Biochemical isolation and mass spectrometric analysis of the CpG island proteome

每个人都起源于一个细胞，一个受精卵。从这些不起眼的开始，这个细胞必须在怀孕期间不断地分裂，从一个复杂的新细胞和特化细胞网络中分裂成支持正常人类生物学的结构、器官和系统。为了实现这种专门化，细胞DNA中编码的一部分基因必须在早期发育的正确时间和地点被表达，或者换句话说，被使用。在发育的早期阶段和以后的生活中，这种控制基因的使用依赖于被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确定的DNA序列，并引导系统导致适当的基因表达。尽管我们对转录因子如何在这些基本过程中发挥作用有了大量的了解，但最近已经清楚的是，潜在的DNA序列及其在细胞内的包装方式也可以深刻地影响转录因子以及基因如何表达。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称为组蛋白的结构蛋白中，组蛋白有助于在我们微小细胞的有限范围内组织DNA。我们和其他人最近表明，一种特殊类型的DNA序列，称为CpG岛，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起着核心作用。如果CpG岛受到干扰，就会导致以基因表达不当为特征的癌症等人类疾病的形成。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，我们对组蛋白化学的改变以及在CpG岛工作的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是初级的，并且是理解这些过程在人类疾病中如何出错的主要障碍。为了解决我们在理解上的这一基本差距，我们将利用跨学科的方法，开发新的基于定量质谱的蛋白质组学方法，以公正地表征细胞内CpG岛DNA的组蛋白化学和蛋白质组成。这将涉及新的分子生物学方法的发展，基因组学的分析工作流程，并提供一个令人兴奋的新基础，在此基础上发现基因表达是如何在发育和整个生物体生理学的背景下被控制的。在这些重要发现的基础上，复杂的遗传扰动研究将使我们了解在没有正常CpG岛功能的情况下细胞如何发生功能障碍，并可能提供重要的新分子线索，解释CpG岛功能异常的人类疾病的病因学。这将为探索我们如何将这些新发现从实验室转化为床边提供未来的机会，并可能在CpG岛生物学受到干扰的疾病中进行药理学干预。

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.