Assessing the functional role of cohesin and transcription in genome topology during Drosophila embryogenesis
The three-dimensional organization of the genome plays an important role in bringing enhancers (E) in close proximity to their cognate promoters (P) via chromatin looping. Such E-P loops occur within topology associated domains (TADs), although how TADs facilitate E-P function is still unresolved. In mammals, TADs are formed by Cohesin mediated loop extrusion. However, it remains unclear how TADs are formed in other species, and how E-P looping is regulated, especially during embryogenesis. In Drosophila, and to a lesser degree also in mouse and humans, many TAD boundaries have features of active transcription such as promoters and the binding of the transcription machinery. Moreover, ectopic insertion of transcriptionally active transposons are able to form a boundary de novo, suggesting that transcription itself can also shape genome topology. We are in a unique position to address these questions thanks to a new optogenetic nuclear depletion system that we developed. Distinguishing cause and consequence between transcription and topology has so far been very difficult using conventional loss-of-function mutants as they often block embryogenesis at very early stages, before TADs are even established. We can now solve these issues using our recently developed iLEXY system, to deplete nuclear proteins in minutes, inside living embryos. We will combine iLEXY with high-resolution genomics to determine the role of cohesin and the basal transcriptional machinery in the establishment and maintenance of TADs and E-P loops during Drosophila embryogenesis. We will target three components each of the cohesin complex (Mau2, Smc3, WapL) and the basal transcriptional machinery (M1bp, Spt4 and Spt5) for depletion. M1bp is essential for the establishment of the pre-initiation complex at a subset of constitutively active genes that are present at TAD boundaries. Spt4 and Spt5 are required for Pol II pausing and its subsequent elongation. By targeting all three, we can thereby distinguish between a role of the PIC assembly, versus Pol II pausing and elongation in TAD boundary formation. Embryos with rapid depletion and repletion of each of these six proteins will be used for low input high resolution genomic assays to quantify (1) chromatin occupancy of proteins via CUT&Tag, (2) changes in TADs and E-P loops via MicroC, and (3) nascent transcription to measure the impact on gene expression. This will reveal if cohesin-mediated loop extrusion is an evolutionarily conserved mechanism in chromatin folding or if alternate mechanisms exist. It will also provide the first functional data on the role of Pol II pausing in embryonic development, as different models have been proposed. Taken together, our proposal has the potential to decouple, for the first time, the interdependency between genome topology and transcription in vivo.
Dr. Eileen E. M. Furlong,Heidelberg
European Molecular Biology Laboratory (EMBL)