Analysis of a DNase I hypersensitive site located -20.9 kb upstream of the CFTR gene

Hugh N. Nuthall, Georges Vassaux, Clare Huxley, Ann Harris*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

25 Scopus citations

Abstract

The cystic fibrosis transmembrane conductance regulator gene (CFTR) shows a tightly regulated pattern of expression with spatial and temporal control. The regulatory elements achieving this appear to lie outside the basal promoter of the gene. We previously identified DNase I hypersensitive sites (DHSS) at -79.S kb and -20.5 kb with respect to the CFTR translational start site which may contain important regulatory elements. We have now investigated further the DHS at -20.5 kb to evaluate its potential function in the regulation of CFTR expression. Finer mapping revealed that the DHS lies at -2029 kb. Deletion of the DHS from a: 310-kb yeast artificial chromosome (YAC) containing the human CFTR gene has shown that this site may be responsible for about 60% of wild-type levels of transcription from the YAC transgene when expressed in Caco2 cells, DNase I footprinting showed several regions of protection within the -20.9 kb region with nuclear extracts from Caco2 cells, but not with extracts from lymphoblastoid cells, which do not show the DHS. Matches to several transcription factor-binding sites were found, but supershift analysis with specific antibodies did not density the transcription factors involved. Two purine/pyrimidine mirror repeat elements within the -20.9kb DHS were shown not to adopt non-B-DNA conformations. Thus, we provide evidence for a role for the -20.9 kb DHS in the transcriptional regulation of the CFTR gene, although the mechanisms mediating this effect remain unclear.

Original languageEnglish (US)
Pages (from-to)431-443
Number of pages13
JournalEuropean Journal of Biochemistry
Volume266
Issue number2
DOIs
StatePublished - Dec 1 1999

Keywords

  • Cystic fibrosis transmembrane conductance regulator (CFTR)
  • DNase I hypersensitive site
  • Gene expression
  • Regulation

ASJC Scopus subject areas

  • Biochemistry

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