Principal Supervisor: Dr Shaun Cowley - Department of Molecular and Cell Biology
Co-supervisor: Dr Salvador Macip
PhD project title: Monitoring the p53 interactome following diverse stimuli using the Biotin-ID assay.
University of Registration: University of Leicester
p53 is a regulator of the cell cycle and functions as a “guardian of the genome”. In response to DNA damage, p53 is post-translationally modified, stabilised and activated. As a transcription factor, p53 then targets the expression of a variety of genes involved in specific processes, such as cycle arrest, apoptosis or senescence, in order to prevent the emergence of transformed cells. p53 interacts with the E3 ubiquitin ligase, MDM2 prior to activation, which keeps p53 in-check by targeting it for proteosomal degradation. Following DNA damage (among many other stimuli), MDM2 binding is lost and p53 is activated by a number of well described kinases, such and ATM and Chk1. However, downstream of these canonical signalling proteins, it unclear which protein interactions are key to p53 function, and whether these are the same for each mode of p53 activation (DNA damage, senescence, hypoxia, heat-shock, etc.).
Recently, more sensitive methods of detecting protein interactions have been developed, including ‘Biotin-ID’. Biotin-ID (Bio-ID) uses a promiscuous version of the biotin ligase, BirA (BirA*) fused to the protein of interest, thus allowing the labelling, and then identification of ‘proximate’ proteins (within approx. 30nm). The advantage of this approach is that it allows both the identification of binding partners and transiently associated proteins, such as kinases (e.g. ATM) which regulate p53 directly. The Cowley lab has used the Bio-ID approach to study the protein demethylase, LSD1 and generated reproducible datasets for known binding partners (CoREST1-3, HDAC1/2, and BHC80), substrates (p53), as well as 23 other novel chromatin proteins (Fig.1A). ￼￼2
We plan to use the Biotin-ID approach to monitor p53 associated proteins following diverse methods of activation.
A human diploid fibroblast cell line (e.g. WI-38) expressing p53-BirA* will be generated, using CRISPR/Cas9 to add BirA* to the endogenous trp53 locus. In our LSD1 study there is relatively little biotinylation in the absence of exogenously added biotin to the media (Fig.1B). We will therefore be able to grow p53-BirA* cells normally, add biotin to the media, and then induce p53 activity in variety of ways: using DNA damaging agents (e.g. doxorubicin, UV light, etc.) inducing hypoxia by culturing cells in low oxygen (<1% O2) and heat-shocking cells. p53 ‘proximate’ proteins will be determined by snap freezing cells, and then preparing protein extracts. Biotinylated proteins will purified using streptavidin, followed by tryptic digest and mass-spectrometric identification (PNACL, proteomics core facility U. of Leicester). These experiments can also be performed over a time-course, 0, 15, 30 mins, etc. so that we are able to capture the range and timing of p53 interactions following activation. The most interesting individual p53 associated proteins (3-6 proteins of high interest) will be validated by co-IP (for the protein of interest) and western blotting. In addition, these can also be monitored in cells using a ‘split GFP’ assay, where p53 and the protein of interest are labelled with the N- or C- terminal portion of GFP, respectively. GFP signal is only reconstituted if the two proteins are able to interact in cells. The addition of GFP will also these interactions to be monitored in real-time (following p53 activation) in live cells using the VisiTech Infinity 3 confocal microscope (advanced imaging facility, U. of Leicester).
We will identify a comprehensive network of p53 associated proteins following different modes of p53 activation using the improved sensitivity of the Bio-ID assay. It will be of great interest to the academic community to know whether the range of activating enzymes and binding proteins is consistent, or different among diverse p53 stimuli.
BBSRC Strategic Research Priority: Molecules, cells and systems
Techniques that will be undertaken during the project:
- Mammalian cell culture
- CRISPR Cas9 gene editing
- Mass-spectrometry and live cell microscopy
Contact: Dr Shaun Cowley, University of Leicester