Figure 6. Chromatin immunoprecipitation (ChIP) analysis of NF-Y, FOXO3 and FOXO4 binding at the LKB1 promoter. Enrichment of PCR products specific for the LKB1 promoter region (upper panel) in comparison to PCR products specific for the LKB1 coding region encompassing exon 4–5 (lower panel). PCR products were amplified from sonified DNA after ChIP, subsequently run on a 1% agarose gel and stained with ethidium bromide. As a positive control (Input), 1/10 of the starting material was used for PCR. Protein-DNA complexes were either incubated with non-specific goat IgG (negative control) or with antibodies against the transcription factors NFY-a, Sp1 (sc-14027 X), FOXO3 or FOXO4. The figure shows a representative of three independent experiments. doi:10.1371/journal.pone.0032590.g006 expression plasmids encoding the different transcription factors were performed (Figure 7). Transient transfection of Sp1 significantly increased luciferase activity of the wild-type promoter when compared with the empty vector. In contrast, no induction could be discerned when Sp1 was co-transfected with the substitution construct (LKB1 Pro V/3) where the Sp1 binding site was mutated (Figure 7A). Ectopic expression of all three NF-Y subunits also activated the LKB1 wild-type promoter, while the deletion construct that lacks all three CCAAT boxes (LKB1 Pro IV/1) could not be induced (Figure 7B). Finally, FOXO proteins also induced reporter gene expression in a sequence specific manner, since the respective mutant LKB1 Pro Mut V/0 was not activated to the same extent after co-transfection with different FOXO expression plasmids (Figure 7C). Although FOXO3 expression significantly increased luciferase activity of the wild- type promoter, induction after FOXO4 transfection was only marginal. One reason for this could be a post-translational modification of the FOXO4 protein. As described above, transcriptional activity of all FOXO proteins is negatively regulated by the protein kinase B (PKB) through direct phosphorylation of three amino acid side chains [24,25]. When mutants of the two FOXO proteins, which lack these PKB phosphorylation sites (FOXO3 A3 and FOXO4 A3) were transfected, LKB1 reporter gene expression was activated even more efficiently (Figure 7C). These results demonstrate that Sp1, NF-Y as well as FOXO3 and FOXO4 have the ability to activate the LKB1 promoter through interaction with their corresponding binding sites. Knockdown of NF-Y and FOXO transcription factors inhibits LKB1 gene expression To further investigate the role of NF-Y and the FOXO proteins in activating endogenous LKB1 gene transcription, siRNA knockdown experiments were performed. While delivery of siRNA against Sp1 resulted in massive cell death, treatment of ‘‘444’’, C33a and IMR-90 cells with NF-YA siRNA significantly diminished endogenous mRNA levels of the alpha subunit of the NF-Y complex (Figure 8A). In contrast, mRNA levels of NF-YB and GAPDH, which were used as internal controls, were not affected. Consistent with the previous experiments, LKB1 mRNA and protein levels were strongly reduced after the NF-YA knockdown, indicating that NF-Y is essential for LKB1 gene expression (Figure 8A and B). Moreover, siRNA knockdown of FOXO3 and FOXO4 also decreased LKB1 expression, both on Transcriptional Regulation of LKB1 mRNA and protein level (Figure 9). Furthermore, phosphorylation of AMPKa, the main substrate of LKB1 was also significantly reduced under these conditions (Figure 8 and 9), indicating that individual knockdown of both NF-YA or the two FOXO proteins FOXO3 and FOXO4 not only diminished LKB1 gene expression, but also interfered with the LKB1-AMPK signalling pathway by reducing AMPK activation. Discussion In the present study we performed a systematic analysis of the LKB1 promoter. Using extensive deletion and mutant analyses, we identified multiple control elements important for LKB1 gene transcription. Three of these elements contained CCAAT boxes and were recognized by the heterotrimeric transcription factor NF-Y [30]. Another evolutionary highly conserved element contained a binding site for forkhead box transcription factors and interacted specifically with the transcription factors FOXO3 and FOXO4 [22]. Besides these factors, whose binding sites were all located upstream of the transcription start site, we found another critical element within the 59-untranslated region (59-UTR). It has been identified as a Sp1 site that was apparently essential for promoter activity in the transient transfection assays (Figure 2). Furthermore, ectopic expression of Sp1 significantly increased LKB1 promoter activity (Figure 7A). Although Sp1 was longtime considered to be a house-keeping transcription factor, the protein is involved in glucose metabolism where Sp1 binding to the acetyl-CoA carboxylase promoter increased transcription in adipocytes [34]. Moreover, Sp1 is also regulated by energy deprivation [35] and plays a role in insulin signaling. However, to which extent Sp1 contributes to endoge- nous LKB1 expression still remains to be elucidated, since siRNA knockdown of Sp1 caused massive cell death in all cell lines investigated. Remarkably, the Sp1 element was not sufficient to activate reporter gene expression alone (Figure 1). It is therefore tempting to speculate that the Sp1 site located within the 59-UTR apparently may cooperate with control elements located upstream of the transcription start site. Indeed, a precedential case has been described for the major histocompatibility complex class II- associated invariant chain, where Sp1 and NF-Y cooperatively activate the gene in cancer cell lines [36]. Consistent with this notion is the fact that siRNA knockdown of the alpha subunit of NF-Y but also of FOXO transcription factors greatly reduced endogenous LKB1 expression (Figure 8 and 9). Moreover, phosphorylation of the alpha subunit of AMPK, the main downstream effector of LKB1, was also diminished under these conditions. This indicates that these proteins, in conjunction with Sp1, are not only indispensible for efficient transcription of the gene, but also affect the activation of downstream targets in the LKB1 signaling pathway. Although we have shown that Sp1, NF-Y and the FOXO transcription factors are necessary for effective LKB1 gene transcription in our cell systems, there also exists the possibility that other functionally important cis-regulatory elements and trans- acting factors exist that influence LKB1 promoter activity in a tissue-specific manner. First, deletion and substitution mutations at other locations had cell line specific but significant effects on promoter activity (Comparison Figure 1 and Figure S1). Second, other cell line specific DNA-binding activities were detected by EMSA. In detail, the liver specific transcription factor FOXA2 (also named HNF-3b) was also able to bind to the FOXO recognition site in the LKB1 promoter (Figure 5). Furthermore, additional studies have shown that different members of this huge PLoS ONE | www.plosone.org 7 March 2012 | Volume 7 | Issue 3 | e32590