Limits...
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.

Show MeSH

Related in: MedlinePlus

NR2F2 binds the variant rs3743462-C oligonucleotide with higher affinity than the rs3743462-T oligonucleotide and the C-allele is associated with a strong decrease of NR2F2 gene expression relative to the T-allele.(A) Multiple alignments of the genomic region between nucleotides −3180 and −3110 of the NR2F2 gene regulatory regions present in the −3210/+873 construct. Deletion is indicated by dashes and points indicate identities. The sequence of the human complementary strand is shown above other sequences. Genomic sequences can be retrieved from GenBank by their accession codes: Homo sapiens (NT_010274.17/:11836273-11840385), Mus musculus (NT_039428.7/Mm7_39468_37:c10507606-10503510; reverse/complementary strand), Rattus norvegicus (NW_047560.2/Rn1_WGA2082_4:c5641306-5636690; reverse/complementary strand). The position of the human SNP is indicated by an asteriskabove the sequences of each species: H. sapiens, −3,138; M. musculus, −3,139; R. norvegicus, −3,152 (where transcription start site is +1). (B) The sense strand sequences (+) of the oligonucleotides used in EMSA are shown. SNP base pairs are shown in lower case letters. (C) The labeled rs3743462-T and rs3743462-C oligonucleotides were incubated with INS-1 832/13 nuclear extracts, and protein binding was analyzed using EMSA. In the representative autoradiograph shown, only the retarded complexes are visible and not the free probe, which was in excess. (D) Comparison of the affinity of protein binding to the rs3743462-T and rs3743462-C variants. The labeled rs3743462-C oligonucleotide (Fig. 2B) was incubated with or without the indicated molar excess of unlabeled rs3743462-T or rs3743462-C oligonucleotide as competitors before addition of INS-1 832/13 nuclear extract. Protein binding was then analyzed using EMSA. In the representative autoradiograph shown, only the retarded complexes are visible and not the free probe, which was in excess. (E) INS-1 832/13 nuclear extracts were incubated with or without of the indicated anti-serum. The labeled oligonucleotide representing the −3153/−3126 NR2F2 regulatory region and containing the rs3743462-C allele was added and protein binding was analyzed using EMSA. In the representative autoradiograph shown, only the retarded complexes are visible and not the free probe, which was in excess. (F) Functional analysis of the rs3743462 alleles in pancreatic β-cells. INS-1 832/13 cells were transiently co-transfected using lipofectamine solution containing either rs3743462 T-allele, C-allele, T-allele with DR-1 mutated site or C-allele with DR-1 mutated site within the context of the 3210/+873 sequences (1.5 µg) and expression vector encoding Renilla luciferase (0.1mg). Cells were then cultured in the presence of 5 mM glucose for 14 h. Results are calculated from the ratio of luciferase/Renilla activity. Means ± SEM of results obtained from at least three independent transfections performed in triplicate are shown. *Significant differences in expression at P<0.05.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3351448&req=5

pone-0035810-g002: NR2F2 binds the variant rs3743462-C oligonucleotide with higher affinity than the rs3743462-T oligonucleotide and the C-allele is associated with a strong decrease of NR2F2 gene expression relative to the T-allele.(A) Multiple alignments of the genomic region between nucleotides −3180 and −3110 of the NR2F2 gene regulatory regions present in the −3210/+873 construct. Deletion is indicated by dashes and points indicate identities. The sequence of the human complementary strand is shown above other sequences. Genomic sequences can be retrieved from GenBank by their accession codes: Homo sapiens (NT_010274.17/:11836273-11840385), Mus musculus (NT_039428.7/Mm7_39468_37:c10507606-10503510; reverse/complementary strand), Rattus norvegicus (NW_047560.2/Rn1_WGA2082_4:c5641306-5636690; reverse/complementary strand). The position of the human SNP is indicated by an asteriskabove the sequences of each species: H. sapiens, −3,138; M. musculus, −3,139; R. norvegicus, −3,152 (where transcription start site is +1). (B) The sense strand sequences (+) of the oligonucleotides used in EMSA are shown. SNP base pairs are shown in lower case letters. (C) The labeled rs3743462-T and rs3743462-C oligonucleotides were incubated with INS-1 832/13 nuclear extracts, and protein binding was analyzed using EMSA. In the representative autoradiograph shown, only the retarded complexes are visible and not the free probe, which was in excess. (D) Comparison of the affinity of protein binding to the rs3743462-T and rs3743462-C variants. The labeled rs3743462-C oligonucleotide (Fig. 2B) was incubated with or without the indicated molar excess of unlabeled rs3743462-T or rs3743462-C oligonucleotide as competitors before addition of INS-1 832/13 nuclear extract. Protein binding was then analyzed using EMSA. In the representative autoradiograph shown, only the retarded complexes are visible and not the free probe, which was in excess. (E) INS-1 832/13 nuclear extracts were incubated with or without of the indicated anti-serum. The labeled oligonucleotide representing the −3153/−3126 NR2F2 regulatory region and containing the rs3743462-C allele was added and protein binding was analyzed using EMSA. In the representative autoradiograph shown, only the retarded complexes are visible and not the free probe, which was in excess. (F) Functional analysis of the rs3743462 alleles in pancreatic β-cells. INS-1 832/13 cells were transiently co-transfected using lipofectamine solution containing either rs3743462 T-allele, C-allele, T-allele with DR-1 mutated site or C-allele with DR-1 mutated site within the context of the 3210/+873 sequences (1.5 µg) and expression vector encoding Renilla luciferase (0.1mg). Cells were then cultured in the presence of 5 mM glucose for 14 h. Results are calculated from the ratio of luciferase/Renilla activity. Means ± SEM of results obtained from at least three independent transfections performed in triplicate are shown. *Significant differences in expression at P<0.05.

