Biochemical isolation and mass spectrometric analysis of the CpG island proteome

CpG 岛蛋白质组的生化分离和质谱分析

• 批准号: 1985785
• 金额: --
• 依托单位: University of Oxford
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2018
• 资助国家: 英国
• 起止时间: 2018 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-1985785
• 关键词: Biochemical; isolation; mass; spectrometric; analysis

Every human being originates from one single cell, a fertilised egg. From these humble beginnings, this cell must divide over and over again during gestation and from a complex network of new and specialised cells which organise into the structures, organs, and systems that support normal human biology. To achieve this specialisation, a subset of the cells genes, which are encoded in its DNA, must be expressed, or in other words used, at the right time and place during early development. This controlled usage of genes during the early stages of development, and in later life, relies on the function of specialised proteins called transcription factors. Transcription factors recognize defined DNA sequences near genes and guide the systems that lead to appropriate gene expression. Although we understand a significant amount about how transcription factors function in these essential processes, it has recently become clear that the underlying DNA sequences and how they are packaged inside cells can also profoundly affect transcription factors and how are genes are expressed. This is because DNA is not found unadorned inside the cell, but is instead is chemically modified and wrapped in structural proteins, called histones, that help to organise it within the limited confines of our microscopic cells. We and others have recently shown that a specific type of DNA sequence, called a CpG island, has a central role in shaping how transcription factors and the gene expression machinery function to ensure the appropriate gene usage. If CpG islands are perturbed this contributes to the formation of human diseases, like cancer, that are characterized by inappropriate gene expression. Interestingly, CpG island DNA sequences appear to regulate gene expression by altering the chemistry of the histones that associate with DNA near genes. However, our understanding of how alterations in histone chemistry and the potential involvement of other proteins that work at CpG islands in shaping normal gene expression remain rudimentary and a major barrier to understanding how these processes go wrong in human disease.To address this fundamental gap in our understanding we will leverage an interdisciplinary approach and develop new quantitative mass spectrometry-based proteomic approaches to unbiasedly characterize the histone chemistry and protein composition of CpG island DNA inside cells. This will involve the development of new molecular biology approaches, omic's analysis workflows, and provide an exciting new basis on which to discover how gene expression is controlled in the context of development and whole organism physiology. Building on these important discoveries sophisticated genetic perturbation studies will allow us to understand how cells malfunction in the absence of normal CpG island function, and could provide essential new molecular leads that explain the aetiology of human disease where CpG island function is abnormal. This will provide future opportunities to explore how we can translate these new finding, from bench to bedside, and possibility intervene pharmacologically in diseases where CpG island biology is perturbed.

每个人都起源于一个单一细胞，即受精卵。从这些卑微的开始，这个细胞必须在妊娠期间一遍又一遍地分裂，并形成一个由新的特化细胞组成的复杂网络，这些细胞组织成支持正常人类生物学的结构、器官和系统。为了实现这种特化，必须在早期发育过程中的正确时间和地点表达或换句话说，使用编码在其 DNA 中的细胞基因的子集。这种在发育早期和后期生命中基因使用的控制依赖于称为转录因子的特殊蛋白质的功能。转录因子识别基因附近的特定 DNA 序列，并指导导致适当基因表达的系统。尽管我们对转录因子如何在这些重要过程中发挥作用有很多了解，但最近我们已经清楚，潜在的 DNA 序列以及它们在细胞内的包装方式也会深刻影响转录因子以及基因的表达方式。这是因为 DNA 在细胞内并不是未经修饰的，而是经过化学修饰并包裹在结构蛋白（称为组蛋白）中，有助于在微观细胞的有限范围内组织 DNA。我们和其他人最近表明，一种称为 CpG 岛的特定类型 DNA 序列在塑造转录因子和基因表达机制如何发挥作用以确保适当的基因使用方面发挥着核心作用。如果 CpG 岛受到干扰，就会导致癌症等人类疾病的形成，这些疾病的特征是基因表达不当。有趣的是，CpG 岛 DNA 序列似乎通过改变与基因附近 DNA 相关的组蛋白的化学性质来调节基因表达。然而，我们对组蛋白化学变化以及 CpG 岛其他蛋白质在塑造正常基因表达中的潜在参与的理解仍然很初级，并且是理解这些过程在人类疾病中如何出错的主要障碍。为了解决我们理解中的这一根本差距，我们将利用跨学科方法并开发新的基于定量质谱的蛋白质组学方法来公正地表征组蛋白化学和蛋白质组成 细胞内的 CpG 岛 DNA。这将涉及新分子生物学方法、omic 分析工作流程的开发，并为发现在发育和整个生物体生理学背景下如何控制基因表达提供令人兴奋的新基础。在这些重要发现的基础上，复杂的遗传扰动研究将使我们能够了解细胞在缺乏正常 CpG 岛功能的情况下如何发生故障，并可以提供必要的新分子线索来解释 CpG 岛功能异常的人类疾病的病因学。这将为探索如何将这些新发现从实验室转化为临床提供机会，并可能对 CpG 岛生物学受到干扰的疾病进行药理学干预。