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3D single-molecule imaging of nuclear proteins and chromatin in pluripotent cells

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SPL - New statistical physics in living matter: non equilibrium states under adaptive control

How pluripotent cells in the mammalian embryo decide their fate is a fundamental question in developmental biology and regenerative medicine. Nuclear proteins that play a key role in these fate decisions have the capacity to form intra-nuclear compartments called condensates and to influence spatiotemporal enhancer-promoter relationships. Using single-cell Hi-C, we recently demonstrated multi-way enhancer-promoter relationships in pluripotent cells enriched for the binding sites of nuclear proteins known to influence pluripotent cell differentiation – transcription factors (SOX2/NANOG) and chromatin regulators (the nucleosome remodelling and deacetylase complex, NuRD, and the H3K4me3 methyltransferase, MLL2 ). However, little is known about the dynamics of these proteins or how they influence enhancer-promoter relationships in live cells. To address this, we develop 3D single-molecule localisation microscopy (SMLM) approaches for tracking of single HaloTag-tagged nuclear proteins within live mammalian nuclei. We then establish machine-learning algorithms that extract biological information from SMLM to determine (1) the chromatin binding kinetics of nuclear proteins; and (2) how nuclear proteins influence chromatin mobility. Finally, we integrate nuclear-scale molecular modelling into our pipelines as well as spatial information from high-density 3D SMLM to reveal whether proteins form condensates in live cells and to characterise condensate properties. Using these approaches, we show that while NANOG and SOX2 bind to enhancers for seconds (with NANOG binding ~3 times more stably than SOX2 ), the NuRD remodeller binds over much longer periods. Although in vitro studies have shown that NANOG and SOX2 can in principle form condensates, we find evidence for only NANOG forming condensates in live pluripotent cells. Moreover, we confirm in live cells our single-cell Hi-C observation that NANOG , SOX2 and NuRD binding sites cluster together. Finally, we show that NuRD and MLL2 influence the range that genes explore within the nucleus. Using chromosome conformation capture, we show that these changes in chromatin mobility are linked to changes in the length-scale over which enhancers activate transcription at nearby genes, and propose that this influences gene activation during pluripotent cell differentiation. Our results highlight the importance of making dynamic live-cell measurements at single-molecule resolution to provide insight into condensates. Furthermore, we have now established our approaches within live mouse embryos, which will lead to future insights into these mechanisms in vivo.

This talk is part of the Isaac Newton Institute Seminar Series series.

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