How enhancers shape and activate the genome

Enhancers are regulatory genomic elements that are responsible for adequate spatio-temporal gene-expression. They may be positioned close to the gene they regulate, but can also be located up to a mega base (Mb) up- or downstream from their target promoter. Communication between promoter and enhancer takes place within the context of the three-dimensional genome. An intricate interplay between the protein complex cohesin and the protein CTCF forms topologically associating domains (TADs), physical domains in which promoters and enhancers communicate. Cohesin is loaded onto DNA and extrudes a growing DNA-loop until it reaches a DNA bound CTCF molecule. CTCF binds DNA at designated sites called CTCF-sites.
In this thesis we have investigated how robust communication between promoter and enhancer is orchestrated. We employed a unique bottom-up approach by integrating ectopic regulatory elements into the genome in order to build various artificial gene expression systems.
In chapter 2 we addressed how gene expression and 3D genome organization are affected by the relative positions of promoters, enhancers and CTCF-sites. We show that expression reduces when the linear distance between promoter and enhancer increases. Yet, CTCF-sites can compensate for this effect. We demonstrate that an enhancer initiates topological contacts and that a CTCF-site restricts these contacts. In chapter 3 we describe the role of cohesin in our observations. With ChIP and knockdown experiments we establish that the enhancer recruits cohesin to convergent CTCF-sites and that cohesin is essential for gene expression exclusively when the concerned gene is regulated by a distal enhancer. In chapter 4 we performed a genetic screen to identify novel factors that, in addition to cohesin, may be required specifically for long distance gene regulation.
Collectively, our data show how proteins and genomic elements cooperate to orchestrate robust gene expression within the 3D genome.