The figure represents linkage disequilibrium based on the HapMap-CEU population data (Data Release 24/phase II Nov08, on NCBI B36 assembly, dbSNP b126) along the FOXE1 locus. The region spans chromosome 9 from coordinates 99,645,254 to 99,668,082 for a total length of 22.82 kilobases. The FOXE1 gene is represented in yellow (exon) and grey (5′ and 3′ untranslated regions). LD is measured by D'/LOD parameters (top panel) and the r-squared correlation value (bottom panel). D' and r-squared values of 1 mean complete LD. The region is tagged by the SNPs shown in the figure, all of which were highly significant in the phase I association study. The functional variant rs1867277 is shown in blue, and the tagSNP rs907577 is shown in red. Specific LD values between these two variants are shown in the figure (four-point star).

(A) Nuclear extracts were prepared from WRO cells for EMSA assays with ³²P-labeled oligonucleotides corresponding to rs1867277 A, rs1867277 G, or the consensus DRE derived from the prodynorphin promoter (DRE-pDYN). The probes were incubated without (lane 1) or with nuclear extracts (lanes 2 to 14). Competition was performed with a 100-fold molar excess of unlabeled related (lanes 3, 6, 9), unrelated (lanes 4, 7, 10,) or alleles A and G (lanes 11 and 12) oligonucleotides, as well as with an anti-DREAM antibody (lane 14). Protein/DNA complexes are indicated by arrows. (B) A ³²P-labeled probe containing the oligonucleotide corresponding to the rs1867277 A allele was incubated alone (lane 1); with WRO nuclear extracts (lanes 2-9); or with TNT translated proteins from empty vector (lane 10), or from USF1 or USF2 expression vectors (lanes 11, 12). Competition was performed with a 100-fold molar excess of unlabeled related (lane 3), unrelated (lane 4), allele G (lane 5), CRE-FOXE1 (lane 6), CRE consensus (lane 7), DRE-pDYN (lane 8), or USF consensus (lane 9) oligonucleotides. (C) A ³²P-labeled probe containing the oligonucleotide corresponding to the rs1867277 A allele was incubated alone (lanes 1, 9); with WRO nuclear extracts (lanes 2–6 and 10–14); or with TNT-translated USF1 (lanes 7, 8) or USF2 protein (lanes 15, 16). Competition was performed with a 100-fold molar excess of unlabeled USF consensus (lanes 3 and 11), unrelated (lanes 4 and 12), or allele G (lanes 5 and 13) oligonucleotides. In addition, supershift assays were performed with specific anti-USF1 (lanes 6 and 8) or anti-USF2 (lanes 14 and 16) antibodies. Protein/DNA complexes are indicated by arrows. The amount of proteins incubated with the probes was 7 µg of WRO nuclear extracts and 3 µl of TNT reaction for in vitro translated proteins.

(Top Panel) The A risk allele is shown in red and marked with an arrow. Nucleotides in blue indicate mismatches with regard to the DREAM consensus sequence, and gaps (indicated by dashes) are introduced into the sequences of both alleles to optimize alignment with the DREAM binding site. The DRE site derived from the human prodynorphin (h-pDYN) promoter is underlined. (Bottom Panel) Alignment of sequences close to the A and G alleles; consensus CRE- and USF-transcription factor binding sites are underlined. Bases in blue indicate differences with the consensus sequences.

(A) The FOXE1 promoter containing the A allele (pGl3b-FOXE1-A) or the G allele (pGl3b-FOXE1-283G) was cotransfected into HeLa cells with 3 µg of the empty expression vector, or with 3 µg of the vector harbouring the cDNAs for DREAM, USF1, USF2, or a dominant negative (DN) form of USF1. When USFs were cotransfected together, only 1.5 µg of each expression vector was used. Promoter activity is expressed as fold induction, relative to the activity observed in the presence of empty expression vector. (B) The FOXE1 promoter containing the A allele was cotransfected into HeLa cells with empty expression vectors or the same vector harbouring the cDNAs for α-CREM, CREB, USF1, USF2, or DREAM in the combinations shown in the figure. Numbers in the left hand panel represent µg of expression vector transfected; in the right hand panel, 3 µg of the indicated transcription factors were cotransfected. Promoter activity is expressed as fold induction, relative to the activity observed in the presence of empty (left) or α-CREM (right) expression vectors. Luciferase activity was normalized to Renilla activity derived from the cotransfected pRL-Tk vector to adjust for transfection efficiency. Results are mean±SD of four independent experiments. (*):P<0.05; (**):P = 0.01–0.001; (***):P<0.001; ns, not significant.