Relationship between chromatin structure and distal regulation

Abstract

Mammalian genomes are organised into topologically associated domains (TADs) and chromatin loops through a loop extrusion process. Structuring of chromatin within these highly conserved TADs is believed to both facilitate gene regulation via distal enhancers whilst insulating their regulatory activity at TAD boundaries. Shh and its regulatory elements are all contained within a highly conserved, well-characterised 1 Mb TAD that has previously been proposed to form an invariant chromatin loop structure across various tissue types. The overall aim of my thesis is to further understand how distal enhancers operate to regulate their target gene during mammalian development. To do this, I used the Shh as a model locus to study the spatiotemporal dynamics of distal enhancers and the mechanisms of long-range gene regulation in relation to three-dimensional chromatin structure. To explore the spatiotemporal regulation of Shh across brain development I first performed ATAC-Seq on dissected, FAC-sorted embryonic brain tissue. I show that the Shh brain enhancers are accessible across embryonic brain development and that the Shh forebrain enhancer, SBE2, shows increased spatial proximity with the Shh gene specifically in SBE2-regulated cells. Next, to investigate the importance of TAD integrity on developmental gene regulation, we have manipulated the Shh TAD by deleting CTCF sites at the TAD boundaries. Both RNA and DNA fluorescence in situ hybridisation assays were used to investigate changes in Shh expression and chromatin conformation that result from these manipulations in single cells of developing embryonic tissue. I found that Shh loses its co-localisation with the tissue-specific enhancers in the CTCF mutant embryos but with no consequence on Shh expression patterns. I then used degron-tagged CTCF and cohesin embryonic stem cell lines to investigate if genome-wide depletion of these TAD architectural proteins affected Shh distal regulation using synthetic transcription factors. I found that cohesin is required for enhancer-driven gene activation, whereas CTCF was dispensable. Finally, I used patient-derived cell lines to study a human-specific SHH TAD boundary deletion found in acheiropodia patients. I show using DNA fluorescence in situ hybridisation that this deletion causes ectopic interactions between the SHH TAD boundary and a novel CTCF peak detected in patient cells, therefore, potentially preventing ZRS from regulating the promoter. Overall, my data shows that the removal of single CTCF sites in mice alters TAD structure but has no readily detectable effect on Shh expression patterns during development and results in no evident phenotypes. This suggests that the developmental regulation of Shh expression is remarkably robust to TAD perturbations. I then go on to show that distal regulation is dependent on cohesin but not CTCF. I propose a model where cohesin extrusion brings together distal elements to induce expression and CTCF functions to stabilise and insulate these interactions. This supports the view that CTCF provides robustness to developmental gene regulation. Ectopic CTCF binding in acheiropodia patients would block extrusion of cohesin and prevent SHH/ZRS interaction during embryonic development.