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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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NF-Y, Sp1 and FOXO transcription factors activate transcription from the LKB1 promoter.Luciferase reporter assays in C33a cells after over-expression of Sp1 (A), NF-Y (B) and FOXO transcription factors (C). 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.
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pone-0032590-g007: NF-Y, Sp1 and FOXO transcription factors activate transcription from the LKB1 promoter.Luciferase reporter assays in C33a cells after over-expression of Sp1 (A), NF-Y (B) and FOXO transcription factors (C). 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.

Mentions: In order to show a function of these factors in LKB1 regulation, co-transfections with LKB1 luciferase reporter constructs and 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.


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)

NF-Y, Sp1 and FOXO transcription factors activate transcription from the LKB1 promoter.Luciferase reporter assays in C33a cells after over-expression of Sp1 (A), NF-Y (B) and FOXO transcription factors (C). 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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3295762&req=5

pone-0032590-g007: NF-Y, Sp1 and FOXO transcription factors activate transcription from the LKB1 promoter.Luciferase reporter assays in C33a cells after over-expression of Sp1 (A), NF-Y (B) and FOXO transcription factors (C). 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.
Mentions: In order to show a function of these factors in LKB1 regulation, co-transfections with LKB1 luciferase reporter constructs and 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.

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