The mechanism of cystic fibrosis transmembrane conductance regulator transcriptional repression during the unfolded protein response.
The unfolded protein response (UPR) aids cellular recovery by increasing the capacity and decreasing the protein load of the endoplasmic reticulum (ER). Although the main pathways of the UPR are known, the mechanisms of UPR-associated transcriptional repression have not been explored in mammalian cells. Previous studies indicate that endogenous cystic fibrosis transmembrane conductance regulator (CFTR) mRNA levels and protein maturation efficiency decrease when the UPR is activated. In the present study, we demonstrate that inhibition of CFTR expression under ER stress leads to reduced cAMP-activated chloride secretion in epithelial monolayers, an indication of diminished CFTR function. Moreover, ER stress and the UPR obliterate endogenous DeltaF508 CFTR mRNA expression in CFPAC-1 cells without affecting recombinant DeltaF508 CFTR mRNA levels or mRNA half-life. These results emphasize that transcriptional repression of CFTR under ER stress, in concert with decreased CFTR maturation efficiency, leads to diminished function. Using human CFTR promoter reporter constructs, we confined the ER stress-associated CFTR transcriptional repression to the minimal promoter. Chromatin immunoprecipitation assays established the binding of the UPR-activated ATF6 transcription factor to this region during ER stress, which links the repression to the UPR. Methylation-specific PCR (MSP) revealed hypermethylation of CpG sites inside and in the vicinity of the MAZ transcription factor binding region of CFTR, demonstrating methylation-dependent repression. Using pharmacological inhibitors, we show that both DNA methylation and histone deacetylation contribute to CFTR transcriptional inhibition. These studies provide novel insight into the mechanism of gene repression during the mammalian UPR.
Bartoszewski
Rafal
R
Department of Cell Biology, University of Alabama, Birmingham, Alabama 35294-0005, USA.
Rab
András
A
Twitty
George
G
Stevenson
Lauren
L
Fortenberry
James
J
Piotrowski
Arkadiusz
A
Dumanski
Jan P
JP
Bebok
Zsuzsa
Z
D000107:Acetylation / Q000187:drug effects; D051702:Activating Transcription Factor 6 / Q000378:metabolism; D002712:Chlorides / Q000378:metabolism; D000242:Cyclic AMP / Q000494:pharmacology; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics* / Q000378:metabolism; D019175:DNA Methylation / Q000187:drug effects; D004721:Endoplasmic Reticulum / Q000473:pathology; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects; D006367:HeLa Cells; D006657:Histones / Q000378:metabolism; D006801:Humans; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding / Q000187:drug effects; D017510:Protein Folding; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000378:metabolism; D013816:Thermodynamics; D014158:Transcription, Genetic
C497111:ATF6 protein, human; D051702:Activating Transcription Factor 6; D002712:Chlorides; D006657:Histones; D012333:RNA, Messenger; D012097:Repressor Proteins; C093349:cystic fibrosis transmembrane conductance regulator delta F508; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D000242:Cyclic AMP