic region.The ends were filled in by Klenow DNA polymerase using standard conditions and religated. The SV40-promoter/enhancedrrivenchloramphenicoalcetyltransferase reporter construct, pCAT-Control(Promega,Madison, WI), wasused as an internal control to normalize for transfection efficiency
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