Biology subjects

Gagliani, E. K.

Publications and source records attributed to Gagliani, E. K..

3 recordsLinked to original sources

bioRxiv2025

A mouse model of Adams-Oliver Syndrome reveals defective Notch1 signaling in endothelial cells as a driver of pathogenesis

Adams-Oliver Syndrome (AOS) is a rare congenital disorder characterized by scalp, limb, and cardiovascular defects. While 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 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 key tissues driving AOS pathogenesis, we employed conditional genetics to isolate the contribution of the vasculature 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 defective Notch1 signaling in the vasculature is a key driver of pathogenesis in this AOS mouse model.

developmental biology↗

bioRxiv2023

Cooperative Gsx2-DNA Binding Requires DNA Bending and a Novel Gsx2 Homeodomain Interface

The conserved Gsx homeodomain (HD) transcription factors specify neural cell fates in animals from flies to mammals. Like many HD proteins, Gsx factors bind A/T-rich DNA sequences prompting the question -- how do HD factors that bind similar DNA sequences in vitro regulate specific target genes in vivo? Prior studies revealed that Gsx factors bind DNA both as a monomer on individual A/T-rich sites and as a cooperative homodimer to two sites spaced precisely seven base pairs apart. However, the mechanistic basis for Gsx DNA binding and cooperativity are poorly understood. Here, we used biochemical, biophysical, structural, and modeling approaches to (1) show that Gsx factors are monomers in solution and require DNA for cooperative complex formation; (2) define the affinity and thermodynamic binding parameters of Gsx2/DNA interactions; (3) solve a high-resolution monomer/DNA structure that reveals Gsx2 induces a 20{degrees} bend in DNA; (4) identify a Gsx2 protein-protein interface required for cooperative DNA binding; and (5) determine that flexible spacer DNA sequences enhance Gsx2 cooperativity on dimer sites. Altogether, our results provide a mechanistic basis for understanding the protein and DNA structural determinants that underlie cooperative DNA binding by Gsx factors, thereby providing a deeper understanding of HD specificity.

biochemistry↗

bioRxiv2021

A Drosophila Su(H) Model of Adams-Oliver Syndrome Reveals Notch Cofactor Titration as a Mechanism Underlying Developmental Defects

Notch signaling is a conserved pathway that converts extracellular receptor-ligand interactions into changes in gene expression via a single transcription factor (CBF1/RBPJ in mammals; Su(H) in Drosophila). In humans, RBPJ variants have been linked to Adams-Oliver syndrome (AOS), a rare autosomal dominant disorder characterized by scalp, cranium, and limb defects. Here, we found that a previously described Drosophila Su(H) allele encodes a missense mutation that alters an analogous residue found in an AOS-associated RBPJ variant. Importantly, genetic studies support a model that Drosophila with a single copy of the AOS-like Su(H) allele behave in an opposing manner as flies with a Su(H) null allele due to a dominant activity of sequestering either the Notch co-activator or the antagonistic Hairless co-repressor. Consistent with this model, AOS-like Su(H) and Rbpj variants decrease DNA binding activity compared to wild type proteins, but these variants do not significantly alter protein binding to the Notch co-activator or the fly and mammalian co-repressors, respectively. Taken together, these data suggest a cofactor sequestration mechanism underlies AOS phenotypes associated with RBPJ variants, whereby a single RBPJ allele encodes a protein with compromised DNA binding activity that retains cofactor binding, resulting in Notch target gene dysregulation.

genetics↗