Activation of the Notch Signaling Pathway In Vivo Elicits Changes in CSL Nuclear Dynamics

Abstract

SummaryA key feature of Notch signaling is that it directs immediate changes in transcription via the DNA-binding factor CSL, switching it from repression to activation. How Notch generates both a sensitive and accurate response—in the absence of any amplification step—remains to be elucidated. To address this question, we developed real-time analysis of CSL dynamics including single-molecule tracking in vivo. In Notch-OFF nuclei, a small proportion of CSL molecules transiently binds DNA, while in Notch-ON conditions CSL recruitment increases dramatically at target loci, where complexes have longer dwell times conferred by the Notch co-activator Mastermind. Surprisingly, recruitment of CSL-related corepressors also increases in Notch-ON conditions, revealing that Notch induces cooperative or “assisted” loading by promoting local increase in chromatin accessibility. Thus, in vivo Notch activity triggers changes in CSL dwell times and chromatin accessibility, which we propose confer sensitivity to small input changes and facilitate timely shut-down.

Decoding the Notch signal

“…Firstly, the evidence indicates a much more dynamic association of CSL with DNA. The CSL-DNA binding affinity appears to be lower than previously measured (Friedmann & Kovall, 2010) and CSL kinetics in Notch off conditions, based on live-imaging experiments, are very dynamic with estimated residence at target sites of circa 1 s ( Figure 2b; Gomez-Lamarca et al, 2018). Second, in vitro measurements revealed that co-repressors and NICD have similar affinities for CSL, or even in some cases repressors have a higher affinity (Collins et al, 2014;Vanderwielen, Yuan, Friedmann, & Kovall, 2011;Yuan et al, 2016), suggesting that a simple subunit exchange would not be thermodynamically favorable, although it remains possible that other factors or protein modifications could modify the protein behaviors.…”

“…The CSL-DNA binding affinity appears to be lower than previously measured(Friedmann & Kovall, 2010)Gomez-Lamarca et al, 2018). In this model, CSL remained stably bound to DNA while the NICD exchange converted it from repression to activation.…”

“…A hint that NICD could promote the formation of hubs comes from the observation, in live imaging experiments, that CSL becomes concentrated around a target locus in vivo(Figure 2a,b;Gomez-Lamarca et al, 2018). A hint that NICD could promote the formation of hubs comes from the observation, in live imaging experiments, that CSL becomes concentrated around a target locus in vivo(Figure 2a,b;Gomez-Lamarca et al, 2018).…”

“…A hint that NICD could promote the formation of hubs comes from the observation, in live imaging experiments, that CSL becomes concentrated around a target locus in vivo(Figure 2a,b;Gomez-Lamarca et al, 2018). In these conditions, CSL binding to DNA remains highly dynamic(Gomez-Lamarca et al, 2018) suggesting that, if the concentration of NICD determines PolII/Med cluster sizes, it must do so via a transient interaction. Interestingly, all TF hubs analyzed to date remain highly dynamic and only transiently interact with nascent sites of transcription, suggesting a "hit and run" mechanism for initiating transcription(Mir et al, 2018), rather than a model where TFs become integrated with stable PolII/Med clusters.…”

Optogenetic inhibition of Delta reveals digital Notch signalling output during tissue differentiation

“…Moreover, once cleaved, NICD molecules move into the nucleus within minutes [4]. While several studies have focused on the mechanisms controlling Notch activation by ligand endocytosis [5][6][7][8][9], knowledge about the activation of Notch targets by cleaved NICD and the dynamics involved remains limited [10][11][12][13][14]. More broadly, the role of signal dynamics in controlling developmental processes and tissue differentiation is only partially understood [15][16][17][18][19].…”