MSc by Research Programme: Does increasing stability of Notch affect formation of the segmented body axis?

This course allows you to work alongside our world renowned experts from the School of Life Sciences and gain a ’real research’ experience. You will have the opportunity to select a research project from a variety of thematic areas of research.

You will be part of our collaborative working environment and have access to outstanding shared facilities such as microscopy and proteomics. Throughout your year, you will develop an advanced level of knowledge on your topic of interest as well as the ability to perform independent research in the topic area. Alongside basic science training in experimental design, data handling and research ethics, we will help you to develop skills in critical assessment and communication. This will be supported by workshops in scientific writing, presentation skills, ethics, laboratory safety, statistics, public engagement and optional applied bioinformatics.

The period of study is one year full-time or two years part-time research, which includes two months to write up the thesis. Please apply via the UCAS postgraduate application form: https://digital.ucas.com/courses/details?coursePrimaryId=c735d826-42b6-ca1f-50db-2a3ac6f68718

Notch is one of the major highly conserved signalling pathways that regulate cell-cell communication which involves gene regulation mechanisms that control multiple processes during development and adult life, including cell fate specification within progenitors. Upon extracellular ligand binding, Notch transmembrane receptors are cleaved, releasing the intracellular domain (NICD) that translocates to the nucleus to regulate expression of specific developmental gene cohorts. NICD is highly labile, and phosphorylation-dependent turnover acts to restrict Notch signalling.

Most canonical Notch activity relies on this regulation of NICD turnover. Moreover, aberrant NICD turnover contributes to numerous cancers and diseases. Despite the multiple impacts of NICD turnover in both development and disease, the molecular mechanism regulating this turnover remains largely uncharacterised. The stability of NICD and therefore duration of the Notch signal is regulated by phosphorylation of the C-Terminal PEST domain which leads to subsequent recruitment of FBXW7, F-Box and WD Repeat Domain Containing 7, (a key component of the SCFSel10/FBXW7 E3 ubiquitin ligase complex). Ultimately, this leads NICD to ubiquitylation and proteasomal degradation. However, the molecular details of NICD degradation mediated by FBXW7 are not well understood.
We recently identified a highly conserved site crucial for NICD recognition by the SCF E3 ligase, which targets NICD for degradation, in in vitro assays. We have used Crispr/CAS9 gene editing technology in mouse embryonic stem cells and human iPS cells to knockin a version of Notch 1 with a point mutation in the residue required for recognition by the SCF E3 ligase.

During early vertebrate development Notch plays a critical role on the progressive formation of the segmented body axis. This process is called somitogenesis and comprises the progressive periodic formation of segments called somites from a tissue called the presomitic mesoderm. The periodicity of the process is regulated by a molecular oscillator acting in the cells of the presomitic medoderm that drives periodic expression of so called clock genes, most of which are Notch target genes.

This project will investigate whether a point mutation in a highly conserved site crucial for affects NICD recognition by the SCF E3 ligase affects the ability of mouse embryonic stem cells and human iPS cells to differentiate into presomitic mesoderm cells and if this has any effect on the periodic expression of the clock genes.

Aims:
1. Does the S2513A Notch1 mutation render NICD non phosphorylatable?
2 Does the S2513A Notch1 mutation affect levels of NICD through inhibiting the interaction with the SCF E3 ligase at endogenous levels?
3 Does the S2513A Notch1 mutation affect the ability of mouse embryonic stem cells and human iPS cells to differentiate into presomitic mesoderm cells?
4 Does the S2513A Notch1 mutation affect the dynamic oscillatory expression of clock genes in the presomitic mesoderm?