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Glucose-dependent regulation of NR2F2 promoter and influence of SNP-rs3743462 on whole body insulin sensitivity.

Boutant M, Ramos OH, Lecoeur C, Vaillant E, Philippe J, Zhang P, Perilhou A, Valcarcel B, Sebert S, Jarvelin MR, Balkau B, Scott D, Froguel P, Vaxillaire M, Vasseur-Cognet M - PLoS ONE (2012)

Bottom Line: The present study aimed to identify the regulatory regions that control NR2F2 gene transcription and to evaluate the effect of NR2F2 promoter variation on glucose homeostasis in humans.The effects of variation at SNP rs3743462 in NR2F2 on quantitative metabolic traits were studied in two European prospective cohorts.The C-allele at rs3743462 was associated with increased NR2F2 binding and decreased NR2F2 gene expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Endocrinology, Metabolism and Cancer, Cochin Institute, CNRS (UMR 8104), Paris, France.

ABSTRACT

Background: The Nuclear Receptor 2F2 (NR2F2/COUP-TFII) heterozygous knockout mice display low basal insulinemia and enhanced insulin sensitivity. We previously established that insulin represses NR2F2 gene expression in pancreatic β-cells. The cis-regulatory region of the NR2F2 promoter is unknown and its influence on metabolism in humans is poorly understood. The present study aimed to identify the regulatory regions that control NR2F2 gene transcription and to evaluate the effect of NR2F2 promoter variation on glucose homeostasis in humans.

Methodology/principal findings: Regulation of the NR2F2 promoter was assessed using gene reporter assays, ChIP and gel shift experiments. The effects of variation at SNP rs3743462 in NR2F2 on quantitative metabolic traits were studied in two European prospective cohorts. We identified a minimal promoter region that down-regulates NR2F2 expression by attenuating HNF4α activation in response to high glucose concentrations. Subjects of the French DESIR population, who carried the rs3743462 T-to-C polymorphism, located in the distal glucose-responsive promoter, displayed lower basal insulin levels and lower HOMA-IR index. The C-allele at rs3743462 was associated with increased NR2F2 binding and decreased NR2F2 gene expression.

Conclusions/significance: The rs3743462 polymorphism affects glucose-responsive NR2F2 promoter regulation and thereby may influence whole-body insulin sensitivity, suggesting a role of NR2F2 in the control of glucose homeostasis in humans.

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Related in: MedlinePlus

Glucose decreases the ability of HNF4α to activate the transcription of the NR2F2 proximal promoter and to bind its chromatin target.(A) INS-1 832/13 cells were transiently co-transfected as described in [7] with a luciferase reporter gene driven by various lengths of the NR2F2 promoter, designated by their 5′ and 3′ end positions relative to the defined NR2F2 gene transcription initiation site [25] and a control vector expressing Renilla luciferase. Cells were cultured in the presence of either 5 mM (white bars) or 20 mM (black bars) glucose. Results were calculated from the ratio of luciferase/Renilla activity. Background expression was subtracted using the mean expression level of empty pGL3-basic. Means ± SEM of results obtained from at least three independent transfections performed in triplicate are shown. *Significant differences, P<0.05. (B) Comparison of mRNA levels of NR2F2 and HNF4α relative to those of cyclophilin determined by real-time RT-QPCR in INS-1 832/13 β-cells stimulated with 5 mM (white bars) or 20 mM glucose (black bars) for 24 h as described in [3] (C) ChIP from INS-1 832/13 cells cultured in the presence of either 5 mM or 20 mM glucose. Targets for QPCR amplifications were the proximal NR2F2 promoter containing the DR1 DNA binding site or a downstream coding region (negative control). Amount of target chromatin precipitated by the HNF4α-specific antibody relative to that precipitated by control IgG (mean of three independent experiments ± SEM). *Significant difference, P<0.05. (D) COS cells were transiently co-transfected with the −328/+873 construct and 50 ng of empty KS vector (white bars), or HNF4α (grey bars) expression vectors. Means ± SEM of results from at least three separate transfections performed in triplicate are shown. *Significant difference, P<0.05. (E) INS-1 832/13 cells were transiently co-transfected with the wild-type or mutated −328/+873 constructs containing the DR1 elements. Cells were cultured in the presence of either 5 mM (white bars) or 20 mM (black bars) glucose. Means ± SEM of results from ten independent transfections performed in triplicate are shown. *Statistically significant differences in percentage of expression (where value for 5 mM glucose is 100% relative to −328/+873 construct) at P<0.05.
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pone-0035810-g001: Glucose decreases the ability of HNF4α to activate the transcription of the NR2F2 proximal promoter and to bind its chromatin target.(A) INS-1 832/13 cells were transiently co-transfected as described in [7] with a luciferase reporter gene driven by various lengths of the NR2F2 promoter, designated by their 5′ and 3′ end positions relative to the defined NR2F2 gene transcription initiation site [25] and a control vector expressing Renilla luciferase. Cells were cultured in the presence of either 5 mM (white bars) or 20 mM (black bars) glucose. Results were calculated from the ratio of luciferase/Renilla activity. Background expression was subtracted using the mean expression level of empty pGL3-basic. Means ± SEM of results obtained from at least three independent transfections performed in triplicate are shown. *Significant differences, P<0.05. (B) Comparison of mRNA levels of NR2F2 and HNF4α relative to those of cyclophilin determined by real-time RT-QPCR in INS-1 832/13 β-cells stimulated with 5 mM (white bars) or 20 mM glucose (black bars) for 24 h as described in [3] (C) ChIP from INS-1 832/13 cells cultured in the presence of either 5 mM or 20 mM glucose. Targets for QPCR amplifications were the proximal NR2F2 promoter containing the DR1 DNA binding site or a downstream coding region (negative control). Amount of target chromatin precipitated by the HNF4α-specific antibody relative to that precipitated by control IgG (mean of three independent experiments ± SEM). *Significant difference, P<0.05. (D) COS cells were transiently co-transfected with the −328/+873 construct and 50 ng of empty KS vector (white bars), or HNF4α (grey bars) expression vectors. Means ± SEM of results from at least three separate transfections performed in triplicate are shown. *Significant difference, P<0.05. (E) INS-1 832/13 cells were transiently co-transfected with the wild-type or mutated −328/+873 constructs containing the DR1 elements. Cells were cultured in the presence of either 5 mM (white bars) or 20 mM (black bars) glucose. Means ± SEM of results from ten independent transfections performed in triplicate are shown. *Statistically significant differences in percentage of expression (where value for 5 mM glucose is 100% relative to −328/+873 construct) at P<0.05.

