Figure 1
MS4A12 promoter activity is governed by a single functional CDX binding element. (A) Proximal 5'-flanking region of the MS4A12 gene. Predicted CDX binding sites are shown in gray, orientation of the elements is depicted by arrows. The TATA box element is boxed, transcription start is underlined. (B) Activation of the MS4A12 promoter measured in luciferase activity assays. The MS4A12 promoter region (-952 to +12) was cloned into the basic pGL4 luciferase reporter vector (Promega). Variants containing mutated CDX binding sites were generated using QuickChange Site-Directed Mutagenesis Kit (Stratagene). 4 × 105 cells were seeded into 6-well plates and transfected with promoter constructs using Lipofectamine (Invitrogen). 24 h after transfection luciferase activity was measured in cell lysates and normalized to fluorescence obtained by co-transfection with eGFP reporter plasmid. All assays were done in triplicates; mean values +/- STD are shown. (C) Binding of CDX1 and CDX2 to the predicted binding site was assessed using NoShift Transcription Factor Assay Kit (Novagen) following the manufacturer's instructions. Biotinylated double-stranded oligonucleotides featuring the wild type (capturewt) (5'-cgt att cca aac ttt aca gtt gca ttt ac-3') or the mutated (capturemut) (5'-cgt att cca aac CCC aca gtt gca ttt ac-3') CDX consensus element in the MS4A12 promoter binding site, and non-biotinylated wild type (competitorwt) or mutated (competitormut) oligonucleotides, were incubated with nuclear extracts of LoVo cells, transferred to a streptavidin-coated 96-well plate and incubated for 1 h at 37°C. Antibodies specific for CDX1 and CDX2 (both from Abcam) were added to the samples and incubated for 1 h at 37°C. The secondary horseradish peroxidase-conjugated antibody was incubated for 30 min at 37°C. TMB substrate was added to the samples to develop colorimetric signals. The reaction was quenched with 1 N HCl and sample absorbance was read at 450 nm on a Wallac Victor2 multi-label counter (Perkin Elmer).