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

验证体内 b-Catenin DamID-seq 系统并阐明脂肪变性和肝细胞癌中的 b-Catenin 靶点

• 批准号: MR/X000877/1
• 负责人: Keisuke Kaji
• 金额: $ 67.39万
• 依托单位: University of Edinburgh
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2023
• 资助国家: 英国
• 起止时间: 2023 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=MR%2FX000877%2F1
• 关键词: Validating; vivo; Catenin; DamID; seq

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.

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单一的细胞，受精卵，并且体内的所有细胞都包含相同的基因组。具有相同基因组的细胞是如何以不同的方式特化的？一组约1,500个称为转录因子(TF)的蛋白质在选择我们基因组中的~20,000个基因中的哪些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特定的功能。每个不同的转铁蛋白可以结合基因组中的10,000个不同的位置。基因附近的转铁蛋白结合决定了该基因是否被激活。因此，了解何时以及哪些转录因子控制哪些基因，对于理解健康的身体是如何在我们的一生中保持的非常重要。β-连环蛋白是婴儿子宫内发育和维持健康成人身体的重要转录因子之一。B-连环蛋白的异常活动会导致多种疾病，包括癌症。像许多其他转录因子一样，b-连环蛋白在基因组中的结合位置因细胞类型的不同而不同。因此，了解每种细胞类型中的b-连环蛋白结合位点可以帮助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的疾病。然而，在我们体内的不同细胞类型的基因组中，识别b-连环蛋白结合位点一直是困难的。目前可用的识别b-连环蛋白结合位点的实验技术需要大量(超过100万)细胞，而且通常不可能纠正来自身体的如此大量的细胞。因此，在这个项目中，我们的目标是建立一种新的技术，称为‘体内b-catenin DamID-Seq’，使我们能够在体内仅用来自不同组织的10,000个细胞识别b-catenin靶标。b-catenin在维持健康的肝功能方面起着关键作用。因此，为了验证这项实验技术，我们将首先研究b-连环蛋白在基因组中与健康肝脏的肝细胞结合的位置。有趣的是，一个转基因小鼠模型显示，当小鼠喂食高脂肪食物时，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)。非酒精性脂肪肝是全球最常见的肝病病因，可导致非酒精性脂肪性肝炎(NASH)和肝硬变，后者是45-65岁人群中第三常见的死亡原因。在英国，据估计约有30%的人患有早期的NAFLD。很明显，b-catenin在NAFLD中起着关键作用，但我们仍然不知道b-catenin控制的哪些基因会加重NAFLD，如果b-catenin在基因组中的结合位置随着食物类型的不同而改变。回答这些问题可能导致逆转NAFLD或防止进展为NASH和肝硬化的新策略。B-连环蛋白在最常见的肝癌--肝细胞癌中也很重要。最近有报道称，b-连环蛋白水平高的肝细胞癌不太可能被我们的免疫细胞清除，这解释了为什么这种肝细胞癌患者往往预后较差。然而，目前还不清楚b-连环蛋白是如何为肝细胞癌提供逃避免疫细胞的能力的。我们将使用体内DAMID-SEQ来解决这些与NAFLD和肝细胞癌中的b-连环蛋白靶标相关的问题。该项目的成功不仅提供了对肝脏生物学和疾病的更好的了解，而且还提供了一种新的实验技术，该技术广泛适用于人体内许多其他类型的细胞和相关疾病，以研究b-连环蛋白的作用，包括各种癌症、阿尔茨海默病和糖尿病。