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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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Deletion-mutant analysis of the LKB1 promoter.(A) 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 http://genome.ucsc.edu/ENCODE/). (B) Luciferase activity of transiently transfected “444” cells with 200 bp deletion constructs (I–VII) of the LKB1 promoter. (C) 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. (D) Comparison of luciferase activity of transiently transfected “444” cells with LKB1 promoter 20 bp deletion constructs (right) and predicted cis-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.
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pone-0032590-g001: Deletion-mutant analysis of the LKB1 promoter.(A) 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 http://genome.ucsc.edu/ENCODE/). (B) Luciferase activity of transiently transfected “444” cells with 200 bp deletion constructs (I–VII) of the LKB1 promoter. (C) 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. (D) Comparison of luciferase activity of transiently transfected “444” cells with LKB1 promoter 20 bp deletion constructs (right) and predicted cis-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.

Mentions: As a first step towards localizing important control regions that activate LKB1 gene expression, we cloned the region flanking the 5′-end of the coding sequence in front of a luciferase reporter gene. The reporter plasmid containing the nucleotide sequence from −1536 to +727 relative to the LKB1 transcription start site (referred to as LKB1 Pro I), was active following transient transfection of “444” cells. As a second step, six 200 bp 5′-deletion mutants (referred to as LKB1 Pro II–VII) were constructed (Figure 1A) and luciferase activity was measured (Figure 1B). Consecutive deletion of the sequence from −1536 to −345 resulted in minimal changes of the promoter activity. Stronger decreases were only observed when the LKB1 Pro III construct was further deleted, indicating that important cis-regulatory elements are located in the area downstream of nucleotide −345. The deletion from −345 to −186 (LKB1 Pro IV) reduced luciferase activity by 50%. The next shorter deletion construct (LKB1 Pro V) showed only 12.5% of the LKB1 Pro III activity. Removing the transcriptional start site [3] by further truncation of LKB1 Pro V, decreased luciferase activity to a level similar to that obtained by transfection with the empty vector. The fact that the residual 550 bp of the 5′-untranslated region (5′-UTR) within LKB1 Pro VI could not activate reporter gene expression alone, indicates that important regulatory elements are located upstream of nucleotide +182.


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)

Deletion-mutant analysis of the LKB1 promoter.(A) 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 http://genome.ucsc.edu/ENCODE/). (B) Luciferase activity of transiently transfected “444” cells with 200 bp deletion constructs (I–VII) of the LKB1 promoter. (C) 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. (D) Comparison of luciferase activity of transiently transfected “444” cells with LKB1 promoter 20 bp deletion constructs (right) and predicted cis-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.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3295762&req=5

pone-0032590-g001: Deletion-mutant analysis of the LKB1 promoter.(A) 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 http://genome.ucsc.edu/ENCODE/). (B) Luciferase activity of transiently transfected “444” cells with 200 bp deletion constructs (I–VII) of the LKB1 promoter. (C) 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. (D) Comparison of luciferase activity of transiently transfected “444” cells with LKB1 promoter 20 bp deletion constructs (right) and predicted cis-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.
Mentions: As a first step towards localizing important control regions that activate LKB1 gene expression, we cloned the region flanking the 5′-end of the coding sequence in front of a luciferase reporter gene. The reporter plasmid containing the nucleotide sequence from −1536 to +727 relative to the LKB1 transcription start site (referred to as LKB1 Pro I), was active following transient transfection of “444” cells. As a second step, six 200 bp 5′-deletion mutants (referred to as LKB1 Pro II–VII) were constructed (Figure 1A) and luciferase activity was measured (Figure 1B). Consecutive deletion of the sequence from −1536 to −345 resulted in minimal changes of the promoter activity. Stronger decreases were only observed when the LKB1 Pro III construct was further deleted, indicating that important cis-regulatory elements are located in the area downstream of nucleotide −345. The deletion from −345 to −186 (LKB1 Pro IV) reduced luciferase activity by 50%. The next shorter deletion construct (LKB1 Pro V) showed only 12.5% of the LKB1 Pro III activity. Removing the transcriptional start site [3] by further truncation of LKB1 Pro V, decreased luciferase activity to a level similar to that obtained by transfection with the empty vector. The fact that the residual 550 bp of the 5′-untranslated region (5′-UTR) within LKB1 Pro VI could not activate reporter gene expression alone, indicates that important regulatory elements are located upstream of nucleotide +182.

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