The Mechanism of Cystic Fibrosis Transmembrane Conductance Regulator Transcriptional Repression during the Unfolded Protein Response

Abstract

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 ⌬F508 CFTR mRNA expression in CFPAC-1 cells without affecting recombinant ⌬F508 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.In eukaryotic cells, the endoplasmic reticulum (ER) 3 is the site of protein folding and assembly. The unfolded protein response (UPR) can result from ER stress brought on by any number of insults (1-3), including depletion of ER Ca 2ϩ stores (1), proteasome blockade (4), increase in the concentration of reactive oxygen species (5, 6), inflammation (7), overexpression of secretory proteins (2, 8), or altered glycosylation (9). In addition to increasing the capacity of the ER by enhancing the synthesis of membrane components and chaperones (10), the UPR also decreases the ER protein load by enhancing ERAD (11) and to some extent by inhibiting transcription and translation (10,12). Although the principal mechanisms of the UPR have been studied extensively, only limited information is available regarding the extent and specificity of transcriptional repression during the UPR. In yeast, the limited number of genes that are transcriptionally repressed by the UPR encode secreted or cell surface proteins (6). Importantly, neither the extent nor the mechanisms of UPRassociated transcriptional repression have been investigated in mammalian cells.The cystic fibrosis transmembrane conductance regulator (CFTR), an integral membrane glycoprotein expressed in the ap...

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