Biology subjects

Roussos, C.

Publications and source records attributed to Roussos, C..

6 recordsLinked to original sources

bioRxiv2026

Transcription factor handover reshapes hub dynamics to coordinate temporal progression of Notch response

To decipher information in space and time, cells require mechanisms to sustain gene expression programmes once an initial signal decays. Localised condensation of transcription complexes into hubs is emerging as a key step in gene regulation and, by combining live imaging of transcription and of transcription complexes at target loci, we have investigated how these evolve to enable a prolonged transcription outcome in response to Notch signalling. Measuring the dynamics of hindsight (hnt) transcription reveals two phases. The first, a probabilistic response similar to E(spl)-C genes, is directly controlled by Notch and the second, a sustained response, is indirectly controlled through the BTB transcription factor Broad. The two phases correlate with a transition in transcription factor enrichment at hnt: complexes of Notch coactivator Mastermind and of Broad are successively enriched. Notably, the lifetime of the hubs formed by each are distinct and correlate with the change in transcription profiles. These reflect the binding dynamics of each complex, with Broad having significantly more stable association with chromatin, forming longer lasting hubs and yielding sustained transcription. Furthermore at intermediate stages we detect hubs containing both Broad and Mam implying an evolution in hub composition. Together our data support that the dynamic properties of transcription hubs are crucial for the probability and dynamics of transcription and point towards a ''hub handover'' in enabling temporal progression in gene expression responses.

developmental biology↗

bioRxiv2026

Notch mediated lateral inhibition is shaped by morphological differences to reinforce bias toward signal-sending or receiving roles

During neurogenesis, neuroblasts are selected from proneural-competent cells through lateral inhibition, a process controlled by the evolutionarily conserved Notch signalling pathway. By tracking transcription from Notch-target genes and cell morphologies in real time, we discovered that the presumptive neuroblast never initiates target-gene transcription. This implies a pre-existing bias directs Notch signalling. The bias correlates with a heterogeneity in apical cell areas which is further reinforced during neuroblast selection. Additionally, the length and duration of neuroblast-neighbour cell contacts predict the likelihood of transcription. Using mathematical modelling we show that lateral inhibition seeded with subtle morphological differences can bias cells toward signal-sending or receiving roles before transcriptional feedback occurs. Notch activation further alters apical cell area, reinforcing the initial bias. We propose that signalling and cell mechanics work together to ensure the robust selection of a single neural precursor.

developmental biology↗

bioRxiv2026

Pioneer-factor activity requires stable chromatin occupancy mediated by both sequence-specific binding and disordered protein domains

Pioneer transcription factors overcome the restrictive barrier imposed by chromatin to drive cell-fate specification, yet how their domains collectively support this activity remains unclear. Here, we use the deeply conserved pioneer factor Grainy head to define the protein-intrinsic features that govern pioneering activity. By integrating biochemistry, genomics and quantitative live-cell imaging, we determined that both the conserved DNA-binding domain and the extended, intrinsically disordered N-terminus are required for the stable chromatin occupancy that supports access to closed chromatin and the induction of chromatin accessibility. The disordered N-terminus supports pioneer activity through interactions that do not rely on strict amino acid sequence but instead overall composition. While our results show that pioneering activity depends on the combinatorial contributions of structured and disordered domains, mitotic retention depends solely on sequence-specific DNA binding. These results support stable chromatin occupancy mediated by multiple protein domains as necessary for pioneering function and that this is separable from the mechanisms required for mitotic retention.

molecular biology↗

bioRxiv2025

Direct observation of Notch signalling induced transcription hubs mediating gene-expression responses

Developmental decisions rely on cells making accurate transcriptional responses to signals they receive, as with Notch pathway activity. Local condensates or transcription factor hubs are a proposed mechanism for facilitating gene activation by nuclear complexes. To investigate their importance in endogenous Notch signalling, we deployed multi-colour live-imaging to measure Notch transcription-complex enrichment at a target gene locus in combination with the transcription dynamics. The co-activator Mastermind (Mam) was present in signalling-dependent nuclear foci, during Notch active developmental stages. Tracking these highly-dynamic Mam hubs together with transcription in the same nucleus, revealed that their condensation precedes and correlates with the profile of transcription and becomes stabilized if transcription is inhibited. Manipulations to signalling levels had concordant effects on hub intensities and transcription profiles, altering their probability and amplitude. Together the results argue that signalling induces the formation of transcription hubs whose properties are instrumental in the quantitative gene expression response to Notch activation.

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

Changes in searching behaviour of CSL transcription complexes in Notch active conditions

During development cells receive a variety of signals, which are of crucial importance to their fate determination. One such source of signal is the Notch signalling pathway, where Notch activity regulates expression of target genes through the core transcription factor CSL. To understand changes in transcription factor behaviour that lead to transcriptional changes in Notch active cells, we have probed CSL behaviours in real time, using in vivo Single Molecule Localisation Microscopy. Trajectory analysis reveals that Notch-On conditions increase the fraction of bound CSL molecules, but also the proportion of molecules with exploratory behaviours. These properties are shared by the co-activator Mastermind. Furthermore, both CSL and Mastermind, exhibit characteristics of local exploration near a Notch target locus. A similar behaviour is observed for CSL molecules diffusing in the vicinity of other bound CSL clusters. We suggest therefore that CSL acquires an exploratory behaviour when part of the activation complex, favouring local searching and retention close to its target enhancers. This change explains how CSL can efficiently increases its occupancy at target sites in Notch-ON conditions.

developmental biology↗