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Gene expression of the tumour suppressor LKB1 is mediated by Sp1, NF-Y and FOXO transcription factors.

Lützner N, De-Castro Arce J, Rösl F - PLoS ONE (2012)

Bottom Line: Overexpression of these factors significantly increased the LKB1 promoter activity.Conversely, small interfering RNAs directed against NF-Y alpha and the two FOXO proteins greatly reduced endogenous LKB1 expression and phosphorylation of LKB1's main substrate AMPK in three different cell lines.Taken together, these results demonstrate that Sp1, NF-Y and FOXO transcription factors are involved in the regulation of LKB1 transcription.

View Article: PubMed Central - PubMed

Affiliation: Research Program Infections and Cancer, German Cancer Research Center (DKFZ), Heidelberg, Germany. n.luetzner@dkfz.de

ABSTRACT
The serine/threonine kinase LKB1 is a tumour suppressor that regulates multiple biological pathways, including cell cycle control, cell polarity and energy metabolism by direct phosphorylation of 14 different AMP-activated protein kinase (AMPK) family members. Although many downstream targets have been described, the regulation of LKB1 gene expression is still poorly understood. In this study, we performed a functional analysis of the human LKB1 upstream regulatory region. We used 200 base pair deletion constructs of the 5'-flanking region fused to a luciferase reporter to identify the core promoter. It encompasses nucleotides -345 to +52 relative to the transcription start site and coincides with a DNase I hypersensitive site. Based on extensive deletion and substitution mutant analysis of the LKB1 promoter, we identified four cis-acting elements which are critical for transcriptional activation. Using electrophoretic mobility shift assays as well as chromatin immunoprecipitations, we demonstrate that the transcription factors Sp1, NF-Y and two forkhead box O (FOXO) family members FOXO3 and FOXO4 bind to these elements. Overexpression of these factors significantly increased the LKB1 promoter activity. Conversely, small interfering RNAs directed against NF-Y alpha and the two FOXO proteins greatly reduced endogenous LKB1 expression and phosphorylation of LKB1's main substrate AMPK in three different cell lines. Taken together, these results demonstrate that Sp1, NF-Y and FOXO transcription factors are involved in the regulation of LKB1 transcription.

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Binding of forkhead box transcription factors FOXO3 and FOXO4 to the LKB1 promoter.(A) The 32P-labeled double-stranded oligonucleotide 5′-GGGGAGGGAGGTAAACAAGATGGCGGC-3′ containing the −28 to −2 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 µg 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 5′-GGGGAGGGAGGTAGCCAAGATGGCGGC-3′. 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.
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pone-0032590-g005: Binding of forkhead box transcription factors FOXO3 and FOXO4 to the LKB1 promoter.(A) The 32P-labeled double-stranded oligonucleotide 5′-GGGGAGGGAGGTAAACAAGATGGCGGC-3′ containing the −28 to −2 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 µg 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 5′-GGGGAGGGAGGTAGCCAAGATGGCGGC-3′. 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.

Mentions: Since mutation of a potential forkhead box transcription factor binding site within the LKB1 promoter significantly reduced promoter activity (Figure 1D), we assumed that FOXO proteins 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.


Gene expression of the tumour suppressor LKB1 is mediated by Sp1, NF-Y and FOXO transcription factors.

Lützner N, De-Castro Arce J, Rösl F - PLoS ONE (2012)

Binding of forkhead box transcription factors FOXO3 and FOXO4 to the LKB1 promoter.(A) The 32P-labeled double-stranded oligonucleotide 5′-GGGGAGGGAGGTAAACAAGATGGCGGC-3′ containing the −28 to −2 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 µg 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 5′-GGGGAGGGAGGTAGCCAAGATGGCGGC-3′. 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.
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pone-0032590-g005: Binding of forkhead box transcription factors FOXO3 and FOXO4 to the LKB1 promoter.(A) The 32P-labeled double-stranded oligonucleotide 5′-GGGGAGGGAGGTAAACAAGATGGCGGC-3′ containing the −28 to −2 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 µg 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 5′-GGGGAGGGAGGTAGCCAAGATGGCGGC-3′. 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.
Mentions: Since mutation of a potential forkhead box transcription factor binding site within the LKB1 promoter significantly reduced promoter activity (Figure 1D), we assumed that FOXO proteins 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.

Bottom Line: Overexpression of these factors significantly increased the LKB1 promoter activity.Conversely, small interfering RNAs directed against NF-Y alpha and the two FOXO proteins greatly reduced endogenous LKB1 expression and phosphorylation of LKB1's main substrate AMPK in three different cell lines.Taken together, these results demonstrate that Sp1, NF-Y and FOXO transcription factors are involved in the regulation of LKB1 transcription.

View Article: PubMed Central - PubMed

Affiliation: Research Program Infections and Cancer, German Cancer Research Center (DKFZ), Heidelberg, Germany. n.luetzner@dkfz.de

ABSTRACT
The serine/threonine kinase LKB1 is a tumour suppressor that regulates multiple biological pathways, including cell cycle control, cell polarity and energy metabolism by direct phosphorylation of 14 different AMP-activated protein kinase (AMPK) family members. Although many downstream targets have been described, the regulation of LKB1 gene expression is still poorly understood. In this study, we performed a functional analysis of the human LKB1 upstream regulatory region. We used 200 base pair deletion constructs of the 5'-flanking region fused to a luciferase reporter to identify the core promoter. It encompasses nucleotides -345 to +52 relative to the transcription start site and coincides with a DNase I hypersensitive site. Based on extensive deletion and substitution mutant analysis of the LKB1 promoter, we identified four cis-acting elements which are critical for transcriptional activation. Using electrophoretic mobility shift assays as well as chromatin immunoprecipitations, we demonstrate that the transcription factors Sp1, NF-Y and two forkhead box O (FOXO) family members FOXO3 and FOXO4 bind to these elements. Overexpression of these factors significantly increased the LKB1 promoter activity. Conversely, small interfering RNAs directed against NF-Y alpha and the two FOXO proteins greatly reduced endogenous LKB1 expression and phosphorylation of LKB1's main substrate AMPK in three different cell lines. Taken together, these results demonstrate that Sp1, NF-Y and FOXO transcription factors are involved in the regulation of LKB1 transcription.

Show MeSH
Related in: MedlinePlus