Defective Notch1 signaling in endothelial cells drives pathogenesis in a mouse model of Adams-Oliver syndrome
Alyssa F. Solano, Kristina Preusse, Brittany Cain, Rebecca Hotz, Parthav Gavini, Zhenyu Yuan, Benjamin Bowen, Gabrielle Maco, Hope Neal, Ellen K. Gagliani, Christopher Ahn, Hee-Woong Lim, Laura Southgate, Rhett A. Kovall, Raphael Kopan, Brian Gebelein
Alyssa F. Solano, Kristina Preusse, Brittany Cain, Rebecca Hotz, Parthav Gavini, Zhenyu Yuan, Benjamin Bowen, Gabrielle Maco, Hope Neal, Ellen K. Gagliani, Christopher Ahn, Hee-Woong Lim, Laura Southgate, Rhett A. Kovall, Raphael Kopan, Brian Gebelein
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Research Article Development Vascular biology

Defective Notch1 signaling in endothelial cells drives pathogenesis in a mouse model of Adams-Oliver syndrome

Abstract

Adams-Oliver syndrome (AOS) is a rare congenital disorder characterized by scalp, limb, and cardiovascular defects. Although variants in the NOTCH1 receptor, DLL4 ligand, and RBPJ transcription factor have been implicated in AOS, the driving tissue types and molecular mechanisms by which these variants cause pathogenesis are unknown. Here, we used quantitative binding assays to show that AOS-associated RBPJ missense variants compromise DNA binding but not cofactor binding. These findings suggest that AOS-associated RBPJ variants do not function as loss-of-function alleles but instead act as dominant-negative proteins that sequester cofactors from DNA. Consistent with this idea, mice carrying an AOS-associated Rbpj allele develop dominant phenotypes that include increased lethality and cardiovascular defects in a Notch1 heterozygous background, whereas Notch1 and Rbpj compound heterozygous null alleles are well tolerated. To facilitate studies into the tissues driving AOS pathogenesis, we employed conditional genetics to isolate the contribution of the vascular endothelium to the development of AOS-like phenotypes. Importantly, our studies show that expression of the Rbpj AOS allele in endothelial cells is both necessary and sufficient to cause lethality and cardiovascular defects. These data establish that reduced Notch1 signaling in the vasculature is a key driver of pathogenesis in this AOS mouse model.

Authors

Alyssa F. Solano, Kristina Preusse, Brittany Cain, Rebecca Hotz, Parthav Gavini, Zhenyu Yuan, Benjamin Bowen, Gabrielle Maco, Hope Neal, Ellen K. Gagliani, Christopher Ahn, Hee-Woong Lim, Laura Southgate, Rhett A. Kovall, Raphael Kopan, Brian Gebelein

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Figure 1

AOS-associated RBPJ variants impair DNA binding.

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AOS-associated RBPJ variants impair DNA binding. (A) Domain map and sequ...
(A) Domain map and sequence alignment of RBPJ orthologs. Conserved residues are highlighted, and AOS-associated variants (*) are denoted by human (blue) and mouse (orange) residue numbers. Black bars indicate DNA-binding regions. NTD = N-terminal domain. BTD = beta-trefoil domain. CTD = C-terminal domain. (Created in BioRender.) (B) Structure of RBPJ on DNA with AOS-associated residue changes denoted by human (blue) and mouse (orange) numbers. (C–H) PyMOL models of structural changes and representative comparative EMSAs of AOS-associated RBPJ variants. Dashed lines within each model denote DNA-residue or residue-residue polar interactions, and red discs indicate steric clash. EMSAs were performed using equimolar concentrations (5, 25, and 125 nM) of WT mouse RBPJ and the R91G (C), K195E (D), E89G (E), Y86C (F), F92V (G), and S358R (H) variants on a DNA probe encoding a high-affinity RBPJ binding site. (I) Graph quantifying the probe depletion for each variant across triplicate EMSAs (see Supplemental Figure 1). A 1-way ANOVA with Tukey’s post hoc correction was used to compare WT RBPJ with each variant. (J) Tabulated ITC data measuring DNA binding affinity of RBPJ variants. Fold-change calculated relative to WT RBPJ. A 2-tailed t test was used to compare KD of WT RBPJ to each variant. *P < 0.05, **P < 0.01, ****P < 0.0001. NS, not significant. N/A, not applicable.