Systematic dissection of enhancer interdependencies

The regulation of gene expression during development and homeostasis is essential to ensure proper formation and function of the body. When and where a gene is activated is regulated by so-called enhancers: genomic sequences that control the activity of a gene from a distance. However, with increasing genomic distance between an enhancer and a promoter, the activation at the promoter decreases. Furthermore, most genes are controlled by multiple active enhancers in the same cell type. However, interaction and cooperativity between individual enhancers in activating the target promoter are poorly understood. We have recently developed a novel synthetic locus that allows for the integration of individual and combinations of enhancers at defined distances to a reporter gene. So far, we have been limited to a single enhancer integration at a time. With this grant we will expand our toolbox and exchange individual landing pads with a recombination-mediated cassette exchange (RMCE). This will allow us to test numerous enhancers in parallel and determine their activation of the target gene, alone or in combination with another enhancer. Once we have implemented such a tool, we will test a library of enhancers at different distances to the promoter and determine whether all enhancers lose efficient activation with increasing distance. Next, we will test whether most enhancers cooperate with other enhancers and increase the expression at the target promoter beyond their individual activities. For the first time, the results from this novel screening approach will test combinations of enhancers at different distances to the promoter and their ability to activate transcription. Together with Alex Stark (IMP, Vienna), we will further explore the enhancers` underlying sequence features using machine learning approaches to identify potential classes of enhancers and determine their cooperative behaviour. Finally, we will build synthetic enhancers with features for ideal cooperativity and test them in our synthetic locus. Once we have identified examples of different enhancers and potential cooperations, we will collaborate with Anton Goloborodko (IMBA) to determine how individual enhancers and combinations of elements shape the 3D genome. We will map the changes in the 3D genome organisation and thereby determine how individual enhancers might increase contact with a promoter. The insights from our work will help to elucidate how enhancers work together to bridge the distance to the promoter and activate it. The lessons learned from our approach will help to identify causal mutations in enhancers that might lead to disease and pave the way for predicting gene expression from sequence alone.