Poising and connectivity of enhancers upon naïve-to-primed transition in human embryonic stem cells (hESCs)

Abstract

Enhancers are non-coding DNAelements that play crucial roles in transcriptional control, particularly in development. Patterns of histone modifications at enhancers are commonly used to infer their activity states and, poised enhancers (PEs) in particular display a ’bivalent’ chromatin state: the ’active’ H3K4me1 and the ’repressive’, Polycombassociated H3K27me3. Typically observed in pluripotent stem cells (PSCs), it was shown that PEs are required for gene activation later during differentiation. However, the function of the poised state of enhancers remains largely unknown. To trace the emergence of PEs in early development, I have extensively optimized a recently developed low-cell number Capture Hi-C protocol to perform Poised Enhancer Capture Hi-C (PECHi-C) in PSCs, in time course upon the naïve-to-primed transition, which is known to associate with a major shift in the localisation of Polycomb proteins, from a broader to a highly focal pattern. PECHi-C revealed that the PE-mediated regulatory circuitry undergoes significant reorganization between the two states. In particular, I detected three predominant patterns of PE-mediated interactions: the UP, DOWN and CONSTANT interaction classes. Integrating these results with Cut&Tag data on histone modifications revealed an interplay between the acquisition of the poised state at enhancers and their interaction dynamics whereby, at least in some cases, the acquisition of the bivalent signature occurs in parallel to the acquisition of their contacts. Moreover, the analyses suggested that day 3 of the transition is a pivotal point of the naïve-to-primed transition for the emergence of PEs. Overall, this thesis provided further insights into the emergence of PE-mediated regulatory circuitry during early embryogenesis. The different patterns of PE connectivity suggest the presence of diverse regulatory mechanisms of PEs, further suggesting that PEs might play a role at earlier stages of embryogenesis, by ensuring the correct transition from the ground state of pluripotency to the primed state.