Binding of NF-Y transcription factor to CCAAT boxes within the LKB1 promoter.

<p>³²P-labeled double-stranded oligonucleotides harbouring the CCAAT boxes (their positions relative to the transcriptional start site, TSS, are indicated) (<b>A</b>) were incubated with 2 µg of nuclear extracts from “444” cells (lanes 1, 7 and 13) or from HepG2 cells (lanes 2–6, 8–12, and 14–18) and separated in a 7% polyacrylamide gel (<b>B</b>). Formation of sequence specific protein complexes was confirmed by competition with unlabeled oligos. Specific bands (indicated by arrows B, C and D) were competed by a 500-fold molar excess of wild-type oligo (WT; lanes 3, 9 and 15) but not by the same excess of mutant oligo (MUT; lanes 4, 10 and 16, respectively). Protein complexes containing the transcription factor NF-Y (arrow A) were further retarded by addition of an antibody against NF-Yα (NFY; lanes 5, 11 and 17) but not by the addition of the same amount of normal goat IgG (IgG; lanes 6, 12 and 18).</p

FOXO transcription factors are required for LKB1 gene expression.

<p>Knockdown of endogenous FOXO3 and FOXO4 in “444”, C33a and IMR-90 cells. Cells were transfected either with scrambled siRNA (Ctr) or with a siRNA targeting all FOXO family members (FOXO). Total RNA was purified and relative mRNA levels of FOXO3, FOXO4, LKB1 and GAPDH were analysed by RT-PCR using ethidium bromide staining (<b>A</b>). Equal amounts of total protein (5 µg) were separated by SDS-PAGE and analysed by western blotting using antibodies against FOXO3, LKB1 phospho-AMPK (Thr172) and actin (<b>B</b>). The data shown is representative of three independent experiments.</p

NF-Yα transcription factor is required for LKB1 gene expression.

<p>Knockdown of endogenous NF-Yα by siRNA inhibits LKB1 expression in “444”, C33a and IMR-90 cells. Cells were transfected either with scrambled siRNA (Ctr) or with a siRNA targeting NF-Yα (NF-YA). Total RNA was purified and mRNA levels of NF-YA, NF-YB, LKB1 and GAPDH were analysed by RT-PCR using ethidium bromide staining (<b>A</b>). Equal amounts of total protein (5 µg) were separated by SDS-PAGE and analysed by western blotting using antibodies against LKB1, phospho-AMPK (Thr172) and actin (<b>B</b>). The data shown is representative of three independent experiments.</p

NF-Y, Sp1 and FOXO transcription factors activate transcription from the LKB1 promoter.

<p>Luciferase reporter assays in C33a cells after over-expression of Sp1 (<b>A</b>), NF-Y (<b>B</b>) and FOXO transcription factors (<b>C</b>). Reporter activity of LKB1 wild-type promoter is indicated in dark grey, while reporter activity of constructs lacking the corresponding transcription factor binding site is indicated in light grey. Luciferase activity (relative light units normalized to renilla luciferase activity) is expressed as the percentage of the signal obtained from co-transfection of 100 ng of the plasmid containing the LKB1 wild-type promoter (LKB1 Pro II, −549 to +727) together with 150 ng of the empty expression vector (CTR). Instead of the empty vector 150 ng of the corresponding transcription factor expression plasmid have been co-transfected. In the case of NF-Y 50 ng of plasmids encoding each subunit NF-Yα, NFY-β and NF-Yγ were transfected together. Each bar represents the means ± standard deviation of three independent experiments.</p

Deletion-mutant analysis of the LKB1 promoter.

