| Abstract |
During development, a multitude of cell signalling pathways control patterning, cell
fate determination and tissue assembly. Notch signalling is one such, highly
conserved, pathway in multicellular animals that regulates a large diversity of
developmental processes. Its function is crucial for normal development and its
misregulation is associated with diseases including cancers. Despite its
fundamental and varied roles, at the core of Notch signalling is a simple pathway.
The Notch trans-membrane receptor itself is cleaved upon ligand binding. This
cleavage releases the Notch intra-cellular domain (NICD) that translocates into the
nucleus. NICD forms an activation complex with the DNA-binding transcription
factor CSL (Su(H) in Drosophila) and the co-activator Mastermind. Binding of the
activation complex promotes target gene expression. An added layer of complexity
to this pathway is that Su(H) can also form a repressor complex with the corepressor
Hairless.
The aim of this PhD is to understand how signal activation and release of NICD lead
to recruitment of the activator complex to the correct targets and to investigate the
role of chromatin complexes in delimiting the transcriptional outcome.
First, to understand how recruitment of the activator complex to target genes
occurs, changes in transcription factor dynamics were monitored upon Notch
activation. Su(H), Mastermind and Hairless behaviours were probed in real time,
using in vivo Single Molecule Localisation Microscopy. Analysis of the behaviour of
these molecules revealed that Notch activation led to an increase of bound Su(H)
molecules, as well as an increase of the fraction of Su(H) molecules with an
exploratory behaviour. Near a target locus, E(spl)-C, Su(H) exhibited characteristics
of local exploration. Importantly all these changes in behaviour were shared by the
co-activator Mastermind but not the co-repressor Hairless. These results showed
that, when incorporated into the activator complex upon NICD release, Su(H)
adopts an exploratory behaviour that promotes local searching and retention near
target enhancers. These findings account for the increased occupancy of CSL at
target sites under Notch-On conditions.
Local changes in behaviour revealed by the study of transcription factor behaviour
suggest a change of local environment at target loci. A second aim was therefore to
investigate the roles of a subset of chromatin factors in shaping the Notch
response. Live-imaging in Drosophila salivary glands revealed recruitment of Moira,
Trithorax-related (Trr) and Mi-2, representative members of 3 diWerent chromatin
complexes, to the Notch target locus E(spl)-C. All three factors were recruited in
Notch active conditions and contributed to the eWicacy of the transcriptional
response. Trr was essential for recruitment of Su(H) and to promote transcription at
E(spl)-C. In contrast, Mi-2 was involved in fine-tuning Notch response by limiting
chromatin opening, co-activator recruitment and ultimately lowering the
probability of target gene transcription. The roles of Mi-2 and Trr were also
examined in a diWerent developmental context, Drosophila egg chambers, where
Notch is endogenously active in follicle cells during a limited period (Stages 5-8). In
this context, single molecule FISH was used to probe transcription of Notch targets.
Unlike in the salivary gland, Trr was dispensable for normal profile of expression in
the follicle cells. Mi-2 proved to play a largely similar role in this tissue, with a
repressive function that contributed to the fine-tuning and shut-down of Notch
response. Additionally, another chromatin repressor complex, Polycom Repressor
Complex 2 (PRC2) was also revealed to be key to ensure proper timing of Notch
response, ensuring shut-down of transcription concomitantly with Mi-2 in late
stages, raising the possibility that they work together. Ultimately it would be of
interest to the understanding of Notch response to investigate whether
perturbations to these chromatin complexes alter the local searching and retention
behaviour of Su(H) at its target loci.
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