The roles of novel mammalian insulator factors in T-cell development

We all start from one cell - the fertilised egg. Our parents endow this cell with a collection of genes (or the genome), which are essentially a set of instructions, a complete manual written in our DNA on how to build a body and how to make it function. For this cell to develop into a functional body, two things need to happen. First it needs to divide many times to grow into the billions of cells that make up our body. With every cell division, the original set of instructions (the genes) is accurately copied, without exception, and carefully packed into each new cell that is produced.
Secondly, as the cells are being multiplied, they need to specialise and assemble into the different tissues and organs, a process called cellular differentiation. This complicates things because a brain cell for example has very different functions from a cell of our immune system, so the two clearly need to access distinctive sets of instructions from their copy of the manual. Both type of cells are being handed the same book, but they need to scour through and highlight only the parts that are relevant to their function while blocking out the rest of the information which could be misleading. This is also referred to as a 'program of gene expression' to describe the fact that although all the cells in our bodies contain the same identical set of genes, only a subset of genes (instructions) are active in a particular type of cell, while the rest are being kept 'silent'.
Being able to accurately direct cells along the different pathways of cellular differentiation is the 'Holy Grail' of regenerative medicine, as it would allow us to replace tissues and organs damaged by accidents or disease. In order to achieve this, we have to understand how the different types of cells in our bodies establish their specific gene expression programs, or in other words how they are able to focus on their characteristic set of instructions and suppress the rest of the information.

I am studying a type of 'regulatory element' which is also written in the DNA but is not part of the genes. These regulatory elements are called 'insulators' or 'boundary elements' and they do not hold direct instructions for the cell. Instead, they are found between genes and demarcate the instructions the cell needs to follow, and the ones it needs to ignore, similar to setting up the boundaries between which you need to draw with a marker pen to highlight a text of interest. Highlighted genes will be active and the instructions they encode followed, while the rest will be ignored. Many diseases, including cancers and autoimmune diseases are believed to be caused by mutations in regulatory elements, as their disruption or deletion can lead to the cell following inappropriate instructions or ignoring necessary ones. Ultimately, the effect is similar to mutations that change the content of the instructions: an aberrant gene expression program, and therefore cell identity or ability to function.
The molecular mechanisms by which insulators work are still poorly understood, partly because we have not yet managed to identify all insulators in the human genome. We have identified all the actual genes but not the elements that decide how the genes are expressed. I have identified a protein factor that can potentially indicate where insulators reside in the human genome. I will use a combination of biochemical techniques combined with maps of the human genome to follow where this protein factor binds to the DNA. This will allow us to map the insulators in the genome. By artificially disrupting this protein in an experimental system, I will block the functions of insulators, which will allow me to investigate the mechanisms by which these regulatory elements control genes. I hope my work will improve our understanding of the molecular mechanisms that control human development, and that one day will help design treatments that can restore aberrant gene expression programs.

Insulators are thought to play important roles in establishing cell type-specific transcription patterns and in genome organisation. Mammals are widely believed to rely on a single type of insulator, defined by the factor CTCF. However, recent studies, including my own work, indicate the distribution and activity of CTCF are not sufficient to explain the compartmentalisation and three-dimensional organisation of the genome, implying significant contribution from alternative insulators. Identifying these elements and the factors that mediate their functions is essential for understanding how gene expression programs are established, maintained and changed during mammalian development.

Using developing T-cells as a model system, I intend to carry out a combination of genome-wide correlative analyses and functional studies to investigate the roles of newly identified putative insulator factors in transcriptional regulation and chromatin organisation. My research plan presents an opportunity to expand the repertoire of mammalian insulators beyond the current monopoly of CTCF, and gain new mechanistic insights into the roles of these regulatory elements in mammalian development. Drawing up the complete landscape of mammalian insulators has direct implications on our ability to understand and diagnose human disease, as recent comprehensive analyses of genome-wide association data revealed more than 50% of disease- and trait-associated genetic variants lie in intergenic regions bearing hallmarks of long-range gene regulatory elements such as enhancers and insulators.

Beneficiaries of my work will include scientists and clinicians with interests in translational research on T-cell lymphoma and autoimmune diseases, as well as medical geneticists. In the long term this work could assist development of diagnosis techniques and treatments.

My experiments will investigate how transcriptional programs are established and regulated during thymocyte differentiation, and will therefore deliver an in-depth analysis of the pathways and mechanisms that drive T-cell development. When these pathways are altered thymocytes can become transformed giving rise to Precursor T-cell Lymphoblastic Lymphoma, which is the most common form of T-cell lymphoma. In addition, recent evidence suggest that autoimmune diseases such as type 1 diabetes and multiple sclerosis are caused by autoreactive T-cells that escape the negative selection process which takes place during thymocyte differentiation. A detailed molecular analysis of the normal thymocyte development and maturation process is essential for understanding the mechanisms that lead to transformation and autoimmunity, and for developing potential intervention strategies.

For medical geneticists, my work will help functionally evaluate disease-associated genetic variants, and thus will contribute to the development of diagnosis strategies and understanding the aetiology of human diseases. Genome-wide association studies (GWAS) are a powerful genetic approach to scan the entire genome for genetic variants most likely to be associated with a certain disease or phenotypical trait. This is a particularly promising approach to identify the genetic bases of common complex diseases, such as heart disease and diabetes, that may arise from the interaction of multiple genes. Hundreds of GWAS have been carried out investigating a diverse range of diseases, and an unexpected finding that emerged is that more than 50% of disease- and trait-associated genetic variants fall within non-coding, intergenic regions at considerable distance from gene bodies. Recent analyses have found these to significantly overlap with DNase I hypersensitive sites (DHS), a hallmark of long-range gene regulatory elements such as enhancers and insulators. Assigning functional significance to disease-associated genetic variants will therefore require comprehensive and accurate mapping of enhancers and insulators. My research plan to characterise new potential mammalian insulator factors will improve our ability to identify insulators in humans and will better define their genome-wide distribution.

Finally, PhD students and academic staff that will contribute to this work will receive a wide range of research training, including important technologies used in functional genomics such as ChIP-sequencing, RNA-sequencing and Hi-C, which will equip them with the necessary skills for a successful career progression.