The role of cis-acting elements in regulating 3D structure and gene expression

Thesis

Precise spatial-temporal gene expression is essential for normal development and differentiation, and, as such, must be tightly controlled. Cis-acting regulatory elements (enhancers, promoters and boundary elements) play a key role in gene regulation, either by enhancing transcription directly or by altering the 3D chromatin conformation to modify interactions between enhancers and promoters. Neither the contributions of individual elements to these processes, nor the complex interplay between them is fully understood. In this thesis, I have specifically investigated the ability of an ectopic CTCF binding site to shape the 3D genome and perturb gene expression. Furthermore, I have studied which aspects of gene activation and transcription depend on the enhancers using a model of the alpha-globin locus from which all enhancers have been removed. The mouse alpha-globin locus provides a well-characterised model of mammalian gene expression. Five enhancer elements regulate the expression of the alpha-globin genes, which lie in a ~65 kb sub-TAD flanked by largely convergent CTCF binding sites. First, I have studied the effect of restricting the 3D interactions of the enhancers by placing an insulator element between the alpha-globin enhancers and genes. This single CTCF binding site significantly reduces enhancer-promoter interactions and strongly decreases the expression of the alpha-globin genes. I created two CTCF insertion models, with the site either oriented to predominantly block loop extrusion from the direction of the enhancers or from the direction of the genes. Gene expression is most strongly affected in the model that is predominantly blocking cohesin coming from the direction of the enhancers. In this orientation, there is a prominent accumulation of cohesin at the insertion site, which is absent in the other orientation. I have also characterized a model in which all five alpha-globin enhancer elements have been deleted. This had a much stronger effect on gene expression than restricting enhancer interactions and sheds light on the role that the enhancers play in activating the locus: for example, the binding of transcription factors and co-factors was almost completely lost from the alpha-globin promoters. This all enhancer knockout model provides a “blank” slate that will be valuable resource for re-building the enhancers and establishing the precise role of each element and their combinations in the future.

Authors: Stolper, R

• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English