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.