LKB1 promoter were identified. FOXO3 and FOXO4 activated LKB1 gene transcription through interaction with their cognate recognition site 59-GTAAACAA-39 [22]. FOXO transcription factors become inactivated by certain growth factors [23] through direct phosphorylation by the protein kinase B (PKB) [24,25]. Since several FOXO target genes are involved in growth control and cell cycle regulation, their inactivation could represent a critical event in malignant transformation [26,27]. Therefore, our findings that functionally link FOXO proteins to the transcrip- tional activation of the tumour suppressor gene LKB1, provide an important step towards a detailed understanding of the complex molecular events that promote carcinogenesis. Results Identification of the LKB1 core promoter As a first step towards localizing important control regions that activate LKB1 gene expression, we cloned the region flanking the 59-end of the coding sequence in front of a luciferase reporter gene. The reporter plasmid containing the nucleotide sequence from 21536 to +727 relative to the LKB1 transcription start site (referred to as LKB1 Pro I), was active following transient transfection of ‘‘444’’ cells. As a second step, six 200 bp 59-deletion mutants (referred to as LKB1 Pro II–VII) were constructed (Figure 1A) and luciferase activity was measured (Figure 1B). Consecutive deletion of the sequence from 21536 to 2345 resulted in minimal changes of the promoter activity. Stronger decreases were only observed when the LKB1 Pro III construct was further deleted, indicating that important cis-regulatory elements are located in the area down- stream of nucleotide 2345. The deletion from 2345 to 2186 (LKB1 Pro IV) reduced luciferase activity by 50%. The next shorter deletion construct (LKB1 Pro V) showed only 12.5% of the LKB1 Pro III activity. Removing the transcriptional start site [3] by further truncation of LKB1 Pro V, decreased luciferase activity to a level similar to that obtained by transfection with the empty vector. The fact that the residual 550 bp of the 59-untranslated region (59-UTR) within LKB1 Pro VI could not activate reporter gene expression alone, indicates that important regulatory elements are located upstream of nucleotide +182. Interestingly, data-base analysis using the human genome browser of the Encyclopedia of DNA Elements (ENCODE) [28] revealed that the area between nucleotide 2345 and the trans- criptional start site coincides with a previously described DNaseI hypersensitive region (Figure 1A). This has been identified by a genome-wide chromatin analysis in different cell types including HepG2 cells, normal human epidermal keratinocytes and normal human lung fibroblasts [29]. Since the sequence within the LKB1 Pro III construct is crucial for reporter gene transcription and co- localize with a nucleosomal structure that is highly accessible to exogenously added DNaseI, favours the notion that the LKB1 core promoter is located downstream of nucleotide 2345. Deletion mutant analysis of the LKB1 promoter identifies regulatory regions In order to localize critical cis-regulatory elements within the LKB1 core promoter more precisely, we constructed a series of 20 bp deletion mutants of LKB1 Pro III (Figure 1C). These constructs (III/ 0–8; IV/0–6 and V/0–5) are schematically outlined in Figure 1D (left). After transient transfection of ‘‘444’’ cells with these 24 different deletion constructs, including those used in the previous assay (LKB1 Pro III/0, IV/0, V/0 and VI/0, see Figure 1A), luciferase activity was measured (Figure 1D, right). While sequence deletions from 2345 to 2246 did not affect promoter activity, further truncation of III/5 to III/6 reduced luciferase activity to 65% of the wild-type promoter. Transcriptional Regulation of LKB1 The next drop in luciferase activity was observed between III/7 and III/8 when nucleotides 2206 to 2187 were deleted. III/8 and IV/0 reached only 57% of the LKB1 Pro III/0 reporter activity and it even dropped to 22% after removing nucleotides 2166 to 2147. Interestingly, all three sequence deletions between nucleotides 2345 and 2147 that decreased LKB1 promoter activity exhibit homology to consensus sequences for CCAAT boxes [30] (Figure 1D, left). Another critical deletion removes a sequence homologous to a transcription factor consensus site was located between nucleotides 228 and 28 (Figure 1D, left). It contains a potential binding site for FOXO transcription factors [22] and reduced luciferase activity of the V/0 construct by 65%. The residual activity was similar to that of the promoterless vector (Figure 1D, right). Likewise, deletions of each CCAAT box and the predicted FOXO binding site also decreased luciferase activity when HPV- negative C33a cells were transfected, although the extent of reduction was slightly different (Figure S1). Two deletions were found to have effects on promoter activity, which were apparently cell type specific. While ‘‘444’’ cells showed an additional drop in activity between IV/3 and IV/4, activity in C33a cells decreased between IV/4 and IV/5. Substitution mutant analyses identify LKB1 promoter elements critical for activated transcription In the course of our 20 bp deletion mutation analysis, we found strong promoter activity within the area downstream of nucleotide 2345 of the LKB1 promoter which is apparently mediated by CCAAT boxes and a FOXO binding site (Figure 1D, left). Interestingly, phylogenetic footprint analysis revealed high homol- ogy of these elements between human, rhesus, mouse, rat and zebrafish, indicating that the sequences required for the binding of activators to the promoter have been conserved, despite having evolved under heterogeneous constraints (Figure 2A). In order to confirm the relevance of these regions in a context in which the promoter length remains unchanged, we generated 21 substitution mutants where 10 bp sequences between positions 2345 to +72 were systematically altered by inserting a NheI restriction site (Figure 2B, referred to as III/1–8, IV/0–6 and V/0–5). After transient transfection of ‘‘444’’ and C33a cells with the mutant promoter constructs, luciferase activity was measured (Figure 2C, ‘‘444’’; Figure S1, C33a) and the result was compared to the phylogenetic footprint analysis of the 2345 to +72 sequence (Figure 2A). The first potential CCAAT box, whose deletion had reduced promoter activity by 35%, was not disrupted by the corresponding substitution mutation III/5. Mutation of the second CCAAT box (III/7), substituting nucleotides 2196 to 2187, caused only a minimal decrease of 16% in luciferase activity in ‘‘444’’ cells. Consistent with the previous analysis (Figure 1D), in which deletion of the site caused only 12% reduction in activity, this suggests a less important function of the second CCAAT box. The first clear reduction that reduced luciferase activity in both cell lines investigated was generated by mutation of the sequence from 2157 to 2148, containing the third potential CCAAT box. The corresponding construct (IV/0) showed only 34% of the wild- type activity in ‘‘444’’ cells. Another critical element was disrupted by the V/0 mutation, which reduced wild type activity to 46% in ‘‘444’’ cells. This element contains the potential FOXO binding site and did also affect promoter activity in the deletion analysis (Figure 1D, right). In addition, we found two other mutations downstream of the FOXO site that reduced promoter activity by greater than 50% in both cell lines. One was disrupting the area around the transcription start site from +3 to +12 where the corresponding construct (V/1) showed 49% of the wild-type activity in ‘‘444’’ cells. The other mutation was disrupting an PLoS ONE | www.plosone.org 2 March 2012 | Volume 7 | Issue 3 | e32590