Morgenstern, C.; (2009) A novel genome-wide approach to identify in vivo targets of Notch signalling. Doctoral thesis , UCL (University College London).

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Abstract

The Notch signalling pathway regulates many developmental processes in metazoan embryos and adults such as cell proliferation, stem cell maintenance, cell fate specification and apoptosis. Despite the importance of this pathway, few targets have been identified, with the Hes (Hairy and Enhancer-of-split) protein family being the best-characterised group of downstream effectors. I have established transgenic mice carrying Biotin Acceptor Peptide (BAP)- tagged versions of Notch1. The tagged protein is fully functional and is biotinylated after crossing to mice expressing the biotinylase from E. coli. Biotinylation was confirmed in a range of different tissues. However, streptavidin chromatin pull-down (bioChIP) experiments from these tissues showed no significant enrichment of known Notch1 target sequences. A possible explanation could be the indirect and transient nature of the interaction between Notch, its DNA binding partner CSL and the promoter of the target gene. A transgenic mouse line expressing a BAP-tagged version of the transcription factor Hes7, a downstream effector of Notch signalling and key regulator of somitogenesis, was similarly generated. Although the tagged Hes7 protein is functional and gets biotinylated in cell culture assays, the transgenic mice exhibit a severe somite/skeletal phenotype indicating that the tagged allele is hypomorphic. A detailed analysis of the phenotype revealed differential axial requirements for Hes7.

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