| Popis: |
Distal enhancer elements regulate the tissue-specific expression of their target genes via the establishment of physical interactions with the gene promoter. In mice, a cluster of five enhancers, jointly classified as a super-enhancer, specifically upregulate α-globin gene expression during erythroid differentiation. Aside from the Nprl3 gene, whose promoter is located inside this enhancer region, expression-levels of other genes within a short distance (<,50kb) of the enhancer region are not affected by the activation of the enhancer in erythroid cells, despite being located within the same sub-TAD in erythroid cells. The CCCTC-binding factor (CTCF) is implicated in the organisation of chromosome topology through the formation of interactions between its binding sites in an orientation-dependent manner. In this thesis, I demonstrate that CTCF functions in vivo as a boundary to maintain α-globin enhancer-promoter specificity in erythroid cells. The study of the local chromatin architecture by next-generation Capture-C reveals that α-globin enhancer and promoter interactions are constrained to a compartment of roughly 70kb. The unidirectional interaction profiles of the α-globin enhancers are delimited by the interactions between two genomic domains flanking the α-globin cluster. Further investigation shows that each of these domains contains several CTCF binding sites orientated in tandem, such that CTCF binding orientation between domains is convergent. Although CTCF binding across the α-globin locus is identical between mouse embryonic stem (ES) cells and erythroid cells, interaction between these domains occurs only in erythroid cells suggesting it is dependent on the formation of tissue-specific α-globin enhancer-promoter interactions. By generating a series of mouse models, deleting CTCF binding sites at the α-globin enhancers singly and in combination, I show that the deletion of two CTCF binding sites directly flanking the enhancer cluster results in a shift in interactions between flanking domains, away from the enhancer region. This leads to an expansion of enhancer interactions to include two genes directly upstream of the α-globin enhancers: Rhbdf1 and Mpg. Despite the Rhbdf1 gene being subject to polycomb group protein-mediated gene repression in erythroid cells, ablation of CTCF binding results in increased interactions between both the Rhbdf1 and Mpg gene promoters and the α-globin enhancers and concurrent strong transcriptional upregulation of both genes. The Rhbdf1 gene promoter acquires the active histone mark H3K4me3, but doesn't lose Polycomb Repressive Complex 2 (PRC2) mark H3K27me3 or binding of its catalytic component Ezh2. Despite the presence of this repressive mark, robust levels of Rhbdf1 expression are detected at levels higher than those in ES cells where this gene is actively expressed under the influence of its own enhancer. I conclude that regulation of the direction of enhancer interactions by CTCF is required for the promoter specificity of enhancers and the maintenance of transcriptional states of nearby genes. |
