Deletion-mutant analysis of the LKB1 promoter.

<p>(<b>A</b>) Regions of high mammalian and vertebrate sequence conservation in the 5′-flanking region of the LKB1 coding sequence are aligned to DNase I hypersensitive sites (DNAse HS) in HepG2, normal human epidermal keratinocytes (NHEK) and normal human lung fibroblasts (NHLF) and to LKB1 200 bp deletion mutants (LKB1 Pro I–VII) extending from nucleotide position −1536 to +727 relative to the transcription start site (modified from the UCSC genome browser at <a href="http://genome.ucsc.edu/ENCODE/" target="_blank">http://genome.ucsc.edu/ENCODE/</a>). (<b>B</b>) Luciferase activity of transiently transfected “444” cells with 200 bp deletion constructs (I–VII) of the LKB1 promoter. (<b>C</b>) 20 bp deletion mutants of the LKB1 promoter Pro III fragment, starting from nucleotide −345 to +727 (III–V) were constructed, digested by the restriction enzymes SacI and XhoI and separated in a 1% agarose gel. The transcriptional start site (TSS) as well as the 5′-untranslated region (5′-UTR) is indicated. (<b>D</b>) Comparison of luciferase activity of transiently transfected “444” cells with LKB1 promoter 20 bp deletion constructs (right) and predicted <i>cis</i>-regulatory elements (left). The positions of the potential CCAAT boxes I–III as well as the forkhead box are indicated. Luciferase activity of all deletion constructs (relative light units normalized against renilla luciferase activity) is expressed as the percentage of the signal obtained with the plasmid containing the LKB1 promoter region downstream of nucleotide −345 (LKB1 Pro III). All assays were performed three times in quadruplicate. The error bars denote mean ± standard deviation.