Transcriptional Regulation of LKB1 Figure 1. Deletion-mutant analysis of the LKB1 promoter. (A) Regions of high mammalian and vertebrate sequence conservation in the 59- 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 21536 to +727 relative to the transcription start site (modified from the UCSC genome browser at http://genome.ucsc.edu/ENCODE/). (B) Luciferase activity of transiently transfected ‘‘444’’ cells with 200 bp deletion constructs (I–VII) of the LKB1 promoter. (C) 20 bp deletion mutants of the LKB1 promoter Pro III fragment, starting from nucleotide 2345 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 59-untranslated region (59-UTR) is indicated. (D) Comparison of luciferase activity of transiently transfected ‘‘444’’ cells with LKB1 promoter 20 bp deletion constructs (right) and predicted cis-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 2345 (LKB1 Pro III). All assays were performed three times in quadruplicate. The error bars denote mean 6 standard deviation. doi:10.1371/journal.pone.0032590.g001 element that was most critical for promoter activity. Notably, this element was located within the 59-UTR between nucleotides +43 to +52. The corresponding mutant (V/3) showed only 10% of the wild-type activity (Figure 2C, marked in red) and lacks apparent similarities to consensus sites for known transcription factors. The transcription factor Sp1 binds to a regulatory element downstream of the transcriptional start site To get insight which transcription factor can bind to this element within the 59-UTR, electrophoretic mobility shift assays (EMSA) were performed. When nuclear extracts of ‘‘444’’ and HepG2 cells were incubated with a DNA probe comprising this LKB1 downstream element (LKB1 DSE), a single protein-DNA complex was observed (Figure 3B, lanes 1–2). To characterize the nature of the corresponding binding factor, we used an excess of different unlabeled competitor oligonucleotides containing con- sensus sequences for known transcription factors (Figure 3A). While the intensity of the band was not affected by the addition of competitor containing AP-1, AP-2, Oct-1, CRE, SRE and E2F sites (Figure 3B, lanes 3–4 and 6–9, respectively), binding was completely inhibited by an excess of a Sp1 consensus site containing oligonucleotide (lane 5). Furthermore, complex forma- tion was competed by a molar excess of the wild-type oligo, but not by the corresponding mutant oligo harbouring the same mutations as the substitution mutant V/3 (Figure 3C, lanes 3 and 4, respectively). Finally, addition of an antibody against the transcription factor Sp1 specifically retarded the complex, while the addition of an antibody against the closely related Sp3 did not (Figure 3C, lanes 5 and 6). Although the LKB1 DSE within the 59- UTR differs from the classical GC box at two nucleotide positions, a similar binding site for Sp1 has been identified previously [31]. These results suggest that the ubiquitously expressed transcription factor Sp1 is involved in LKB1 regulation. Binding of NF-Y to three CCAAT boxes within the LKB1 promoter Based on the results of the LKB1 promoter mutant analysis, oligonucleotides containing the potential CCAAT boxes were also PLoS ONE | www.plosone.org 3 March 2012 | Volume 7 | Issue 3 | e32590