Mammalian embryo development: Towards a single-molecule perspective.

Abstract

Revealing how pluripotent cells generate distinct cell lineages in the early mammalian embryo is central to uncovering the mechanisms that drive mammalian development and for unlocking the potential of pluripotent cells for regenerative medicine. The objective of my lab is to provide single-molecule insights into nuclear protein complexes that influence the ability of pluripotent cells to differentiate. Although recent studies have characterised the enzymatic activities of some of these protein complexes, little is known about their intra-nuclear dynamics or how they influence spatiotemporal 3D enhancer-promoter relationships. To investigate these dynamics, we have established tools for live-cell 3D tracking of single HaloTag-tagged nuclear proteins and inactive dCas9-labelled genes. We have also developed machine-learning algorithms to infer properties about the resulting trajectories, allowing us to determine the chromatin binding kinetics of key nuclear protein complexes and also how they control chromatin movement at specific enhancers/promoters. Using these approaches, we reveal the chromatin binding kinetics of two major protein complexes that regulate differentiation - the chromatin remodeller NuRD and the histone methyltransferase KMT2B. Furthermore, we show that both NuRD and KMT2B influence the range genes explore within the nucleus. We then use single-cell transcriptomics (scRNAseq) and chromosome conformation capture (Hi-C) to show that these changes in chromatin movement are linked to the length-scale over which enhancers activate transcription at nearby genes. We propose a model linking the movement of enhancers to gene activation as pluripotent cells differentiate. Our results highlight the importance of making dynamic measurements at single-cell and single-molecule resolution to provide insight into transcriptional control during differentiation. We are currently establishing these technologies within live mouse embryos and we believe this will help the field understand the underlying molecular mechanisms of cell-fate decisions in vivo.