An NKX-COUP-TFII morphogenetic code directs mucosal endothelial addressin expression

Abstract

Immunoglobulin family and carbohydrate vascular addressins encoded by Madcam1 and St6gal1 control lymphocyte homing into intestinal tissues, regulating immunity and inflammation. The addressins are developmentally programmed to decorate endothelial cells lining gut post-capillary and high endothelial venules (HEV), providing a prototypical example of organ- and segment-specific endothelial specialization. We identify conserved NKX-COUP-TFII composite elements (NCCE) in regulatory regions of Madcam1 and St6gal1 that bind intestinal homeodomain protein NKX2-3 cooperatively with venous nuclear receptor COUP-TFII to activate transcription. The Madcam1 element also integrates repressive signals from arterial/capillary Notch effectors. Pan-endothelial COUP-TFII overexpression induces ectopic addressin expression in NKX2-3⁺ capillaries, while NKX2-3 deficiency abrogates expression by HEV. Phylogenetically conserved NCCE are enriched in genes involved in neuron migration and morphogenesis of the heart, kidney, pancreas and other organs. Our results define an NKX-COUP-TFII morphogenetic code that targets expression of mucosal vascular addressins.

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.

Fig. 2. NKX2-3 and COUP-TFII heterodimerize via the tinman (TN) domain to activate MAdCAM1.

A Sequence of wildtype and mutant CE probes for EMSA. B EMSA showing migration of recombinant NKX2-3 and COUP-TFII bound to the CE probe (left) and to CE_ΔC_A probe that lacks the COUP-TFII “A” site (right). Co-incubation of NKX2-3 and COUP-TFII yields a distinct band (blue arrows) reflecting an NKX2-3:COUP-TFII DNA complex that is supershifted by anti-NKX2-3 antibody but not by control antibody. C EMSA of protein complexes formed by incubation of the indicated TFs with a mutant CE probe lacking the NKX2-3 and the COUP-TFII “A” binding sites (CE_ΔN_ΔC_A). The probe binds COUP-TFII (lane 7) and not NKX2-3 (lane 2), but seeds a COUP-TFII:NKX2-3:DNA complex (blue arrow) when co-incubated with both TFs. The heterodimer-DNA complex is supershifted by anti-NKX2-3 antibody (red star). NKX2-3^ΔTN failed to heterodimerize with COUP-TFII (lane 6). D Disruption of the NKX2-3:COUP-TFII heterodimer-DNA complex by a competing NKX2-3 TN domain peptide (WT) or by a scrambled peptide (SP). Data are expressed as percent of probe bound by NKX2-3:COUP-TFII heterodimer. E Madcam1 expression in TNFα-treated DN-MAML bEnd.3 cells stably overexpressing Nkx2-3 or the Nkx2-3 TN domain, as evaluated by real-time PCR. Values are mean ± SEM of three biological replicates. Two tailed t-test, paired. **: p value < 0.01. F Proximity ligation assay of NKX2-3 and COUP-TFII in TNFα-treated DN-MAML bEnd.3 cells stained for MAdCAM1 (blue) and ERG (green), with the proximity ligation signal shown in red. Inset shows a negative control with isotype-matched primary antibodies. Scale bar: 20$\mu$m.

Fig. 3. NKX2-3 regulates St6gal1 expression via a conserved NCCE.

A Schematic of the St6gal1 gene and an NCCE in the second intron. B EMSA showing migration of NKX2-3, COUP-TFII-GST (red arrows), and NKX2-3:COUP-TFII heterodimer (blue arrow) bound to an St6gal1 CE probe. C St6gal1 expression in DN-MAML bEnd.3 cells stably transfected with Nkx2-3 or Nr2f2 shRNA, evaluated by real-time PCR. D St6gal1 expression in DN-MAML bEnd.3 cells stably transfected with NKX2-3 or the NKX2-3 TN domain. E Activity of luciferase reporter driven by St6gal1 NCCE-containing promoter (St6Gal1-LUC; gray bars) or St6gal1 NCCE-containing promoter with mutated COUP-TFII binding sequence (St6Gal1_ΔCOUP-LUC; white bars), when co-transfected with Nr2f2 and/or Nkx2-3 in 293T cells. F Immunofluorescence of alpha-2,6-sialic acid binding lectin SNA staining in PP HEV in WT vs Nkx2-3 deficient mice (red). HEV are marked by AF450-labeled anti-addressin antibodies MECA79, MECA89, and MECA367. Autofluorescence is shown in green. Scale bars: 50 µm. Results in C-E are shown as mean ± SEM from three independent transfections. Two tailed t-test, paired. *: p value<0.05; **: p value<0.01; ***: p value < 0.005.

Fig. 4. Pan-endothelial COUP-TFII expression induces ectopic Madcam1 and St6gal1 in intestinal capillaries.

A Experimental timeline for induction of COUP-TFII in EC. B UMAP plot of PP BEC colored by EC subset (left) or by genotype (right). C Violin plots showing expression of select genes in EC-iCOUP^OE vs control BEC subsets. Dots represent mean gene expression of separate cohorts (male and female cohorts from two independent experiments), and are presented with SEM. D Confocal imaging showing MAdCAM1 expression in lamina propria capillaries in EC-iCOUP^OE (arrowheads) but not control (arrows) mice. Scale bars: 50 µm, EC stained by i.v. injection of directly conjugated antibodies. 20×, whole mount. See also Supplementary Fig. 6A for MAdCAM1 extension into capillary segments in PP.

Fig. 5. Genome-wide NCCE are associated with organ morphogenesis.

A Select GO terms enriched in NCCE+ genes compared to controls. NCCE+ genes are defined as genes nearest to conserved NCCE, where the NCCE are defined as having a 6–16 bp spacing between the centers of a COUP-TFII and an NKX HD motif. Control 1: genes associated with conserved COUP-TFII and NKX HD motifs separated by larger spacings (30–60 bp). Controls 2–4: genes associated with conserved NKX motifs 6–16 bp from conserved scrambled motifs GTAC(G/C), AGTC(G/C), or TGGA(C/T), respectively. B NCCE+ genes in select pathways associated with heart development, neuronal functions, and organ morphogenesis.