Transcriptional Regulation of LKB1 Figure 2. Substitution mutant analysis of the LKB1 promoter. Match of phylogenetic footprint with substitution mutant analysis of the LKB1 promoter region ranging from nucleotide position 2345 to +75. (A) Mammalian base-wise conservation (blue and red bars) is displayed together with sequence alignments of different species, predicted transcription factor (TF)-binding sites (red boxes) and positions of mutations within the corresponding substitution mutants of the LKB1 promoter reporter constructs. Conserved sequences (‘‘Mammal Cons’’) are indicated in blue, non- conserved sequences in red. The height of the corresponding bars represents the degree of conservation. Mutations which reduce reporter activity in the substitution mutant analysis by more than 50% are highlighted in a light red background, while mutations without influence on reporter activity are coloured in a light blue background. Display of alignments and mammalian conservation were modified from the UCSC genome browser (see Fig. 1). The positions of the potential CCAAT/Forkhead/Sp1 boxes are indicated as red rectangles. (B) Substitution mutants of the LKB1 promoter Pro II fragment, encompassing nucleotides 2549 to +727, were constructed by introducing a 10 bp mutation, comprising a NheI restriction site, within the area of 2345 to +75 by PCR and religation with the respective deletion mutant. The presence of the mutation within the LKB1 promoter was monitored by agarose gel electrophoresis following restriction enzyme digestion with SacI and NheI. (C) Luciferase activity of transiently transfected ‘‘444’’ cells with LKB1 promoter 10 bp substitution mutants reveals a critical role of four cis-acting elements regulating LKB1 transcription (red bars). Activity of substitution mutants (relative light units normalized against renilla luciferase activity) is expressed as the percentage of the signal obtained with the plasmid containing the LKB1 wild-type promoter (LKB1 Pro II, 2549 to +727). Each bar represents the means 6 standard deviation of three independent experiments made in quadruplicate. doi:10.1371/journal.pone.0032590.g002 examined by EMSA (Figure 4A). Nuclear extracts from ‘‘444’’ and HepG2 cells were incubated with three different radiolabeled double-stranded oligonucleotides (LKB1 CCAAT-1, 2 and 3, Figure 4B). Both extracts generated one predominant protein- DNA complex with all three oligonucleotides (‘‘complex B’’). In addition, both cell lines formed a second complex (complex D) only with the LKB1 CCAAT-3 oligo. Another complex (‘‘complex C’’) was only formed in HepG2 cells when oligos CCAAT-2 and 3 were examined. Specificity was confirmed by the absence of complex formation when a 500-fold molar excess of the unlabeled wild-type oligo was included (lanes 3, 9 and 15, respectively). In contrast, competition with a mutant oligo had no effect on complex formation (lanes 4, 10 and 16). The heterotrimeric transcription factor NF-Y has been described to bind CCAAT boxes [30]. To test whether these protein-DNA complexes contain NF-Y proteins, antibody incubation experiments PLoS ONE | www.plosone.org 4 March 2012 | Volume 7 | Issue 3 | e32590