cis-Acting elements within CFTR 5'-flanking DNA are not sufficient to decrease gene expression in response to phorbol ester.

Peter J Mogayzel; Muneer Esmail; Traci L Wagner; Pamela L Zeitlin; Melissa Ashlock

doi:10.1016/s0167-4781(02)00395-0

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PubMed · 12084577

cis-Acting elements within CFTR 5'-flanking DNA are not sufficient to decrease gene expression in response to phorbol ester.

Peter J Mogayzel·Muneer Esmail·Traci L Wagner·Pamela L Zeitlin·Melissa Ashlock

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Abstract

The cystic fibrosis transmembrane conductance regulator gene (CFTR) is regulated in a tissue-specific and developmental fashion. Although it has been known for some time that phorbol esters decrease CFTR expression in cell lines that have high CFTR mRNA levels, the cis-acting elements that control this down-regulation remain ill-defined. The role of cis-acting elements within the CFTR minimal promoter in modulating responses to phorbol 12-myristate 13-acetate (PMA) and forskolin was assessed using luciferase reporter gene (luc)-containing plasmids transfected into Calu-3 and HT-29 cells. PMA treatment had no effect on luciferase activity in Calu-3 cells transiently transfected with plasmids containing luc driven by up to 2.3 kb of CFTR 5'-flanking DNA. PMA increased luciferase activity in transfected HT-29 cells. A more extensive region of DNA was evaluated using a yeast artificial chromosome (YAC) containing luc driven by approximately 335 of CFTR 5'-flanking DNA (y5'luc) stably introduced into HT-29 cells. Clonal cell lines containing y5'luc were created and assessed for luciferase activity at baseline and in response to forskolin and PMA. There was a wide range of baseline luciferase activities among the clones (42-1038 units/microg protein) that was not entirely due to the number of luc copies present within the cells. Treatment with both PMA and forskolin led to increased luciferase activity in six randomly selected clonal cell lines. As expected, endogenous CFTR expression increased in response to forskolin and decreased in response to PMA. These studies demonstrate that luc-containing YAC vectors can be used to study CFTR expression in human cells. In addition, these data suggest that important regulatory elements responsible for decreased CFTR expression in response to PMA are not located upstream of CFTR in the approximately 335 kb 5'-flanking sequence included in this YAC construct.

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5' Flanking Region Adenocarcinoma Animals Colforsin Cystic Fibrosis Transmembrane Conductance Regulator Electrophoresis, Gel, Pulsed-Field Gene Expression Regulation Genes, Reporter Humans Luciferases Lung Neoplasms Promoter Regions, Genetic Recombinant Fusion Proteins Tetradecanoylphorbol Acetate Tumor Cells, Cultured Journal Article Research Support, Non-U.S. Gov't

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Peter J Mogayzel, Muneer Esmail, Traci L Wagner, Pamela L Zeitlin, Melissa Ashlock. 2002-07-19. cis-Acting elements within CFTR 5'-flanking DNA are not sufficient to decrease gene expression in response to phorbol ester.. https://doi.org/10.1016/s0167-4781(02)00395-0

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