Biology subjects

Bray, S. J.

Publications and source records attributed to Bray, S. J..

7 recordsLinked to original sources

bioRxiv2026

The Core Promoter has an active role in interpreting patterning information

Precise spatiotemporal control of gene expression is conventionally attributed to distal enhancers. The extent to which different core promoters actively shape transcriptional output from enhancers remains unclear, especially in the context of genes that contain alternate promoters. We have explored these questions using the Drosophila grainy head (grh) locus, and tested the activities conferred by its alternate promoters in combination with a well characterised Notch responsive enhancer. We find that, of the alternate promoters tested, only the grh short promoter is competent to drive neuroblast-specific expression. Notably, when paired with identical enhancers, grh-short and grh-medium promoters drive distinct expression patterns. The activity of other core promoters, such as hsp70, is also selective. Together the results establish that the core promoter acts alongside enhancers to confer specificity and robustness on gene expression profiles.

developmental biology↗

bioRxiv2023

Dynamic modes of Notch transcription hubs conferring memory and stochastic activation revealed by live imaging the co-activator Mastermind

Developmental programming involves the accurate conversion of signaling levels and dynamics to transcriptional outputs. The transcriptional relay in the Notch pathway relies on nuclear complexes containing the coactivator Mastermind (Mam). By tracking these complexes in real time, we reveal that they promote formation of a dynamic transcription hub in Notch ON nuclei which concentrates key factors including the Mediator CDK module. The composition of the hub is labile and persists after Notch withdrawal conferring a memory that enables rapid reformation. Surprisingly, only a third of Notch ON hubs progress to a state with nascent transcription, that correlates with Polymerase II and core Mediator recruitment. This probability is increased by a second signal. The discovery that target-gene transcription is probabilistic has far-reaching implications because it implies that stochastic differences in Notch pathway output can arise downstream of receptor activation.

developmental biology↗

bioRxiv2023

OptIC Notch reveals mechanism that regulates receptor interactions with CSL.

Active Notch signalling is elicited through receptor-ligand interactions that result in release of the Notch intracellular domain (NICD), which translocates into the nucleus. NICD activates transcription at target genes forming a complex with the DNA-binding transcription factor CSL (CBF1/Su(H)/Lag-1) and co-activator Mastermind. Despite this, CSL lacks its own nuclear localisation sequence, and it remains unclear where the tripartite complex is formed. To probe mechanisms involved, we designed an optogenetic approach to control NICD release (OptIC-Notch) and monitored consequences on complex formation and target gene activation. Strikingly we observed that, when uncleaved, OptIC-Notch sequestered CSL in the cytoplasm. Hypothesising that exposure of a juxta membrane {Phi}W{Phi}P motif is key to sequestration, we masked this motif with a second light sensitive domain in OptIC-Notch{{omega}}, which was sufficient to prevent CSL sequestration. Furthermore, NICD produced by light-induced cleavage of OptIC-Notch or OptIC-Notch{{omega}} chaperoned CSL into the nucleus and induced target gene expression, showing efficient light controlled activation. Our results demonstrate that exposure of the {Phi}W{Phi}P motif leads to CSL recruitment and suggest this can occur in the cytoplasm prior to nuclear entry. Summary statementLight controlled exposure of the Notch {Phi}W{Phi}P motif leads to CSL sequestration and suggest this interaction could occur prior to nuclear entry during normal signalling.

developmental biology↗

bioRxiv2021

Delta C2 Domain β1-2 loop contributes to robust Notch signalling

Accurate Notch signalling is critical for organism development and homeostasis. Fine-tuning of Notch-ligand interactions have substantial impact on signalling-outputs. Recent structural studies identified a conserved N-terminal C2 domain in human Notch ligands which conferred phospholipid binding in vitro. Here we show that Drosophila ligands adopt the same C2 domain structure with analogous variations in the loop regions, including the so-called {beta}1-2 loop that has been associated with phospholipid binding. Mutations in the {beta} 1-2 loop of Delta C2 domain retain Notch binding but have impaired ability to interact with phospholipids in vitro. To investigate its role in vivo we deleted five residues within the {beta} 1-2 loop of endogenous Delta by CRISPR/Cas9 gene editing. Strikingly, this change compromised ligand function. The modified Delta enhanced phenotypes produced by Delta loss of function alleles and suppressed that of Notch alleles. As the modified protein was present on the cell surface in normal amounts, these results argue that C2 domain phospholipid-binding is necessary for robust signalling in vivo where the balance of cis and trans ligand-receptor interactions is finely tuned.