<p>(<b>A</b>) Regions of high mammalian and vertebrate sequence conservation in the 5′-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 −1536 to +727 relative to the transcription start site (modified from the UCSC genome browser at <a href="http://genome.ucsc.edu/ENCODE/" target="_blank">http://genome.ucsc.edu/ENCODE/</a>). (<b>B</b>) Luciferase activity of transiently transfected “444” cells with 200 bp deletion constructs (I–VII) of the LKB1 promoter. (<b>C</b>) 20 bp deletion mutants of the LKB1 promoter Pro III fragment, starting from nucleotide −345 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 5′-untranslated region (5′-UTR) is indicated. (<b>D</b>) Comparison of luciferase activity of transiently transfected “444” cells with LKB1 promoter 20 bp deletion constructs (right) and predicted <i>cis</i>-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 −345 (LKB1 Pro III). All assays were performed three times in quadruplicate. The error bars denote mean ± standard deviation.</p

Binding of Sp1 to a non-canonical GC box downstream of the transcription start site.

<p>(<b>A</b>) Overview of oligonucleotides used for EMSA. LKB1 DSE: downstream element, position +35 to +65 within the LKB1 5′-UTR and the corresponding mutant (LKB1 DSE Mut). Sequences of oligonuceotides containing consensus sites of different transcription factors used for competition are given below. (<b>B</b>) The radioactively labeled LKB1 DSE was incubated with 4 µg of nuclear extracts obtained from “444” cells (lane 1) or HepG2 cells (lane 2–9). A 500-fold molar excess of indicated unlabeled competitor oligos was added (lanes 3–9) and separated in a 7% polyacrylamide gel. (<b>C</b>) Complex formation (Sp1 S) of the same probe with 4 µg of nuclear extracts from “444” cells (lane 1) or from HepG2 cells (lane 2–6) was self-competed by a 500-fold molar excess of wild-type oligo (WT; lanes 3) but not by a mutant oligo (LKB1 DSE MUT; lane 4). Addition of an antibody against Sp1 (sc-59 X) (lane 5) retarded the specific complex (Sp1 SS) while addition of an antibody against Sp 3 (lane 6) did not.</p

Chromatin immunoprecipitation (ChIP) analysis of NF-Y, FOXO3 and FOXO4 binding at the LKB1 promoter.

<p>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-α, Sp1 (sc-14027 X), FOXO3 or FOXO4. The figure shows a representative of three independent experiments.</p

Substitution mutant analysis of the LKB1 promoter.

<p>Match of phylogenetic footprint with substitution mutant analysis of the LKB1 promoter region ranging from nucleotide position −345 to +75. (<b>A</b>) 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032590#pone-0032590-g001" target="_blank">Fig. 1</a>). The positions of the potential CCAAT/Forkhead/Sp1 boxes are indicated as red rectangles. (<b>B</b>) Substitution mutants of the LKB1 promoter Pro II fragment, encompassing nucleotides −549 to +727, were constructed by introducing a 10 bp mutation, comprising a NheI restriction site, within the area of −345 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. (<b>C</b>) Luciferase activity of transiently transfected “444” cells with LKB1 promoter 10 bp substitution mutants reveals a critical role of four <i>cis</i>-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, −549 to +727). Each bar represents the means ± standard deviation of three independent experiments made in quadruplicate.</p

Oligonucleotides used in EMSA.

<p>FOXO3 core binding motive is indicated in bold. Parts of adjacent FOXO3 binding sites within the same oligonucleotide are underlined.</p

Glucocorticoid-induced transcriptional activation of FOXO3 is further enhanced by AMPK activation.

<p>(A) MCF-10A cells were treated with 1 mM of specific AMPK activator AICAR in combination with either vehicle (Ctrl), 1 µM dexamethasone (DEX) or a mixture of 1 µM dexamethasone and 1 µM RU-486 (DEX/RU) for 3, 6, 12 and 24 h. (B) MCF-10A cells were treated with vehicle (Ctrl), 1 µM dexamethasone (DEX) or a combination of 1 µM dexamethasone and 1 mM AICAR (DEX+AICAR) in the presence or absence of 10 µg/ml cycloheximide (CHX) for 6 h. Relative mRNA levels of FOXO3 were analysed by quantitative real-time PCR (mean ±SD, n = 3). Relative protein levels of FOXO3 in comparison to actin as internal control were analysed by western blotting in the presence or absence of cycloheximide.</p