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

Research output: Contribution to journalArticlepeer-review


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 ∼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/μg 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 ∼335 kb 5′-flanking sequence included in this YAC construct.

Original languageEnglish (US)
Pages (from-to)306-315
Number of pages10
JournalBiochimica et Biophysica Acta - Gene Structure and Expression
Issue number3
StatePublished - Jul 19 2002


  • CFTR expression
  • Cystic fibrosis
  • Phorbol ester

ASJC Scopus subject areas

  • Structural Biology
  • Biophysics
  • Biochemistry
  • Genetics


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