developmental biology↗

bioRxiv2020

Loss of epithelial polarity redirects Notch signaling and triggers a Xrp1 response during neoplastic growth in Drosophila

Aggressive neoplastic growth can be initiated by a limited number of genetic alterations, such as the well-established cooperation between loss of cell architecture and hyperactive signaling pathways. However, our understanding of how these different alterations interact and influence each other remains very incomplete. Using Drosophila paradigms of imaginal wing disc epithelial growth, we have monitored the changes in Notch pathway activity according to the polarity status of cells (scrib mutant). We show that the scrib mutation impacts the direct transcriptional output of the Notch pathway, without altering the global distribution of Su(H), the Notch dedicated transcription factor. The Notch-dependent neoplasms require however, the action of a group of transcription factors, similar to those previously identified for Ras/scrib neoplasm (namely AP-1, Stat92E, Ftz-F1, and bZIP factors), further suggesting the importance of this transcription factor network during neoplastic growth. Finally our work highlights some Notch/scrib specificities, in particular the role of the PAR domain containing bZIP transcription factor and Notch direct target Pdp1 for neoplastic growth.

developmental biology↗

bioRxiv2019

Notch signaling coordinates ommatidial rotation in the Drosophila eye via transcriptional regulation of the EGF-Receptor ligand Argos

In all metazoans, a small number of evolutionarily conserved signaling pathways are reiteratively used during development to orchestrate critical patterning and morphogenetic processes. Among these, Notch (N) signaling is essential for most aspects of tissue patterning where it mediates the communication between adjacent cells to control cell fate specification. In Drosophila, Notch signaling is required for several features of eye development, including the R3/R4 cell fate choice and R7 specification. Here we show that hypomorphic alleles of Notch - belonging to the Nfacet class - reveal a novel phenotype: while photoreceptor specification in the mutant ommatidia is largely normal, defects are observed in ommatidial rotation (OR), a planar cell polarity (PCP)-mediated morphogenetic cell motility process. We demonstrate that during OR Notch signaling is specifically required in the R4 photoreceptor to upregulate the transcription of argos (aos), an inhibitory ligand to the EGFR, to fine-tune the activity of Egfr signaling. Consistently, the loss-of-function defects of Nfacet alleles and EGFR-signaling pathway mutants are largely indistinguishable. A Notch-regulated aos enhancer confers R4 specific expression arguing that aos is directly regulated by Notch signaling in this context via Su(H)- Mam dependent transcription.

developmental biology↗

bioRxiv2018

SWI/SNF chromatin remodeling controls Notch-responsive enhancer accessibility

Notch signaling plays a key role in many cell fate decisions during development by directing different gene expression programs via the transcription factor CSL, known as Su(H) in Drosophila. Which target genes are responsive to Notch signaling is influenced by the chromatin state of enhancers, yet how this is regulated is not fully known. Detecting an increase in the histone variant H3.3 in response to Notch signaling, we tested which chromatin remodelers or histone chaperones were required for the changes in enhancer accessibility to Su(H) binding. This revealed a crucial role for the Brahma SWI/SNF chromatin remodeling complex in conferring enhancer accessibility and enabling the transcriptional response. The Notch-responsive regions had high levels of nucleosome turnover which were dependent on the Brahma complex, increased with Notch signaling and primarily involved histone H3.3. Together these results highlight the importance of SWI/SNF-mediated nucleosome turnover in rendering enhancers responsive to Notch.

developmental biology↗