Mentions: In order to assess part of the functional basis of NR2F2 in relation to glucose homeostasis in humans, we have tested the hypothesis that variations in the allelic distribution of common SNPs at the NR2F2 locus impact glucose metabolism-related quantitative traits. Three common SNPs (rs3743462, rs1807198 and rs11045, with a minor allele frequency (MAF) >0.10), located in a 12.5-kbp genomic interval at the NR2F2 locus on human chromosome 15q26, were analyzed as previously described [9]. We first measured their association in a subset of 654 normoglycemic non-obese individuals selected from the prospective DESIR cohort [10]. Only one SNP, rs3743462, showed trends of association with lower fasting insulin plasma concentrations, lower indices of basal insulin secretion (HOMA-B) and insulin resistance (HOMA-IR) (P≤0.002). This gene variant is located in an upstream regulatory region of the NR2F2 gene, at −3,138 bp from the transcription start site (Fig. 2A) and the effect allele is characterized by the substitution of a thymidine by a cytosine. No strong linkage disequilibrium (LD) (r2<0.50 from the HapMap3 database) was seen between rs3743462 and 14 other common SNPs present over a 500 kbp region encompassing the NR2F2 locus.


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)

NR2F2 binds the variant rs3743462-C oligonucleotide with higher affinity than the rs3743462-T oligonucleotide and the C-allele is associated with a strong decrease of NR2F2 gene expression relative to the T-allele.(A) Multiple alignments of the genomic region between nucleotides −3180 and −3110 of the NR2F2 gene regulatory regions present in the −3210/+873 construct. Deletion is indicated by dashes and points indicate identities. The sequence of the human complementary strand is shown above other sequences. Genomic sequences can be retrieved from GenBank by their accession codes: Homo sapiens (NT_010274.17/:11836273-11840385), Mus musculus (NT_039428.7/Mm7_39468_37:c10507606-10503510; reverse/complementary strand), Rattus norvegicus (NW_047560.2/Rn1_WGA2082_4:c5641306-5636690; reverse/complementary strand). The position of the human SNP is indicated by an asteriskabove the sequences of each species: H. sapiens, −3,138; M. musculus, −3,139; R. norvegicus, −3,152 (where transcription start site is +1). (B) The sense strand sequences (+) of the oligonucleotides used in EMSA are shown. SNP base pairs are shown in lower case letters. (C) The labeled rs3743462-T and rs3743462-C oligonucleotides were incubated with INS-1 832/13 nuclear extracts, and protein binding was analyzed using EMSA. In the representative autoradiograph shown, only the retarded complexes are visible and not the free probe, which was in excess. (D) Comparison of the affinity of protein binding to the rs3743462-T and rs3743462-C variants. The labeled rs3743462-C oligonucleotide (Fig. 2B) was incubated with or without the indicated molar excess of unlabeled rs3743462-T or rs3743462-C oligonucleotide as competitors before addition of INS-1 832/13 nuclear extract. Protein binding was then analyzed using EMSA. In the representative autoradiograph shown, only the retarded complexes are visible and not the free probe, which was in excess. (E) INS-1 832/13 nuclear extracts were incubated with or without of the indicated anti-serum. The labeled oligonucleotide representing the −3153/−3126 NR2F2 regulatory region and containing the rs3743462-C allele was added and protein binding was analyzed using EMSA. In the representative autoradiograph shown, only the retarded complexes are visible and not the free probe, which was in excess. (F) Functional analysis of the rs3743462 alleles in pancreatic β-cells. INS-1 832/13 cells were transiently co-transfected using lipofectamine solution containing either rs3743462 T-allele, C-allele, T-allele with DR-1 mutated site or C-allele with DR-1 mutated site within the context of the 3210/+873 sequences (1.5 µg) and expression vector encoding Renilla luciferase (0.1mg). Cells were then cultured in the presence of 5 mM glucose for 14 h. Results are calculated from the ratio of luciferase/Renilla activity. Means ± SEM of results obtained from at least three independent transfections performed in triplicate are shown. *Significant differences in expression at P<0.05.