Mitchell, Jennifer ATaylor, Tiegh2024-11-132024-11-132024-11http://hdl.handle.net/1807/141340Gene expression relies on a complex interplay between regulatory elements dispersed across the genome, and chromatin structure guiding functional interactions. Precise spatiotemporal regulation of organism development and tissue patterning involves enhancer-gene interactions, yet mechanisms directing enhancers to their target genes are poorly understood. Enhancers influence gene expression by recruiting transcription factors and establishing long-range contacts with target genes. This targeting process involves an interplay between transcriptional regulatory complexes which are functionally directed or segregated by the action of architectural proteins such as CTCF and Cohesin. In my thesis, I investigate the regulatory interplay among chromatin architecture, genomic distance, and enhancer activity using CRISPR-Cas9 mediated genomic deletions on two model loci. I used the Sox2 loci as a model to study the relationship between enhancer activity and its contact with a target gene. Sox2 is regulated by a cluster of enhancer elements which are located at a distance downstream from it; I systematically removed clusters of transcription factor bound regions to identify that the chromatin topology within this locus is decoupled from the activity of the enhancer and distributed across almost 40 kilobases of chromatin. Within the Lefty locus, the Lefty1 and Lefty2 genes are paralogs which directly interact with one another, but I have identified that they are regulated by separate enhancer elements. Systematic removal of elements between these two genes revealed that the regulatory elements driving gene expression within this locus are insulated from one another in a distributed manner by the genomic distance between the genes, even though they reside close together in the 3D nuclear space. Finally, to better assess the relationship between chromatin distance, architecture, and enhancer activity, I have used the Sox2 locus to develop a high-throughput assay to screen putative regulatory elements for their activity over a moderate genomic distance from the Sox2 gene. Using this system I have identified requirements within synthetically created enhancer sequences for their activity at a distance. Taken together my thesis has identified that enhancers are differentially affected by the chromatin landscape surrounding them, highlighting that there are yet unknown sequence determinants of enhancer-gene targeting encoded within the genome.Attribution 4.0 Internationalhttp://creativecommons.org/licenses/by/4.0/ChromatinCRISPREnhancerLeftyPluripotencySox20369Thesis