Non-Alcoholic Fatty Liver Disease (NAFLD) caused by a high fat diet (HFD) is a growing major public health problem world-wide. About 30% of people are in the early stage of NAFLD in UK, and about 10-30% of people who have NAFLD develop Non-Alcohol Related Steatohepatitis (NASH), a more serious condition of NAFLD, which can lead to cirrhosis, the third most common cause of death in people aged 45-65 years. A pathological feature of adult NAFLD typically starts with triglyceride accumulation in hepatocytes near the central veins. However, it is unclear why triglycerides are preferentially deposited in this area of the liver. Understanding the underlying molecular mechanism may lead to the development of strategies to treat, diagnose and ultimately develop a preventative medicine.
The pericentral area near the central veins in the liver is where Wnt/b-catenin signaling is active. Notably, hepatocyte specific b-catenin knockout mice were shown to have very small triglyceride accumulation in the liver whereas transgenic mice developed a severe NAFLD phenotype under a HFD condition (Behari, Am. J. Pathol, 2014). However, several known direct b-catenin target genes in the liver under a normal diet condition cannot explain the role of b-catenin in NAFLD. Since b-catenin targets change depending on expression of other TFs and/or activity of other signaling pathways even within the same cells, we hypothesized that b-catenin changes its targets in response to the HFD condition and activates genes involved in NAFLD.
In this project, we aim to detect genome-wide b-catenin binding sites and identify direct target genes critical for NAFLD development. Currently, the most common strategy to identify direct transcription factor (TF) binding sites is ChIP-seq. However, b-catenin ChIP-seq has been difficult to successfully perform. Thus, we apply an alternative technique called DNA adenine methyltransferase identification with sequencing (DamID-seq) which we have recently optimized in mouse cells (Tosti, Genome Research, 2017). DamID-seq is based on the expression of a TF tethered to the DNA adenine methyltransferase (Dam) from E.Coli and does not require immunoprecipitation or crosslinking, thus it is suited for proteins which bind to DNA indirectly and/or for which ChIP-seq is difficult (Vogel, Nature Protocol, 2007).
We will first establish an in vivo b-catenin DamID-seq system generating mouse lines with inducible b-catenin-Dam expression in a wide range of cell types. Using this system, we will identify direct b-catenin binding sites in pericentral and periportal hepatocytes isolated from mice fed a normal or high fat diet. We will also perform RNA-seq with the same hepatocyte subpopulations. Together with RNA-seq datasets from the 1000 liver tissue samples in the SteatoSITE project lead by Prof Fallowfield (https://steatosite.com/
) we will identify b-catenin binding sites and candidate genes critical for NAFLD development and progression. Finally we will validate the importance of these gene and their regulatory regions in an in vitro 3D organoid NAFLD model (Kozyra, Scientific Reports, 2018), using the CRISPR/Cas9-mediated genome modification.
This project involves a wide range of cellular and molecular biology techniques including DamID-seq, 3D organoid culture, CRISPR/Cas9-mediated genome modification, as well as mouse handling, histological analyses, flow cytometry, and imaging. Under the supervision of the basic scientist, Prof Kaji, and the clinical scientist, Prof Fallowfield, the PhD student will learn all these techniques as well as biology of the liver. Dr Tomlinson, a Senior Lecturer in Bioinformatics, and the bioinformatics course at MRC CRM will offer training for the next generation sequencing data processing. We therefore expect the student will be fully equipped with biological and computational knowledge as well as interpersonal work skills to start their career as a researcher after their PhD.
This MRC programme is joint between the Universities of Edinburgh and Glasgow. You will be registered at the host institution of the primary supervisor detailed in your project selection.
All applications should be made via the University of Edinburgh, irrespective of project location. For those applying to a University of Glasgow project, your application along with any supporting documents will be shared with University of Glasgow. http://www.ed.ac.uk/studying/postgraduate/degrees/index.php?r=site/view&id=919
Please note, you must apply to one of the projects and you must contact the primary supervisor prior to making your application. Additional information on the application process is available from the link above.
For more information about Precision Medicine visit: http://www.ed.ac.uk/usher/precision-medicine