Dynamics of enhancer-promoter communication and transcription in mouse pseudo-embryos

Objective

Embryonic development requires the coordinated execution of sequential gene expression programmes in space and time. This complex regulation is achieved by enhancers, cis-regulatory elements that relay spatiotemporal regulatory information to target promoter(s) in response to transcription factor (TF) binding. While enhancers (E) and promoters (P) are often separated by large linear genomic distances, they can come into close 3D proximity through E-P contacts. However, the mechanisms regulating the temporal dynamics of E-P communication and transcription remain elusive. In the nucleus, chromatin is folded by loop extrusion; disrupting this process abrogates large-scale architecture, yet has only minor effects on E-P contacts and gene expression. Also, the contribution of TFs bound at enhancers and promoters to E-P communication is still unclear. Importantly, previous studies mostly investigated these mechanisms in model systems where chromatin structure and transcription are at steady state, thus neglecting their dynamics. This proposal aims to investigate the causal role of loop extrusion and TF binding in the temporal dynamics of E-P communication and transcription during mammalian embryogenesis, using gastruloids (cultured pseudo-embryos) as a paradigm. This will be achieved through two objectives: (1) mapping how E-P contacts, TF occupancy and transcription evolve over developmental time; and (2) perturbing loop extrusion and TF occupancy with targeted protein degradation at specific developmental time windows. As a readout, I will combine genomic and imaging technologies. Genomics will provide a genome-wide view of changes in E-P contacts and transcription averaged across cells, while imaging will determine at selected loci whether these changes co-occur within individual nuclei. The mechanistic insights revealed by this dual approach will have broad implications for understanding gene regulation in both physiological and pathological developmental processes.