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Title: Notch targets and EGFR pathway regulation
Author: Housden, B.
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2011
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Previous work in the lab identified potential Notch targets in a muscle related Drosophila cell line using a combination of genome wide expression array, chromatin immunoprecipitation (ChIP) and bioinformatics approaches. My project was focussed on investigating the regulation of EGFR pathway components by Notch signalling and unravelling mechanisms by which this crosstalk is dependent on context. Previous genetic studies have demonstrated extensive crosstalk between the Notch and EGFR pathways. The unexpected result from the genome wide studies was the overrepresentative of EGFR pathway components that were direct targets. This group of nine targets included both positive and negative regulators of the pathway. One of my first goals was to validate these as direct targets, for which I used a combination of luciferase and in-vivo reporter assays. To address the question of how activation and inhibition of the EGFR pathway is resolved into a final effect on EGFR output the temporal profiles of the different components was investigated. Initial results show that there are distinct temporal activation profiles for different EGFR related genes following Notch activation. This is predicted to lead to an initial inhibition of EGFR signalling (due to fast initial inhibitor accumulation) followed by an activation of signalling (due to inhibitor decrease and activator production). Current work on this project is focussed on identifying different classes of temporal response at the genome wide scale and asking whether there is any consistency in the types of genes that adopt specific profiles. One of the challenges in dissecting the response to Notch is understanding why genes respond only in certain contexts. Argos, which encodes an EGFR inhibitor, is one example of a gene whose response to Notch signalling is context dependent. My results show that argos is positively regulated in the muscle progenitors. However, in the wing pouch, argos expression is reduced upon Notch activation. I have mapped these contrasting responses to separable enhancers, one active in the muscle precursors that I have shown to be directly regulated by Notch and the other active in the wing pouch where it integrates both EGFR and Notch signals. The latter is mediated by the Notch target E(spl)mβ and is therefore indirectly affected by Notch. Further studies to distinguish the components acting through the different enhancers revealed that the bHLH protein Twist is required for Notch activation of the muscle precursor enhancer and the wing pouch enhancer activity appears to involve an interplay between general activation and restricted repressors.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available