Mentions: To map the cis-regulatory elements responsible for glucose responsiveness in NR2F2, we transiently co-transfected luciferase plasmid reporters in a INS-1 832/13 β-cell line with various deleted sections of the NR2F2 regulatory region. This corresponds to a fragment of 4 kbp that encompasses the upstream NR2F2 regulatory region that was shown to be sufficient to direct NR2F2 expression in pancreatic β-cells [8]. In comparison with a concentration of 5 mM, a glucose concentration of 20 mM provoked a 45% reduction of luciferase activity (P<0.05) in INS-1 832/13 β-cells co-transfected with the full length NR2F2 promoter construct −3210/+873) (Fig. 1A). These measured impacts were consistent with previous observations on endogenous reduction of NR2F2 mRNA abundance by high glucose concentrations (Fig. 1B and [3]). The deletion analysis further established that the fragment from −328 to +873 was the minimal region required to confer a significant inhibition by high glucose concentrations (Fig. 1A). In this proximal promoter, we previously showed that HNF4α binds the conserved direct repeat-1 (DR-1) hormone response element (HRE) [7]. We also showed that HNF4α is able to activate the endogenous NR2F2 gene in INS-1 832/13 β-cells [7]. As shown in figure 1B, we observed the same reduction of HNF4α mRNA levels as observed for NR2F2 mRNA levels in the presence of high glucose levels in INS-1 cells suggesting that this transcription factor could be involved in the glucose responsiveness of NR2F2. As show in figure 1C, the antibodies for HNF4α immunoprecipitated with the promoter region of NR2F2, revealing the presence of endogenous HNF4α on this DNA binding site. A high glucose concentration (20 mM) in comparison to a concentration of 5 mM induced a significant reduction of immunoprecipitation with anti-HNF4α antibodies. HNF4α bound to the DR-1 DNA binding site in a glucose-dependent manner. In COS-7 cells, that lack endogenous expression of HNF4α, the co-transfection with an HNF4α expression vector and the −328/+873 luciferase reporter plasmid induced a 4-fold increase in luciferase activity (Fig. 1D). Moreover, when the DR-1 DNA binding site was mutated in the −328/+873 construct, we measured a 70% reduction in luciferase activity (Fig. 1E). This suggests that a nuclear receptor is a transactivator of the NR2F2 promoter in β-cells cultured in 5 mM glucose, a condition that allows maximal expression of NR2F2. Furthermore the mutations at the DR-1 binding site led to a significantly weaker repression by 20 mM glucose (Fig. 1E). Altogether these results suggest that HNF4α is a transcription factor required for inhibition of NR2F2 promoter activity by high concentrations of glucose. They also suggest a more complex pattern of regulation with additional factors involved in the inhibition of NR2F2 transcription activity by high concentrations of glucose.


