Dissecting distance in enhancer-mediated gene regulation

Abstract

In this thesis it is described how the genomic distance between enhancers and promoters contributes to gene regulation. In chapter 1, the current knowledge on long-range enhancer-promoter communication is highlighted, including the proteins involved, and the interplay with the structural organization of the DNA in the nucleus. Chapter 2 reviews novel technologies that enable more complex analysis of 3D-genome organization in cells. In chapter 3, we studied the contribution of different proteins to enhancer-mediated gene activation in a distance-dependent manner. Using a CRISPRi screen, we found that the cohesin complex is specifically necessary for gene regulation over long distances. Then, we validated these findings using acute depletion of cohesin and nascent transcriptome analysis, combined with computational predictions of endogenous enhancer-promoter distance. We confirmed that cohesin has a specific role in mediating long-range enhancer-dependent transcription regulation. This is build further on in chapter 4, where we employed similar assays to dissect the contribution of other factors, such as mediator and LDB1. Mediator was found to play a distance-biased role, with long-range enhancer-connected genes being most affected by its depletion. LDB1 was found to accumulate more on distal enhancers, sensitizing them to perturbation. Together, these data suggested that long-range enhancers need an optimal trans-factor environment to successfully activate their target genes. Finally, we tested the hypothesis that enhancer-promoter distance contributes to gene regulation in chapter 5, by investigating how the linear separation between the globin enhancers and promoters impacts upon correct transcriptional silencing of these developmentally regulated genes. This shows that decreasing the linear distance between regulatory elements enables an overwrite of their promoter-encoded silencing, potentially offering an alternative therapeutic strategy for reactivating fetal globin expression to compensate for hemoglobin-associated diseases. Finally, in chapter 6, we discuss and contextualize these results and propose how to further dissect the relationship between enhancer-promoter distance and gene regulation.