Validating an in vivo b-Catenin DamID-seq system and illuminating b-Catenin targets in steatosis and hepatocellular carcinoma

Our bodies are made up of around 300 different types of cells, each with a different, specialized role. However, all the cell types arise from a single cell, a fertilized egg, and all cells in the body contain the same genome. How are the cells with the same genome specialized in different ways? A group of about 1,500 proteins called transcription factors (TFs) play important roles to select which of the ~20,000 genes in our genome to be used in particular cell types. This allows cells to perform their specialized functions. Each distinct TF can bind >10,000 different positions in the genome. The combination of TF binding near the gene determines if that gene is activated or not. Thus knowing when and which TFs control which genes is very important to understand how healthy bodies are maintained throughout our life.

b-Catenin is one of important TFs for baby's development in womb and maintenance of a healthy adult body. Abnormal activities of b-Catenin cause multiple diseases, including cancer. Like many other TFs, where in the genome b-Catenin binds is different in depending on cell types. Thus, knowing b-Catenin binding sites in each cell type can allow us to understand why b-Catenin over activation in a particular cell type causes a particular disease. However, it has been difficult to identify b-Catenin binding sites in the genome of various cell types in our body. The currently available experimental techniques to identify b-Catenin binding sites require a large amount (over 1 million) of cells, and it is often impossible to correct such large number of cells from a body. Thus, in this project, we aim to establish a novel technique called 'in vivo b-Catenin DamID-Seq', which enables us to identify b-Catenin targets with only 10,000 cells from different tissues in a body.

b-Catenin plays critical roles in maintaining healthy liver function. Thus, we will first investigate where in the genome b-Catenin binds in hepatocytes in the healthy liver in order to validate this experimental technique. Interestingly, a genetically modified mouse model showed that over activation of b-catenin in the liver caused severe non-alcoholic fatty liver disease (NAFLD) when the mice were fed on a high fat diet. NAFLD is the most common cause of liver disease worldwide and can lead to Non-alcohol related steatohepatitis (NASH) and Cirrhosis, the third most common cause of death in people aged 45-65 years. In UK, it is estimated that about 30% of people have early stage of NAFLD. It is clear b-Catenin plays a critical role in NAFLD, but we still do not know which genes controlled by b-Catenin aggravate NAFLD, if b-Catenin binding positions in the genome change depending on the type of food. Answering these questions could lead to novel strategies to reverse NAFLD or prevent the progression to NASH and Cirrhosis. b-Catenin is also important in hepatocellular carcinoma (HCC), the most common type of liver cancer. It has recently been reported that HCC with a high level of b-Catenin are less likely being removed by our immune cells, explaining why patients with such HCC tend to have poor prognosis. Nevertheless, it is still not clear how b-Catenin provides the HCC with the ability to escape from the immune cells. We will address these questions associated with b-Catenin targets in NAFLD and HCC using in vivo DamID-Seq.

Success of this project offer not only better understanding of liver biology and diseases, but also the novel experimental technique that is widely applicable in many other cell types in a body and associated diseases to investigate the roles of b-Catenin, including various cancers, Alzheimer's disease and diabetes.

Canonical Wnt/b-Catenin signalling plays critical roles in various biological contexts including development, stem cells, cancers and other diseases. To understand mechanisms underlying the context specific responses to Wnt, identifying genome-wide b-Catenin binding sites is critical. However, a conventional strategy to detect transcription factor binding sites, ChIP-seq, requires >10^6 cells, and collecting such number of specific cells in vivo is often difficult. While more recently developed techniques Cut&Run/Tag usually require fewer (~10^5) cells, they often demand cell type-dependent optimization and successful b-Catenin Cut&Run/Tag with in vivo cells have not been reported to date. We recently confirmed an alternative strategy, DamID-seq, works well for b-Catenin with 10,000 mouse ES cells. Nevertheless, DamID-seq needs transgene expression and has not readily used for mouse cells in vivo. Thus, we have generated transgenic mouse lines with inducible b-Catenin DamID system that enables genome-wide identification of b-Catenin binding sites with 10,000 cells from many different cell types in vivo. In this project, we aim to validate this system using hepatocytes, and demonstrate its value by revealing the roles of b-Catenin in two critical liver diseases, non-alcoholic fatty liver disease (NAFLD) and hepatocellular carcinoma (HCC). Accumulated data suggest that b-Catenin enhances hepatic lipid accumulation under high fat diet and aggravate NAFLD, although it suppresses lipogenic genes under normal diet. While loss-of-function mutations of APC, a negative regulator of b-Catenin, are rare in HCC, HCC with other b-Catenin stabilizing mutations is very common and has a strong immune evasion phenotype and bad prognosis. We will reveal molecular mechanisms of these complex phenotypes using in vivo b-Catenin DamID, and offer the b-Catenin DamID mice as a widely applicable powerful research tool for the community.