Abstract
Interphase chromosomes are mainly shaped by loop extrusion and compartmentalisation mechanisms. However, their temporal component and cause-effect relationships remain largely unknown. In this study, we use acute degradation of WAPL, CTCF and cohesin in mouse embryonic stem cells to investigate the dynamics of loop extrusion and its relationship to compartmentalisation. Stabilisation of cohesin on chromatin by depletion of WAPL results in the formation of extended loops and promotes looping between non-convergent CTCF sites. Loss of WAPL also results in a rapid decrease in compartmentalisation, which is reversed by subsequent removal of cohesin, directly demonstrating the opposite role of extrusion on compartmentalisation. Using combined depletion of WAPL and CTCF, we identify fountains, a feature of chromosome organisation that emanates from enhancer regions and exhibits strong cohesin binding. Fountains form rapidly after mitosis and early in mammalian development. Cohesin depletion confirms that fountains are cohesin dependent, and their disruption leads to the downregulation of fountain-proximal genes, suggesting a role in gene regulation. Taken together, by exploiting the temporal precision of acute protein depletion, our study reveals fountains as an extrusion-mediated, fast-forming feature of 3D genome organisation.