Biochemical isolation and mass spectrometric analysis of the CpG island proteome

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.