In vivo analysis of DNase I hypersensitive sites in the human CFTR gene.
BACKGROUND
The cystic fibrosis transmembrane conductance regulator gene (CFTR) shows a complex pattern of expression. The regulatory elements conferring tissue-specific and temporal regulation are thought to lie mainly outside the promoter region. Previously, we identified DNase I hypersensitive sites (DHS) that may contain regulatory elements associated with the CFTR gene at -79.5 and at -20.5 kb with respect to the ATG and at 10 kb into the first intron.


METHODS
In order to evaluate these regulatory elements in vivo we examined these DHS in a human CFTR gene that was introduced on a yeast artificial chromosome (YAC) into transgenic mice. The 310 kb human CFTR YAC was shown to restore the pheno-type of CF-null mice and so is likely to contain most of the regulatory elements required for tissue-specific expression of CFTR.


RESULTS
We found that the YAC does not include the -79.5 kb region. The DHS at -20.5 kb is present in the chromatin of most tissues of the transgenic mice, supporting its non-tissue-specific nature. The DHS in the first intron is present in a more restricted set of tissues in the mice, although its presence does not show complete concordance with CFTR expression. The intron I DHS may be important for the higher levels of expression found in human pancreatic ducts and in lung submucosal glands.


CONCLUSIONS
These data support the in vivo importance of these regulatory elements.
Moulin
D S
DS
Institute of Molecular Medicine, Oxford University, John Radcliffe Hospital, U.K.
Manson
A L
AL
Nuthall
H N
HN
Smith
D J
DJ
Huxley
C
C
Harris
A
A
D000818:Animals; D018244:Chromosomes, Artificial, Yeast; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000096:biosynthesis / Q000235:genetics*; D003850:Deoxyribonuclease I / Q000378:metabolism; D015870:Gene Expression; D005816:Genetic Complementation Test; D006801:Humans; D051379:Mice; D008822:Mice, Transgenic; D011485:Protein Binding; D012045:Regulatory Sequences, Nucleic Acid; D015183:Restriction Mapping; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013045:Species Specificity; D014018:Tissue Distribution
C505032:CFTR protein, human; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D003850:Deoxyribonuclease I