can bind to this element. To test this assumption, EMSA using a radiolabeled double-stranded oligonucleotide, containing the potential FOXO binding site of the LKB1 promoter was carried out (Figure 5). Purified GST-tagged FOXO3 protein (lanes 2–6), but not GST interacted specifically with the oligo (lane 1) resulting in the formation of a single DNA-protein complex (Figure 5A). Sequence specificity of the complex was further confirmed by oligonucleotide competition and antibody incubation experiments. Here, complex formation was both effectively competed by an excess of unlabeled wild-type but not with mutant oligo and supershifted by the addition of a GST specific antibody. In further experiments, nuclear extracts from ‘‘444’’ cells after transfection with either expression plasmids encoding different FOXO proteins or siRNA directed against all FOXO factors in non-transfected cells were incubated with the probe in the presence of an excess of mutated competitor (Figure 5B). Complex formation was inhibited in cells treated with siRNA against FOXO proteins (lane 1). In contrast, ectopic expression of FOXO4 (lane 3) or mutant FOXO4 A3 (lane 4) that is constantly localized within the nucleus [32], as well as FOXO3 (lane 7) increased formation of the FOXO containing complexes when compared to transfections with the corresponding empty vectors (indicated a ‘‘V’’, see lanes 2 and 6). Moreover, an antibody against FOXO4 altered the mobility of the FOXO4 containing complex (FOXO4 supershift, SS; lane 5) and an antibody against FOXO3 disrupted the FOXO3 containing complex (lane 8). In addition, incubation of the oligonucleotide with extracts from HepG2 cells resulted in the formation of a different complex (lane 10). It is unlikely that this complex is formed by another FOXO member, since none of the used cell lines expressed FOXO1 or FOXO6 (data not shown). Since HepG2 cells express Transcriptional Regulation of LKB1 the liver specific transcription factor FOXA2, also known as HNF- 3b [33], it can be assumed that the observed protein-DNA complex may contain this factor. After ectopic expression of FOXA2 in ‘‘444’’ cells, known to lack this factor endogenously, complex formation with similar mobility could be discerned (lane 9), indicating that also other forkhead box transcription factors can bind to this element in a tissue specific manner. However, as deduced from EMSA analysis, FOXO3 and FOXO4 seem to be the key player in LKB1 gene regulation (see below). Chromatin immunoprecipitation demonstrates binding of NFY, Sp1 and FOXO proteins to the endogenous LKB1 promoter In order to examine whether NF-Y, Sp1 and the FOXO proteins also bind to the LKB1 promoter in vivo, chromatin immunoprecipitation (ChIP) assays were performed (Figure 6). All antibodies against NF-YA, Sp1, FOXO3 and FOXO4 specifically enriched the region containing the LKB1 core promoter in comparison to a non-specific antibody. In contrast, no enrichment was observed for the LKB1 coding sequence. Taken together, the ChIP analysis confirms the interaction between NF-Y, FOXO3 and FOXO4 with the endogenous LKB1 promoter and further supports a critical role of these factors in LKB1 gene expression. Ectopic expression of Sp1, NF-Y and the FOXO transcription factors FOXO3 and FOXO4 activates the LKB1 promoter In order to show a function of these factors in LKB1 regulation, co-transfections with LKB1 luciferase reporter constructs and Figure 5. Binding of forkhead box transcription factors FOXO3 and FOXO4 to the LKB1 promoter. (A) The 32P-labeled double-stranded oligonucleotide 59-GGGGAGGGAGGTAAACAAGATGGCGGC-39 containing the 228 to 22 region of the LKB1 core promoter was incubated with either 25 ng of recombinant GST (lane1) or GST-tagged FOXO3 protein (lane 2–6) and separated in a 4% polyacrylamide gel. (B) The same oligonucleotide as described in (A), but incubated with 4 mg of nuclear extracts from ‘‘444’’ cells (lanes 1–9) or from HepG2 cells (lane 10) in the presence of a 500-fold molar excess of the mutant unlabeled oligo 59-GGGGAGGGAGGTAGCCAAGATGGCGGC-39. Protein complexes containing the transcription factors FOXO3, FOXO4 and FOXA2 are indicated by arrows. Cells were transfected with either siRNA against the FOXO family members (lane1) or with expression plasmids encoding FOXO4 (lane 3), mutant FOXO4 A3 (lane 4 and 5), FOXO3 (lane 7 and 8), FOXA2 (lane 9) or with the corresponding empty vectors (V) (lane 2 and 6). Addition of an antibody against FOXO4 (lane 5) resulted in further retardation of the FOXO4 containing complex (FOXO4 SS), while addition of the FOXO3 antibody (lane 8) inhibited formation of the FOXO3 complex. doi:10.1371/journal.pone.0032590.g005 PLoS ONE | www.plosone.org 6 March 2012 | Volume 7 | Issue 3 | e32590