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1. Enhancers with cooperative Notch binding sites are more resistant to regulation by the Hairless co-repressor

   https://doi.org/10.1371/journal.pgen.1009039  

   Kuang, Yi; Pyo, Anna; Eafergan, Natanel; Cain, Brittany; Gutzwiller, Lisa M.; Axelrod, Ofri; Gagliani, Ellen K.; Weirauch, Matthew T.; Kopan, Raphael; Kovall, Rhett A.; et al (September 2021, PLOS Genetics)

   Aster, Jon Christopher (Ed.)

   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. 

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2. Conserved Gsx2/Ind homeodomain monomer versus homodimer DNA binding defines regulatory outcomes in flies and mice

   https://doi.org/10.1101/gad.343053.120  

   Salomone, Joseph; Qin, Shenyue; Fufa, Temesgen D.; Cain, Brittany; Farrow, Edward; Guan, Bin; Hufnagel, Robert B.; Nakafuku, Masato; Lim, Hee-Woong; Campbell, Kenneth; et al (January 2021, Genes & Development)

   null (Ed.)

   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. 

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3. Heterogeneous NF-κB activation and enhancer features shape transcription in Drosophila immunity

   https://doi.org/10.1101/2025.05.19.654881  

   Nawar, Noshin; Rits, Emma; Cohen, Lianne; Wunderlich, Zeba (May 2025, bioRxiv)

   Conserved NF-κB signaling pathways shape immune responses in animals. In mammals, NF-κB activation patterns and downstream transcription vary with stimulus, cell type, and stochastic differences among identically treated cells. Whether animals without adaptive immunity exhibit similar heterogeneity or rely on distinct immune strategies remains unknown. We engineered Drosophila melanogaster S2* reporter cells as an immune-responsive model to monitor the dynamics of an NF-κB transcription factor, Relish, and downstream transcription in single, living cells. Following immune stimulation, Relish exhibits diverse nuclear localization dynamics that fall into distinct categories, with both the fraction of responsive cells and their activation speed rising with stimulus dose. Pre-stimulus features, including Relish nuclear fraction, predict a cell's responsiveness to stimulation. Simultaneous measurement of Relish and downstream transcription revealed that the probability of transcriptional bursts from immune-responsive enhancers correlates with Relish nuclear fraction. The number of NF-κB binding sites tunes transcriptional activity among immune enhancers. Our study uncovers heterogeneity in NF-κB activation and target gene expression within Drosophila, illustrating how dynamic NF-κB behavior and enhancer architecture tune gene regulation. 

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4. A Drosophila Su(H) model of Adams-Oliver Syndrome reveals cofactor titration as a mechanism underlying developmental defects

   https://doi.org/10.1371/journal.pgen.1010335  

   Gagliani, Ellen K.; Gutzwiller, Lisa M.; Kuang, Yi; Odaka, Yoshinobu; Hoffmeister, Phillipp; Hauff, Stefanie; Turkiewicz, Aleksandra; Harding-Theobald, Emily; Dolph, Patrick J.; Borggrefe, Tilman; et al (August 2022, PLOS Genetics)

   Wappner, Pablo (Ed.)

   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. 

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5. Unveiling dynamic enhancer–promoter interactions in Drosophila melanogaster

   https://doi.org/10.1042/BST20220325  

   Deng, Hao; Jin, Gaochen; Lim, Bomyi (November 2022, Biochemical Society Transactions)

   Proper enhancer–promoter interactions are essential to maintaining specific transcriptional patterns and preventing ectopic gene expression. Drosophila is an ideal model organism to study transcriptional regulation due to extensively characterized regulatory regions and the ease of implementing new genetic and molecular techniques for quantitative analysis. The mechanisms of enhancer–promoter interactions have been investigated over a range of length scales. At a DNA level, compositions of both enhancer and promoter sequences affect transcriptional dynamics, including duration, amplitude, and frequency of transcriptional bursting. 3D chromatin topology is also important for proper enhancer–promoter contacts. By working competitively or cooperatively with one another, multiple, simultaneous enhancer–enhancer, enhancer–promoter, and promoter–promoter interactions often occur to maintain appropriate levels of mRNAs. For some long-range enhancer–promoter interactions, extra regulatory elements like insulators and tethering elements are required to promote proper interactions while blocking aberrant ones. This review provides an overview of our current understanding of the mechanism of enhancer–promoter interactions and how perturbations of such interactions affect transcription and subsequent physiological outcomes. 

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