A genetic dissection of TAD formation during embryogenesis

Regulation of gene expression is a complex process that requires the action of DNA elements such as promoters, enhancers and insulators. In eukaryotic genomes, these elements are organized in self-interacting units, the Topologically-Associating Domains (TADs). The role of TADs in regulating gene expression is increasingly appreciated, though still poorly understood. Genetic dissection of TADs and their boundaries have shown that TADs can restrict enhancer- promoter interactions, but also promote enhancer-mediated activation. It is not clear by which mechanisms TADs can form, although insulator protein binding at TAD boundaries has been implicated. TADs are largely conserved between different cell types and an intriguing question is when they form during embryogenesis and whether they can help drive developmental gene expression programs during. Using Drosophila melanogaster as a model organism, I decided to first investigate the timing of chromatin topology establishment in the early embryo. I observed that 3D spatial proximity across different loci appeared simultaneously with the onset of zygotic transcription, consistent with TADs being established during that stage. To interrogate the mechanisms regulating TAD establishment, I depleted individual insulator proteins (BEAF-32, CTCF and CP190) during the first steps of embryogenesis. Most TADs could form in the absence of those proteins. However, a mild loss of TAD insulation throughout the genome was detected. Specific loci appeared more severely affected upon loss of CP190, although it is not clear what distinguishes those loci from unaffected loci. Insulator depletion also caused gene expression defects that are potentially linked to topological alterations. Deletion of specific DNA elements within a TAD boundary revealed that transcription and insulator protein binding may both be required for the full insulation potential of certain boundaries. To systematically investigate which factors determine TAD boundary function, I inserted several TAD boundaries into two TADs containing distinct properties. Boundary insertions disrupted local chromatin topology in nearly half of the cases, revealing a strong context-specificity for boundary function. While some boundaries altered chromatin topology in a locus- and orientation-specific manner, other boundaries affected both TADs. Some insertions caused developmental lethality, indicating effects on gene expression associated with topological alterations. Overall, the findings presented here indicate a remarkable context-specificity of mechanisms and factors shaping the genome topology in Drosophila, possibly being required for the precise regulation of highly complex gene expression programs during embryonic development