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专门作用。然而，所有的细胞类型都来自一个单一的细胞，一个受精卵，体内的所有细胞都含有相同的基因组。具有相同基因组的细胞如何以不同的方式特化？一组约1,500种称为转录因子（TF）的蛋白质在选择我们基因组中约20，000个基因中的哪一个用于特定细胞类型方面发挥着重要作用。这使得细胞能够执行其专门的功能。每个不同的TF可以结合基因组中> 10，000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非常重要。β-连环蛋白（b-Catenin）是胎儿在子宫内发育和维持成人健康的重要转铁蛋白之一。B-连环蛋白的异常活性导致多种疾病，包括癌症。像许多其他TF一样，在基因组中，b-连环蛋白结合的位置取决于细胞类型而不同。因此，了解每种细胞类型中的b-连环蛋白结合位点可以让我们理解为什么特定细胞类型中的b-连环蛋白过度激活会导致特定疾病。然而，很难在我们体内的各种细胞类型的基因组中识别b-连环蛋白结合位点。目前可用的鉴定b-连环蛋白结合位点的实验技术需要大量（超过100万）细胞，并且通常不可能从身体中校正如此大量的细胞。因此，在这个项目中，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个细胞来识别b-Catenin靶标。因此，我们将首先研究在基因组中b-连环蛋白在健康肝脏的肝细胞中结合的位置，以验证这种实验技术。有趣的是，转基因小鼠模型显示，当小鼠喂食高脂肪饮食时，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是全球肝病最常见的原因，可导致非酒精相关性脂肪性肝炎（NASH）和肝硬化，这是45-65岁人群的第三大常见死因。在英国，据估计约有30%的人患有早期NAFLD。很明显，b-Catenin在NAFLD中起着关键作用，但我们仍然不知道由b-Catenin控制的哪些基因会加重NAFLD，如果基因组中的b-Catenin结合位置根据食物类型而变化。解决这些问题可能会导致逆转NAFLD或预防NASH和肝硬化进展的新策略。联蛋白在肝细胞癌（HCC）中也很重要，HCC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，这解释了为什么患有此类HCC的患者往往预后不良。然而，目前尚不清楚b-连环蛋白如何为HCC提供逃离免疫细胞的能力。我们将使用体内DamID-Seq解决与NAFLD和HCC中的b-连环蛋白靶点相关的这些问题。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，而且还提供了广泛适用于体内许多其他细胞类型和相关疾病的新实验技术，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.