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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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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-α, Sp1 (sc-14027 X), FOXO3 or FOXO4. The figure shows a representative of three independent experiments.
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pone-0032590-g006: 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-α, Sp1 (sc-14027 X), FOXO3 or FOXO4. The figure shows a representative of three independent experiments.

Mentions: In order to examine whether NF-Y, Sp1 and the FOXO proteins also bind to the LKB1 promoter in vivo, chromatin immunoprecipitation (ChIP) assays were performed (Figure 6). All antibodies against NF-YA, Sp1, FOXO3 and FOXO4 specifically enriched the region containing the LKB1 core promoter in comparison to a non-specific antibody. In contrast, no enrichment was observed for the LKB1 coding sequence. Taken together, the ChIP analysis confirms the interaction between NF-Y, FOXO3 and FOXO4 with the endogenous LKB1 promoter and further supports a critical role of these factors in LKB1 gene expression.


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)

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-α, Sp1 (sc-14027 X), FOXO3 or FOXO4. The figure shows a representative of three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3295762&req=5

pone-0032590-g006: 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-α, Sp1 (sc-14027 X), FOXO3 or FOXO4. The figure shows a representative of three independent experiments.
Mentions: In order to examine whether NF-Y, Sp1 and the FOXO proteins also bind to the LKB1 promoter in vivo, chromatin immunoprecipitation (ChIP) assays were performed (Figure 6). All antibodies against NF-YA, Sp1, FOXO3 and FOXO4 specifically enriched the region containing the LKB1 core promoter in comparison to a non-specific antibody. In contrast, no enrichment was observed for the LKB1 coding sequence. Taken together, the ChIP analysis confirms the interaction between NF-Y, FOXO3 and FOXO4 with the endogenous LKB1 promoter and further supports a critical role of these factors in LKB1 gene expression.

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