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0035810-g002: NR2F2 binds the variant rs3743462-C oligonucleotide with higher affinity than the rs3743462-T oligonucleotide and the C-allele is associated with a strong decrease of NR2F2 gene expression relative to the T-allele.(A) Multiple alignments of the genomic region between nucleotides −3180 and −3110 of the NR2F2 gene regulatory regions present in the −3210/+873 construct. Deletion is indicated by dashes and points indicate identities. The sequence of the human complementary strand is shown above other sequences. Genomic sequences can be retrieved from GenBank by their accession codes: Homo sapiens (NT_010274.17/:11836273-11840385), Mus musculus (NT_039428.7/Mm7_39468_37:c10507606-10503510; reverse/complementary strand), Rattus norvegicus (NW_047560.2/Rn1_WGA2082_4:c5641306-5636690; reverse/complementary strand). The position of the human SNP is indicated by an asteriskabove the sequences of each species: H. sapiens, −3,138; M. musculus, −3,139; R. norvegicus, −3,152 (where transcription start site is +1). (B) The sense strand sequences (+) of the oligonucleotides used in EMSA are shown. SNP base pairs are shown in lower case letters. (C) The labeled rs3743462-T and rs3743462-C oligonucleotides were incubated with INS-1 832/13 nuclear extracts, and protein binding was analyzed using EMSA. In the representative autoradiograph shown, only the retarded complexes are visible and not the free probe, which was in excess. (D) Comparison of the affinity of protein binding to the rs3743462-T and rs3743462-C variants. The labeled rs3743462-C oligonucleotide (Fig. 2B) was incubated with or without the indicated molar excess of unlabeled rs3743462-T or rs3743462-C oligonucleotide as competitors before addition of INS-1 832/13 nuclear extract. Protein binding was then analyzed using EMSA. In the representative autoradiograph shown, only the retarded complexes are visible and not the free probe, which was in excess. (E) INS-1 832/13 nuclear extracts were incubated with or without of the indicated anti-serum. The labeled oligonucleotide representing the −3153/−3126 NR2F2 regulatory region and containing the rs3743462-C allele was added and protein binding was analyzed using EMSA. In the representative autoradiograph shown, only the retarded complexes are visible and not the free probe, which was in excess. (F) Functional analysis of the rs3743462 alleles in pancreatic β-cells. INS-1 832/13 cells were transiently co-transfected using lipofectamine solution containing either rs3743462 T-allele, C-allele, T-allele with DR-1 mutated site or C-allele with DR-1 mutated site within the context of the 3210/+873 sequences (1.5 µg) and expression vector encoding Renilla luciferase (0.1mg). Cells were then cultured in the presence of 5 mM glucose for 14 h. Results are calculated from the ratio of luciferase/Renilla activity. Means ± SEM of results obtained from at least three independent transfections performed in triplicate are shown. *Significant differences in expression at P<0.05.
Mentions: In order to assess part of the functional basis of NR2F2 in relation to glucose homeostasis in humans, we have tested the hypothesis that variations in the allelic distribution of common SNPs at the NR2F2 locus impact glucose metabolism-related quantitative traits. Three common SNPs (rs3743462, rs1807198 and rs11045, with a minor allele frequency (MAF) >0.10), located in a 12.5-kbp genomic interval at the NR2F2 locus on human chromosome 15q26, were analyzed as previously described [9]. We first measured their association in a subset of 654 normoglycemic non-obese individuals selected from the prospective DESIR cohort [10]. Only one SNP, rs3743462, showed trends of association with lower fasting insulin plasma concentrations, lower indices of basal insulin secretion (HOMA-B) and insulin resistance (HOMA-IR) (P≤0.002). This gene variant is located in an upstream regulatory region of the NR2F2 gene, at −3,138 bp from the transcription start site (Fig. 2A) and the effect allele is characterized by the substitution of a thymidine by a cytosine. No strong linkage disequilibrium (LD) (r2<0.50 from the HapMap3 database) was seen between rs3743462 and 14 other common SNPs present over a 500 kbp region encompassing the NR2F2 locus.

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