Three-dimensional interrogation of gene regulation using next generation chromatin conformation capture and super-resolution imaging.

To form the cells and tissues that make up our bodies requires exquisite control of the activity of our genes. The protein products of genes form the functional and structural components of our cells. Gene expression is controlled by a subgroup of proteins called transcription factors which bind specific sequences in our DNA (also called the "genome"). If this control is altered by sequence changes in the genome then the correct function of our cells is affected and can lead to disease: the particular disease or diseases depend on the types of cells affected. Scientists have become very good at finding the ~ 2% of our DNA that encodes for protein, however it has become clear that other parts of our genome (called regulatory elements) play a critical role in controlling our genes.

Regulatory elements are scattered throughout the genome and novel methods that identify such elements show that it is very difficult to understand which gene they control as they do not necessarily control nearby genes, but may affect quite distant genes. A technique called chromosome conformation capture can link regulatory elements to the genes they control by mapping their physical interactions, which are necessary for their activity, but this still does not explain why certain elements can contact and regulate some specific genes but not others. This is an important question because changes in the genome sequence can prevent regulatory elements talking to the correct genes or cause them to talk to the wrong genes with very damaging consequences. The mechanism that makes these interactions specific is unknown, but is thought to be due to the three dimensional arrangement of the genome in the nucleus. The work in this proposal adapts technologies developed by a collaboration between molecular biologists, chemists and computational scientists to visualize these interactions in 3D to understand how certain regulatory elements work with specific genes and how changes in the genome can alter this and cause disease.

Next Generation Sequencing has allowed us to investigate the structure and function of the genome at unprecedented detail. In particular, chromatin accessibility assays (DNAse-seq and ATAC-seq) produce comprehensive maps of the active regions of the genome and ChIP-seq allows sub-classification by chromatin signature which infers potential function. This has revealed an unsuspected level of complexity in the regulatory landscape of the genome with estimates of ~ 2M potential regulatory elements to control ~20,000 genes. GWAS studies have mapped the majority of disease susceptibility variants to the non-coding portion of the genome suggesting that regulatory elements may be their major targets. However finding which genes these regulatory elements are controlling and how this is achieved is a major challenge, mainly due to the unpredictability of both their number and distribution around their cognate genes. It has been shown that although these elements affect multiple genes, they may "ignore" neighbouring genes in favour of more distal genes. Chromosome conformation capture-based technologies link regulatory elements and genes via their physical interaction, but do not explain why some interactions are favoured and others prevented. As the physical interactions of these elements are thought to be required for their function, this represents a fundamental mechanism for specificity and selectivity. Such interactions, in turn, are likely related to the overall 3D structure of the genome. As small changes in non-coding sequence can have major effect on gene expression, and considering the amount of observed genome variation, it is essential to understand the mechanisms by which regulatory elements work. We propose to develop new technologies to visualize specific regulatory interactions at unprecedented levels of sensitivity and resolution, so that regulatory elements can be seen interacting within the context of the surrounding chromosomal landscape.

Beneficiaries of this research will be:
1. Research scientists in academia who are working to develop a better understanding of biology in healthy cells and organisms.
2. Biomedical scientists in academia and industry who are endeavouring to understand the biological processes that occur in the development and progression of human diseases.
3. In particular the project will train a highly skilled postdoctoral researcher who will develop new inter-disciplinary skills.
4. Scientists in the pharmaceutical and biotech industries who are working in discovery biology to determine new targets for diagnostics and therapy in a wide range of diseases.
5. Companies that manufacture and supply oligonucleotide probes to academia and industry for use in the above applications.
6. The UK economy through pharmaceutical and industrial development enhancing business revenue and innovative capacity.
7. The general public who could benefit from new and improved diagnostics and therapies.
8. The general public in gaining knowledge from the modelling and visualisation outcomes proposed.
9. Schools and colleges through outreach activities.