Fig. 1: An evolutionarily conserved composite element (CE) in the Madcam1 promoter integrates NKX2-3, COUP-TFII, and HEY1 to regulate transcription.

A Sequence of the CE in indicated species, and schematic of the CE. Conserved E-box (HEY1 binding site), homeodomain (NKX2-3 binding site) and COUP-TFII binding sites are highlighted in red, purple and green respectively. B Nkx2-3 or cooperative Nkx2-3 and Nr2f2 enhance TNFα stimulated luciferase reporter activity driven by CE-containing Madcam1 promoter (CE_NFκB-LUC) in 293T cells. Mutation of the homeodomain motif (in CE_ΔN-NFκB-LUC) inhibits activity. Data normalized to CE_NFκB-LUC activity. C HEY1 dose-dependently suppresses Nkx2-3 (light gray bars) or cooperative Nr2f2 and Nkx2-3 (dark gray bars) transcriptional activation from the CE-containing Madcam1 promoter in TNFα stimulated 293T cells. Data normalized to basal reporter activity without TNFα stimulation (dashed line). D Effects of mutation of COUP-TFII “A” (CE_ΔC_A-LUC) or “B” (CE_ΔC_B-LUC) sites on cooperative Nr2f2 and Nkx2-3 activation of luciferase reporter in 293T cells. Results were normalized to control CE-containing reporter activity. Vector contains three tandem copies of CE. E Induction of Madcam1 expression in TNFα stimulated bEnd.3 cells overexpressing Nkx2-3, evaluated by real-time PCR. Results are normalized to basal Madcam1 levels in bEnd.3 cells. F Knockdown of Nkx2-3 or Nr2f2 by shRNA inhibits Madcam1 expression in bEnd.3 cells. Data were normalized to β-actin and then to expression in cells transduced with control shRNA. G Endogenous Madcam1 expression in bEnd.3 cells or bEnd.3 cells stably transduced with Hey1 or DN-MAML, evaluated by real-time PCR. Results are normalized to basal Madcam1 in unstimulated bEnd.3 cells. Inset: immunofluorescence staining showing MAdCAM1 expression (green) in TNFα-stimulated bEnd.3 vs DN-MAML bEnd.3 cells. Scale bars: 20\(\mu\)m. H Upper panel: schematic of the Madcam1 promoter with (CE_NFκB-LUC) or without (NFκB-LUC) the CE. Lower panel: luciferase reporter activity driven by the Madcam1 promoter with (CE_NFκB-LUC; gray bars) or without (NFκB-LUC; white bars) the CE. CE_NFκB-LUC activity is normalized to the activity of NFκB-LUC. All TNFα-stimulations were done for 36 h. Values are mean ± SEM of three independent experiments unless stated otherwise. *: p value < 0.05; **: p value < 0.01; ***: p value < 0.005; ****: p value < 0.001. Two tailed t-test, paired.