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J. Biol. Chem., Vol. 283, Issue 18, 12154-12165, May 2, 2008

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

Rafal Bartoszewski, András Rab¹, George Twitty, Lauren Stevenson, James Fortenberry, Arkadiusz Piotrowski^¶, Jan P. Dumanski^¶, and Zsuzsa Bebök²

From the Departments of Cell Biology and ^¶Genetics and Cystic Fibrosis Research Center, University of Alabama, Birmingham, Alabama 35294-0005

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.

Received for publication, September 11, 2007 , and in revised form, February 28, 2008.

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Present address: Dept. of Laboratory Medicine, University of Pécs Medical School, 7624 Pécs, Hungary.

² To whom correspondence should be addressed: Dept. of Cell Biology, University of Alabama at Birmingham, 1918 University Blvd., MCLM 760, Birmingham, AL 35294-0005. Tel.: 205-975-5449; Fax: 205-934-7593; E-mail: bebok{at}uab.edu.

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