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Townson, J. M.

Publications and source records attributed to Townson, J. M..

2 recordsLinked to original sources
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↗