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

Peter J Mogayzel1, Muneer Esmail, Traci L Wagner

  • 1Eudowood Division of Pediatric Respiratory Sciences, The Johns Hopkins Medical Institutions, 600 North Wolfe Street, Park 316, Baltimore, MD 21287-2533, USA. mogayzel@mail.jhmi.edu

Biochimica Et Biophysica Acta
|June 27, 2002
PubMed
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Researchers investigated how phorbol esters affect cystic fibrosis transmembrane conductance regulator (CFTR) gene expression. They found that regulatory elements controlling PMA-induced CFTR down-regulation are not within the tested 5’-flanking DNA sequence.

Area of Science:

  • Molecular Biology
  • Genetics
  • Cell Biology

Background:

  • The cystic fibrosis transmembrane conductance regulator (CFTR) gene exhibits tissue-specific and developmental regulation.
  • Phorbol esters are known to decrease CFTR expression in certain cell lines, but the underlying cis-acting elements are not well-defined.

Purpose of the Study:

  • To investigate the role of cis-acting elements within the CFTR minimal promoter in response to phorbol 12-myristate 13-acetate (PMA) and forskolin.
  • To assess the utility of yeast artificial chromosome (YAC) vectors for studying CFTR gene regulation in human cells.

Main Methods:

  • Luciferase reporter gene assays were performed using plasmids transfected into Calu-3 and HT-29 cells.
  • A YAC vector containing a large region of CFTR 5'-flanking DNA was stably introduced into HT-29 cells.

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  • Luciferase activity and endogenous CFTR expression were measured following treatment with PMA and forskolin.

    • Main Results:

    • PMA affected luciferase activity differently in Calu-3 and HT-29 cells.
    • In HT-29 cells, PMA and forskolin increased luciferase activity, and endogenous CFTR expression responded as expected (increased with forskolin, decreased with PMA).
    • A wide range of baseline luciferase activities was observed among clonal cell lines containing the YAC construct.

    Conclusions:

    • Luciferase-containing YAC vectors are effective tools for studying CFTR expression in human cells.
    • The key regulatory elements responsible for PMA-induced CFTR down-regulation are not located within the approximately 335 kb of 5'-flanking CFTR DNA analyzed in this study.