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EASTBIO Developing in vivo and ex vivo inducible beta-catenin DamID-seq systems


College of Medicine and Veterinary Medicine

Prof K Kaji , Dr S Tomlinson Wednesday, January 06, 2021 Competition Funded PhD Project (Students Worldwide)

About the Project

Canonical Wnt/b-catenin signalling plays critical roles in various biological contexts including development, stem cells, and diseases. To understand mechanisms underlying the context specific responses to Wnt, identifying b-catenin binding sites is important. However, b-catenin ChIP-seq has been difficult and only a limited number of cell types has been used for b-catenin ChIP-seq to date. ChIP-seq for transcription factors usually requires >10^6 cells, and collecting such number of specific cells in vivo is often difficult. Recently we have succeeded in genome-wide identification of b-catenin targets with 10,000 mouse ES cells using an alternative technique, DamID-seq, and generated transgenic mouse liens ready to perform in vivo inducible b-catenin DamID (iDamID).

In this project, we aim to validate the in vivo b-catenin iDamID using pericentral hepatocytes, in which Wnt/b-catenin is known to control metabolic gene expression. Subsequently we will perform the same in vivo b-catenin iDamID in a non-alcoholic fatty liver disease (NAFLD) model with high fat diet (HFD). NAFLD is the most common cause of liver disease, strongly associated with the metabolic syndrome, including obesity, insulin resistance, type 2 diabetes and dyslipidemia, a growing problem in modern society with a $20 billion to $35 billion per year drug market value by 2025. Hepatocytes with high Wnt/b-catenin signalling preferentially accumulates lipid droplets under HFD, and hepatocyte-specific b-catenin knockout (KO) and overexpression (OE) results in milder and severer hepatic steatosis under HFD, respectively. However, gene expression of b-catenin KO or OE hepatocytes under normal diet indicates that Wnt/b-catenin signalling rather suppresses lipogenesis, and it is not clear why active Wnt/b-catenin signalling correlates with severer lipid droplets accumulation in NAFLD. Thus, we hypothesize that direct Wnt/b-catenin targets in hepatocytes changes depending on diet, and address this hypothesis with in vivo iDamID.

In parallel, we will also develop lentiviral b-catenin iDamID system which is applicable to human primary cell culture, and complement the mouse in vivo DamID system. Once the system is established and validated with mouse ES cells, we will apply it to 3D primary human hepatocyte spheroid culture, in the presence and absence of hepatic steatosis inducers, free fatty acids and insulin, known to activate Wnt/b-catenin signalling. These human hepatocytes b-catenin DamID-seq data will be compared to the mouse in vivo data, and used to identify liver disease associated SNPs overlap with the b-catenin binding sites.

In summary, this project will provide the research community with powerful and widely applicable tools to identify direct Wnt/b-catenin targets with minimal cell numbers, demonstrating their application in fundamental liver biology.

Apply Now

This 4 year PhD project is part of a competition funded by EASTBIO BBSRC Doctoral Training Partnership (DTP) http://www.eastscotbiodtp.ac.uk/how-apply-0.

EASTBIO Application, Equality, Diversity and Inclusion (EDI) survey and Reference Forms can be downloaded via http://www.eastscotbiodtp.ac.uk/how-apply-0

Please send your completed EASTBIO Application Form and EDI survey along with a copy of your academic transcripts to before the deadline.

You should also ensure that two references have been sent to by the deadline using the EASTBIO Reference Form.

Please refer to UKRI website and Annex B of the UKRI Training Grant Terms and Conditions for full eligibility criteria.

Funding Notes

This opportunity is open to UK and international students and provides funding to cover stipend and UK level tuition fees. The University of Edinburgh will cover the difference between home and international fees meaning that the EASTBIO DTP will offer fully-funded studentships to all appointees. However there is a cap on the number of international students the DTP can recruit. It is therefore important for us to know from the outset which fees status category applicants will fall under when formally applying for entry to our university.

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