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5 Janelia Publications
Showing 1-5 of 5 resultsOur ability to unambiguously image and track individual molecules in live cells is limited by packing of multiple copies of labeled molecules within the resolution limit. Here we devise a universal genetic strategy to precisely control copy number of fluorescently labeled molecules in a cell. This system has a dynamic titration range of >10,000 fold, enabling sparse labeling of proteins expressed at different abundance levels. Combined with photostable labels, this system extends the duration of automated single-molecule tracking by 2 orders of magnitude. We demonstrate long-term imaging of synaptic vesicle dynamics in cultured neurons as well as in intact zebrafish. We found axon initial segment utilizes a "waterfall" mechanism gating synaptic vesicle transport polarity by promoting anterograde transport processivity. Long-time observation also reveals that transcription factor hops between clustered binding sites in spatially-restricted sub-nuclear regions, suggesting that topological structures in the nucleus shape local gene activities by a sequestering mechanism. This strategy thus greatly expands the spatiotemporal length scales of live-cell single-molecule measurements, enabling new experiments to quantitatively understand complex control of molecular dynamics in vivo.
Transcription factor (TF)-directed enhanceosome assembly constitutes a fundamental regulatory mechanism driving spatiotemporal gene expression programs during animal development. Despite decades of study, we know little about the dynamics or order of events animating TF assembly at cis-regulatory elements in living cells and the long-range molecular "dialog" between enhancers and promoters. Here, combining genetic, genomic, and imaging approaches, we characterize a complex long-range enhancer cluster governing Krüppel-like factor 4 (Klf4) expression in naïve pluripotency. Genome editing by CRISPR/Cas9 revealed that OCT4 and SOX2 safeguard an accessible chromatin neighborhood to assist the binding of other TFs/cofactors to the enhancer. Single-molecule live-cell imaging uncovered that two naïve pluripotency TFs, STAT3 and ESRRB, interrogate chromatin in a highly dynamic manner, in which SOX2 promotes ESRRB target search and chromatin-binding dynamics through a direct protein-tethering mechanism. Together, our results support a highly dynamic yet intrinsically ordered enhanceosome assembly to maintain the finely balanced transcription program underlying naïve pluripotency.
50 years ago, Vincent Allfrey and colleagues discovered that lymphocyte activation triggers massive acetylation of chromatin. However, the molecular mechanisms driving epigenetic accessibility are still unknown. We here show that stimulated lymphocytes decondense chromatin by three differentially regulated steps. First, chromatin is repositioned away from the nuclear periphery in response to global acetylation. Second, histone nanodomain clusters decompact into mononucleosome fibers through a mechanism that requires Myc and continual energy input. Single-molecule imaging shows that this step lowers transcription factor residence time and non-specific collisions during sampling for DNA targets. Third, chromatin interactions shift from long range to predominantly short range, and CTCF-mediated loops and contact domains double in numbers. This architectural change facilitates cognate promoter-enhancer contacts and also requires Myc and continual ATP production. Our results thus define the nature and transcriptional impact of chromatin decondensation and reveal an unexpected role for Myc in the establishment of nuclear topology in mammalian cells.
Animal development is orchestrated by spatio-temporal gene expression programmes that drive precise lineage commitment, proliferation and migration events at the single-cell level, collectively leading to large-scale morphological change and functional specification in the whole organism. Efforts over decades have uncovered two 'seemingly contradictory' mechanisms in gene regulation governing these intricate processes: (i) stochasticity at individual gene regulatory steps in single cells and (ii) highly coordinated gene expression dynamics in the embryo. Here we discuss how these two layers of regulation arise from the molecular and the systems level, and how they might interplay to determine cell fate and to control the complex body plan. We also review recent technological advancements that enable quantitative analysis of gene regulation dynamics at single-cell, single-molecule resolution. These approaches outline next-generation experiments to decipher general principles bridging gaps between molecular dynamics in single cells and robust gene regulations in the embryo.
Our ability to unambiguously image and track individual molecules in live cells is limited by packing of multiple copies of labeled molecules within the resolution limit. Here we devise a universal genetic strategy to precisely control protein copy number in a cell. This system has a dynamic titration range of more than 10,000 fold, enabling sparse labeling of proteins expressed at widely different levels. Combined with fluorescence signal amplification tags, this system extends the duration of automated single-molecule tracking by 2 orders of magnitude. We demonstrate long-term imaging of synaptic vesicle dynamics in cultured neurons as well as in live zebrafish. We found that axon initial segment utilizes a waterfall mechanism gating synaptic vesicle transport polarity by promoting anterograde transport processivity. Long-time observation also reveals that transcription factor Sox2 samples clustered binding sites in spatially-restricted sub-nuclear regions, suggesting that topological structures in the nucleus shape local gene activities by a sequestering mechanism. This strategy thus greatly expands the spatiotemporal length scales of live-cell single-molecule measurements for a quantitative understanding of complex control of molecular dynamics in vivo.
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