Glucose-dependent regulation of NR2F2 promoter and influence of SNP-rs3743462 on whole body insulin sensitivity.

Boutant M, Ramos OH, Lecoeur C, Vaillant E, Philippe J, Zhang P, Perilhou A, Valcarcel B, Sebert S, Jarvelin MR, Balkau B, Scott D, Froguel P, Vaxillaire M, Vasseur-Cognet M - PLoS ONE (2012)

Glucose decreases the ability of HNF4α to activate the transcription of the NR2F2 proximal promoter and to bind its chromatin target.(A) INS-1 832/13 cells were transiently co-transfected as described in [7] with a luciferase reporter gene driven by various lengths of the NR2F2 promoter, designated by their 5′ and 3′ end positions relative to the defined NR2F2 gene transcription initiation site [25] and a control vector expressing Renilla luciferase. Cells were cultured in the presence of either 5 mM (white bars) or 20 mM (black bars) glucose. Results were calculated from the ratio of luciferase/Renilla activity. Background expression was subtracted using the mean expression level of empty pGL3-basic. Means ± SEM of results obtained from at least three independent transfections performed in triplicate are shown. *Significant differences, P<0.05. (B) Comparison of mRNA levels of NR2F2 and HNF4α relative to those of cyclophilin determined by real-time RT-QPCR in INS-1 832/13 β-cells stimulated with 5 mM (white bars) or 20 mM glucose (black bars) for 24 h as described in [3] (C) ChIP from INS-1 832/13 cells cultured in the presence of either 5 mM or 20 mM glucose. Targets for QPCR amplifications were the proximal NR2F2 promoter containing the DR1 DNA binding site or a downstream coding region (negative control). Amount of target chromatin precipitated by the HNF4α-specific antibody relative to that precipitated by control IgG (mean of three independent experiments ± SEM). *Significant difference, P<0.05. (D) COS cells were transiently co-transfected with the −328/+873 construct and 50 ng of empty KS vector (white bars), or HNF4α (grey bars) expression vectors. Means ± SEM of results from at least three separate transfections performed in triplicate are shown. *Significant difference, P<0.05. (E) INS-1 832/13 cells were transiently co-transfected with the wild-type or mutated −328/+873 constructs containing the DR1 elements. Cells were cultured in the presence of either 5 mM (white bars) or 20 mM (black bars) glucose. Means ± SEM of results from ten independent transfections performed in triplicate are shown. *Statistically significant differences in percentage of expression (where value for 5 mM glucose is 100% relative to −328/+873 construct) at P<0.05.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3351448&req=5

