It is now well established that genome organization plays a major role in its functioning. Enhancers act as genetic switches to activate transcription at gene promoters, a process which forms chromatin loops. Other loops prevent enhancers from communicating with the wrong genes, for example by forming insulating structures known as topologically-associating domains. Understanding how chromatin loops and domains are formed, and the role of these structures in regulating gene expression, is the central theme of the work presented in this thesis. A key player in genome organization is the versatile architectural protein and transcription factor known as CCCTC-binding factor (CTCF), which attaches to chromatin at specific DNA binding sites with an asymmetric sequence motif. We examined the role of CTCF binding sites and other DNA regulatory elements in the first two practical chapters presented, while the third is focused on new fluorescence microscopy techniques designed to quantify enhancer-promoter contacts in real time. In mouse embryonic stem cells (ESCs), we found that both deleting and inverting the orientation of CTCF binding motifs at loop anchor regions is sufficient to disrupt chromatin loops, and sometimes alters the expression levels of nearby genes. Building further on this work, we examined how different DNA regulatory elements, including CTCF binding sites, structure chromatin and control gene expression at the murine Prdm14 super enhancer domain. We demonstrate that two topologically distinct structures, achieved by genome editing of a CTCF boundary, are both able to functionally insulate the Prdm14 super enhancer. We show that the wild-type “insulated” Prdm14 super enhancer is partially responsible for expression of the gene Slco5a1, which is located in an adjacent domain, while deletion of the intervening boundary leads to fusion of the two domains and strong upregulation of Slco5a1. Deletion of the downstream domain boundary has the opposite effect on gene expression: both Slco5a1 and Prdm14 were downregulated when the right boundary was deleted, revealing functionally distinct roles for the two CTCF boundaries in this locus. In a separate study, we set up tools to quantify the dynamic interactions and transcriptional output of an active enhancer-promoter pair in single, living human cells. This work provides proof-of-concept to enable studying the link between enhancer-promoter contacts and transcription, based on simultaneous DNA and RNA labelling techniques. We further establish a method enabling future evaluation of enhancer-promoter contacts, based on the principle of co-diffusion. Overall, we provide evidence that enhancer-promoter interactions and their dependent gene transcription can be affected by (CTCF-directed) looping. We further provide proof-of-concept for state-of-the-art imaging studies to image and quantify enhancer-promoter interactions using new DNA labelling techniques. We expect deciphering gene control in 3D to help in understanding the principles of life and disease and to guide future therapies.
|31 Oct 2019
|Published - 31 Oct 2019