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

我们的身体由大约300种不同类型的细胞组成，每种细胞都有不同的特殊作用。然而，所有的细胞类型都来自一个细胞，一个受精卵，并且身体中的所有细胞都含有相同的基因组。具有相同基因组的细胞是如何以不同的方式特殊化的？一组大约1500种被称为转录因子（tf）的蛋白质在我们基因组中大约2万个基因中选择哪些基因用于特定的细胞类型方面发挥着重要作用。这允许细胞执行它们的特殊功能。每一个不同的TF可以结合基因组中1万个不同的位置。基因附近TF结合的组合决定了该基因是否被激活。因此，了解何时以及哪些tf控制哪些基因对于了解我们一生中如何保持健康的身体是非常重要的。b-连环蛋白是胎儿在子宫内发育和维持健康成人身体的重要tf之一。b-连环蛋白的异常活动导致多种疾病，包括癌症。像许多其他tf一样，在基因组中b-连环蛋白的结合是不同的，取决于细胞类型。因此，了解每种细胞类型中的b-Catenin结合位点可以让我们理解为什么b-Catenin在特定细胞类型中过度激活会导致特定疾病。然而，在我们体内各种细胞类型的基因组中，很难确定b-Catenin的结合位点。目前可用的鉴定b-Catenin结合位点的实验技术需要大量（超过100万）细胞，并且通常不可能从一个身体中校正如此大量的细胞。因此，在本项目中，我们的目标是建立一种名为“体内b-Catenin DamID-Seq”的新技术，该技术使我们能够仅用来自体内不同组织的10,000个细胞识别b-Catenin靶点。b-连环蛋白在维持健康的肝功能中起着关键作用。因此，我们将首先研究基因组中b-Catenin在健康肝脏中与肝细胞结合的位置，以验证该实验技术。有趣的是，一种转基因小鼠模型显示，当小鼠被喂食高脂肪饮食时，肝脏中b-连环蛋白的过度激活会导致严重的非酒精性脂肪性肝病（NAFLD）。NAFLD是世界范围内最常见的肝脏疾病原因，可导致非酒精相关性脂肪性肝炎（NASH）和肝硬化，这是45-65岁人群中第三大常见死亡原因。在英国，估计约有30%的人患有早期NAFLD。显然b-Catenin在NAFLD中起着至关重要的作用，但我们仍然不知道b-Catenin控制的哪些基因加重了NAFLD，如果b-Catenin在基因组中的结合位置随着食物类型的变化而变化。回答这些问题可能会导致新的策略来逆转NAFLD或防止进展为NASH和肝硬化。b-连环蛋白在肝细胞癌中也很重要，肝细胞癌是最常见的肝癌类型。最近有报道称，具有高水平b-连环蛋白的HCC不太可能被我们的免疫细胞清除，这解释了为什么这类HCC患者往往预后较差。然而，目前尚不清楚b-Catenin如何为HCC提供逃离免疫细胞的能力。我们将使用体内DamID-Seq来解决这些与NAFLD和HCC中b-Catenin靶点相关的问题。该项目的成功不仅提供了对肝脏生物学和疾病的更好理解，而且还提供了一种新的实验技术，可广泛应用于体内许多其他细胞类型和相关疾病中，以研究b-Catenin的作用，包括各种癌症，阿尔茨海默病和糖尿病。

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.