pone-0035810-g001: Glucose decreases the ability of HNF4α to activate the transcription of the NR2F2 proximal promoter and to bind its chromatin target.(A) INS-1 832/13 cells were transiently co-transfected as described in [7] with a luciferase reporter gene driven by various lengths of the NR2F2 promoter, designated by their 5′ and 3′ end positions relative to the defined NR2F2 gene transcription initiation site [25] and a control vector expressing Renilla luciferase. Cells were cultured in the presence of either 5 mM (white bars) or 20 mM (black bars) glucose. Results were calculated from the ratio of luciferase/Renilla activity. Background expression was subtracted using the mean expression level of empty pGL3-basic. Means ± SEM of results obtained from at least three independent transfections performed in triplicate are shown. *Significant differences, P<0.05. (B) Comparison of mRNA levels of NR2F2 and HNF4α relative to those of cyclophilin determined by real-time RT-QPCR in INS-1 832/13 β-cells stimulated with 5 mM (white bars) or 20 mM glucose (black bars) for 24 h as described in [3] (C) ChIP from INS-1 832/13 cells cultured in the presence of either 5 mM or 20 mM glucose. Targets for QPCR amplifications were the proximal NR2F2 promoter containing the DR1 DNA binding site or a downstream coding region (negative control). Amount of target chromatin precipitated by the HNF4α-specific antibody relative to that precipitated by control IgG (mean of three independent experiments ± SEM). *Significant difference, P<0.05. (D) COS cells were transiently co-transfected with the −328/+873 construct and 50 ng of empty KS vector (white bars), or HNF4α (grey bars) expression vectors. Means ± SEM of results from at least three separate transfections performed in triplicate are shown. *Significant difference, P<0.05. (E) INS-1 832/13 cells were transiently co-transfected with the wild-type or mutated −328/+873 constructs containing the DR1 elements. Cells were cultured in the presence of either 5 mM (white bars) or 20 mM (black bars) glucose. Means ± SEM of results from ten independent transfections performed in triplicate are shown. *Statistically significant differences in percentage of expression (where value for 5 mM glucose is 100% relative to −328/+873 construct) at P<0.05.
Mentions: To map the cis-regulatory elements responsible for glucose responsiveness in NR2F2, we transiently co-transfected luciferase plasmid reporters in a INS-1 832/13 β-cell line with various deleted sections of the NR2F2 regulatory region. This corresponds to a fragment of 4 kbp that encompasses the upstream NR2F2 regulatory region that was shown to be sufficient to direct NR2F2 expression in pancreatic β-cells [8]. In comparison with a concentration of 5 mM, a glucose concentration of 20 mM provoked a 45% reduction of luciferase activity (P<0.05) in INS-1 832/13 β-cells co-transfected with the full length NR2F2 promoter construct −3210/+873) (Fig. 1A). These measured impacts were consistent with previous observations on endogenous reduction of NR2F2 mRNA abundance by high glucose concentrations (Fig. 1B and [3]). The deletion analysis further established that the fragment from −328 to +873 was the minimal region required to confer a significant inhibition by high glucose concentrations (Fig. 1A). In this proximal promoter, we previously showed that HNF4α binds the conserved direct repeat-1 (DR-1) hormone response element (HRE) [7]. We also showed that HNF4α is able to activate the endogenous NR2F2 gene in INS-1 832/13 β-cells [7]. As shown in figure 1B, we observed the same reduction of HNF4α mRNA levels as observed for NR2F2 mRNA levels in the presence of high glucose levels in INS-1 cells suggesting that this transcription factor could be involved in the glucose responsiveness of NR2F2. As show in figure 1C, the antibodies for HNF4α immunoprecipitated with the promoter region of NR2F2, revealing the presence of endogenous HNF4α on this DNA binding site. A high glucose concentration (20 mM) in comparison to a concentration of 5 mM induced a significant reduction of immunoprecipitation with anti-HNF4α antibodies. HNF4α bound to the DR-1 DNA binding site in a glucose-dependent manner. In COS-7 cells, that lack endogenous expression of HNF4α, the co-transfection with an HNF4α expression vector and the −328/+873 luciferase reporter plasmid induced a 4-fold increase in luciferase activity (Fig. 1D). Moreover, when the DR-1 DNA binding site was mutated in the −328/+873 construct, we measured a 70% reduction in luciferase activity (Fig. 1E). This suggests that a nuclear receptor is a transactivator of the NR2F2 promoter in β-cells cultured in 5 mM glucose, a condition that allows maximal expression of NR2F2. Furthermore the mutations at the DR-1 binding site led to a significantly weaker repression by 20 mM glucose (Fig. 1E). Altogether these results suggest that HNF4α is a transcription factor required for inhibition of NR2F2 promoter activity by high concentrations of glucose. They also suggest a more complex pattern of regulation with additional factors involved in the inhibition of NR2F2 transcription activity by high concentrations of glucose.

Bottom Line: The present study aimed to identify the regulatory regions that control NR2F2 gene transcription and to evaluate the effect of NR2F2 promoter variation on glucose homeostasis in humans.The effects of variation at SNP rs3743462 in NR2F2 on quantitative metabolic traits were studied in two European prospective cohorts.The C-allele at rs3743462 was associated with increased NR2F2 binding and decreased NR2F2 gene expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Endocrinology, Metabolism and Cancer, Cochin Institute, CNRS (UMR 8104), Paris, France.

ABSTRACT

Background: The Nuclear Receptor 2F2 (NR2F2/COUP-TFII) heterozygous knockout mice display low basal insulinemia and enhanced insulin sensitivity. We previously established that insulin represses NR2F2 gene expression in pancreatic β-cells. The cis-regulatory region of the NR2F2 promoter is unknown and its influence on metabolism in humans is poorly understood. The present study aimed to identify the regulatory regions that control NR2F2 gene transcription and to evaluate the effect of NR2F2 promoter variation on glucose homeostasis in humans.

Methodology/principal findings: Regulation of the NR2F2 promoter was assessed using gene reporter assays, ChIP and gel shift experiments. The effects of variation at SNP rs3743462 in NR2F2 on quantitative metabolic traits were studied in two European prospective cohorts. We identified a minimal promoter region that down-regulates NR2F2 expression by attenuating HNF4α activation in response to high glucose concentrations. Subjects of the French DESIR population, who carried the rs3743462 T-to-C polymorphism, located in the distal glucose-responsive promoter, displayed lower basal insulin levels and lower HOMA-IR index. The C-allele at rs3743462 was associated with increased NR2F2 binding and decreased NR2F2 gene expression.

Conclusions/significance: The rs3743462 polymorphism affects glucose-responsive NR2F2 promoter regulation and thereby may influence whole-body insulin sensitivity, suggesting a role of NR2F2 in the control of glucose homeostasis in humans.

Show MeSH
Related in: MedlinePlus