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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 heterozygous Drosophila with the AOS-like Su(H) allele behave in an opposing manner to heterozygous 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 have decreased 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 the AOS-associated RBPJ allele encodes a protein with compromised DNA binding activity that retains cofactor binding, resulting in Notch target gene dysregulation. 

Notch signaling controls many developmental processes by regulating gene expression. Notch-dependent enhancers recruit activation complexes consisting of the Notch intracellular domain, the C bf/ S u(H)/ L ag1 (CSL) transcription factor (TF), and the Mastermind co-factor via two types of DNA sites: monomeric CSL sites and cooperative dimer sites called S u(H) p aired s ites (SPS). Intriguingly, the CSL TF can also bind co-repressors to negatively regulate transcription via these same sites. Here, we tested how synthetic enhancers with monomeric CSL sites versus dimeric SPSs bind Drosophila Su(H) complexes in vitro and mediate transcriptional outcomes in vivo . Our findings reveal that while the Su(H)/Hairless co-repressor complex similarly binds SPS and CSL sites in an additive manner, the Notch activation complex binds SPSs, but not CSL sites, in a cooperative manner. Moreover, transgenic reporters with SPSs mediate stronger, more consistent transcription and are more resistant to increased Hairless co-repressor expression compared to reporters with the same number of CSL sites. These findings support a model in which SPS containing enhancers preferentially recruit cooperative Notch activation complexes over Hairless repression complexes to ensure consistent target gene activation. 

How homeodomain proteins gain sufficient specificity to control different cell fates has been a long-standing problem in developmental biology. The conserved Gsx homeodomain proteins regulate specific aspects of neural development in animals from flies to mammals, and yet they belong to a large transcription factor family that bind nearly identical DNA sequences in vitro. Here, we show that the mouse and fly Gsx factors unexpectedly gain DNA binding specificity by forming cooperative homodimers on precisely spaced and oriented DNA sites. High-resolution genomic binding assays revealed that Gsx2 binds both monomer and homodimer sites in the developing mouse ventral telencephalon. Importantly, reporter assays showed that Gsx2 mediates opposing outcomes in a DNA binding site-dependent manner: Monomer Gsx2 binding represses transcription, whereas homodimer binding stimulates gene expression. In Drosophila , the Gsx homolog, Ind, similarly represses or stimulates transcription in a site-dependent manner via an autoregulatory enhancer containing a combination of monomer and homodimer sites. Integrating these findings, we test a model showing how the homodimer to monomer site ratio and the Gsx protein levels defines gene up-regulation versus down-regulation. Altogether, these data serve as a new paradigm for how cooperative homeodomain transcription factor binding can increase target specificity and alter regulatory outcomes. 

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 unexpectedly found that inserting an enhancer containing pioneer transcription factor sites coupled to Notch dimer sites can induce a subset of Notch haploinsufficiency phenotypes in Drosophila with wild type Notch gene dose. Using Drosophila genetics, we show that this enhancer induces Notch phenotypes in a Cdk8-dependent, transcription-independent manner. We further combined mathematical modeling with quantitative trait and expression analysis to build a model that describes how changes in Notch signal production versus degradation differentially impact cellular outcomes that require long versus short signal duration. Altogether, these findings support a ‘bind and discard’ mechanism in which enhancers with specific binding sites promote rapid Cdk8-dependent Notch turnover, and thereby reduce Notch-dependent transcription at other loci and sensitize tissues to gene dose based upon signal duration.