In our bodies genes need to be activated at the right time and the right place. Many of these genes are activated by pieces of DNA that are located far away in the genome. The folding of our genome plays an important role in the correct regulation of these genes. We will investigate which factors are important in this process. This will lead to a better understanding how genes are regulated during embryonal development and what happens in developmental disorders.

A cell is often likened to a bowl of spaghetti, in which each chromosome reflects a single entity of pasta. Remarkably though, the different chromosomes are not intermingled, as each chromosome is confined to its own region. What actually keeps the chromosomes apart? And what purpose does this serve? Molecular machines known as condensin play a central role in these processes. We have discovered how cells activate condensin such that it can correct structure our chromosomes.

Transcription factors are the essential drivers of gene expression. We have recently discovered an exciting new role for transcription factors in the spatial organisation of the genome. Within the linear genome there are regions that contain a high-density of lineage specific transcription factor binding, so-called transcription factor clusters. These clusters bring together genes in the nucleus, that are expressed in a tissue-specific manner. In the current proposal we aim to shed light on the functional role of these tissue-specific 3D genome conformations. The project will be a close collaboration between molecular perturbation experiments and computational analysis. We will perform genome-wide 3D genome measurements in mouse embryonic stem cells after inducible depletion of the pluripotency specific factors Oct4 and Nanog. Based on these results we will choose transcription factor clusters in which we will mutate individual transcription factor binding sites using genome editing. Mutated transcription factor clusters will be disrupted in their long-range DNA interactions. By measuring the transcription factor binding and expression level of distally contacted regions we can determine the contribution of the 3D genome to gene expression. Furthermore, we have preliminary data showing that transcription factors are also associated with specific genome conformations in primary tissues. We will generalise our observations in ESCs by expanding our dataset to primary mouse tissues. We will perform extensive data mining on existing Hi-C datasets in human cell lines and extend our Hi-C analysis to pleural effusion breast cancer samples to understand the role transcription factor mediated genome organisation in human disease. We are beginning to uncover the role of transcription factors in 3D genome organisation. With the current proposal we can start to tease apart the functional role of this remarkable genome structure.

Transcription factors turn on genes at the correct moment in the correct cell type. In this study, we will apply advanced microscopy techniques to visualize individual transcription factor molecules inside cells, while simultaneously measuring when genes are activated. The results will reveal how transient transcription factor binding to DNA regulates the dynamics of gene expression.