Biology subjects

Cain, B.

Publications and source records attributed to Cain, B..

5 recordsLinked to original sources

bioRxiv2025

Decoding the mechanisms of cooperative DNA binding by the Paired-like homeodomain family

The 36 Paired-like homeodomain transcription factors are required for the development of many cell types, tissues, and organs as missense variants in 24 genes are associated with a variety of diseases and developmental disorders. How these factors identify distinct genomic targets using highly similar DNA binding domains is not fully understood. Here, we focus on determining how the Paired-like homeodomain factors gain DNA binding specificity by cooperatively binding palindromic sites spaced three base pairs apart (P3 site). Through structural, biochemical, and bioinformatic approaches, we define 11 rules that describe homeodomain residues that are critical, permissive, and inhibitory to cooperativity on the P3 site. Applying these rules, we successfully altered the cooperative behavior of Paired-like factors, identified residues that prevent the related Antennapedia class of homeodomains from binding cooperatively, and predict that thirty-eight disease-associated missense variants across ten Paired-like proteins alter cooperativity. Using quantitative DNA binding assays, we confirmed eleven of twelve of these disease-associated variants impact cooperativity but not DNA binding affinity. These findings reveal the importance of cooperativity in defining DNA binding specificity and highlight how missense variants associated with as many as fifteen different diseases can selectively disrupt cooperative DNA binding.

biochemistry↗

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↗

bioRxiv2024

Structural and biochemical characterization of the ALX4 dimer reveals novel insights into how disease alleles impact ALX4 function

1.How homeodomain proteins gain sufficient DNA binding specificity to regulate diverse processes has been a long-standing question. Here, we determine how the ALX4 Paired-like protein achieves DNA binding specificity for a TAAT - NNN - ATTA dimer site. We first show that ALX4 binds this motif independently of its co-factor, TWIST1, in cranial neural crest cells. Structural analysis identified seven ALX4 residues that participate in dimer binding, many of which are conserved across the Paired-like family, but not other homeodomain proteins. Unexpectedly, the two ALX4 proteins within the dimer use distinct residues to form asymmetric protein-protein and protein-DNA interactions to mediate cooperativity. Moreover, we found that ALX4 cooperativity is required for transcriptional activation and that ALX4 disease variants cause distinct molecular defects that include loss of cooperativity. These findings provide new insights into how Paired-like factors gain DNA specificity and show how disease variants can be stratified based on their molecular defects.

molecular 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↗

bioRxiv2019

Enhancer architecture sensitizes cell specific responses to Notch gene dose via a bind and discard mechanism

Notch pathway haploinsufficiency can cause severe developmental syndromes with highly variable penetrance. Currently, we have a limited mechanistic understanding of phenotype variability due to gene dosage. Here, we show that inserting a single enhancer containing pioneer transcription factor sites coupled to Notch dimer sites can unexpectedly induce a subset of Drosophila Notch haploinsufficiency phenotypes in an animal with wild type Notch gene dose. Mechanistically, this enhancer couples Notch DNA binding to degradation in a Cdk8-dependent, transcription-independent manner. Using mathematical modeling combined with quantitative trait and expression analysis, we show that tissues requiring long duration Notch signals are more sensitive to perturbations in Notch degradation compared to tissues relying upon short duration processes. These findings support a novel \"bind and discard\" mechanism in which enhancers with specific binding sites promote rapid Notch turnover, reduce Notch-dependent transcription at other loci, and thereby sensitize tissues to gene dose based upon signal duration.

developmental biology↗