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Liu Lab / Publications
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24 Publications

Showing 1-10 of 24 results
01/29/18 | Visualizing transcription factor dynamics in living cells.
Liu Z, Tjian R
The Journal of Cell Biology. 2018 Jan 29:. doi: 10.1083/jcb.201710038

The assembly of sequence-specific enhancer-binding transcription factors (TFs) at cis-regulatory elements in the genome has long been regarded as the fundamental mechanism driving cell type-specific gene expression. However, despite extensive biochemical, genetic, and genomic studies in the past three decades, our understanding of molecular mechanisms underlying enhancer-mediated gene regulation remains incomplete. Recent advances in imaging technologies now enable direct visualization of TF-driven regulatory events and transcriptional activities at the single-cell, single-molecule level. The ability to observe the remarkably dynamic behavior of individual TFs in live cells at high spatiotemporal resolution has begun to provide novel mechanistic insights and promises new advances in deciphering causal-functional relationships of TF targeting, genome organization, and gene activation. In this review, we review current transcription imaging techniques and summarize converging results from various lines of research that may instigate a revision of models to describe key features of eukaryotic gene regulation.

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12/04/17 | Visualizing long-term single-molecule dynamics in vivo by stochastic protein labeling.
Liu H, Dong P, Ioannou MS, Li L, Shea J, Pasolli HA, Grimm JB, Rivlin PK, Lavis LD, Koyama M, Liu Z
Proceedings of the National Academy of Sciences of the United States of America. 2017 Jan 09;115(2):343-8. doi: 10.1073/pnas.1713895115

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 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.

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09/01/17 | A dynamic interplay of enhancer elements regulates Klf4 expression in naïve pluripotency.
Xie L, Torigoe SE, Xiao J, Mai DH, Li L, Davis FP, Dong P, Marie-Nelly H, Grimm J, Lavis L, Darzacq X, Cattoglio C, Liu Z, Tjian R
Genes & Development. 2017 Sep 01;31(17):1795-1808. doi: 10.1101/gad.303321.117

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.

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07/28/17 | Myc Regulates Chromatin Decompaction and Nuclear Architecture during B Cell Activation.
Kieffer-Kwon K, Nimura K, Rao SS, Xu J, Jung S, Pekowska A, Dose M, Stevens E, Mathe E, Dong P, Huang S, Ricci MA, Baranello L, Zheng Y, Ardori FT, Resch W, Stavreva D, Nelson S, McAndrew M, Casellas A, Finn E, Gregory C, St Hilaire BG, Johnson SM, Dubois W, Cosma MP, Batchelor E, Levens D, Phair RD, Misteli T, Tessarollo L, Hager G, Lakadamyali M, Liu Z, Floer M, Shroff H, Aiden EL, Casellas R
Molecular Cell. 2017 Jul 28;67(4):566-78. doi: 10.1016/j.molcel.2017.07.013

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.

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05/01/17 | Shaping development by stochasticity and dynamics in gene regulation.
Dong P, Liu Z
Open Biology. 2017 May;7(5):. doi: 10.1098/rsob.170030

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.

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03/13/17 | Stochastic protein labeling enables long-term single molecule observation in vivo.
Liu H, Dong P, Ioannou MS, Li L, Shea J, Pasolli HA, Grimm JB, Rivlin PK, Lavis LD, Koyama M, Liu Z
bioRxiv. 2017 Mar 13:. doi: 10.1101/116186

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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08/03/16 | Real-time imaging of Huntingtin aggregates diverting target search and gene transcription.
Li L, Liu H, Dong P, Li D, Legant WR, Grimm JB, Lavis LD, Betzig E, Tjian R, Liu Z
eLife. 2016 Aug 03;5:. doi: 10.7554/eLife.17056

The presumptive altered dynamics of transient molecular interactions in vivo contributing to neurodegenerative diseases have remained elusive. Here, using single-molecule localization microscopy, we show that disease-inducing Huntingtin (mHtt) protein fragments display three distinct dynamic states in living cells - 1) fast diffusion, 2) dynamic clustering and 3) stable aggregation. Large, stable aggregates of mHtt exclude chromatin and form 'sticky' decoy traps that impede target search processes of key regulators involved in neurological disorders. Functional domain mapping based on super-resolution imaging reveals an unexpected role of aromatic amino acids in promoting protein-mHtt aggregate interactions. Genome-wide expression analysis and numerical simulation experiments suggest mHtt aggregates reduce transcription factor target site sampling frequency and impair critical gene expression programs in striatal neurons. Together, our results provide insights into how mHtt dynamically forms aggregates and disrupts the finely-balanced gene control mechanisms in neuronal cells.

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07/29/16 | Bright photoactivatable fluorophores for single-molecule imaging.
Lavis LD, Grimm JB, English BP, Choi H, Muthusamy AK, Mehl BP, Dong P, Brown TA, Lippincott-Schwartz J, Liu Z, Lionnet T
bioRxiv. 2016 Jul 29:066779. doi: 10.1101/066779

Small molecule fluorophores are important tools for advanced imaging experiments. The development of self-labeling protein tags such as the HaloTag and SNAP-tag has expanded the utility of chemical dyes in live-cell microscopy. We recently described a general method for improving the brightness and photostability of small, cell-permeable fluorophores, resulting in the novel azetidine-containing "Janelia Fluor" (JF) dyes. Here, we refine and extend the utility of the JF dyes by synthesizing photoactivatable derivatives that are compatible with live cell labeling strategies. These compounds retain the superior brightness of the JF dyes once activated, but their facile photoactivation also enables improved single-particle tracking and localization microscopy experiments.

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03/21/16 | Emerging imaging and genomic tools for developmental systems biology.
Liu Z, Keller PJ
Developmental Cell. 2016 Mar 21;36(6):597-610. doi: 10.1016/j.devcel.2016.02.016

Animal development is a complex and dynamic process orchestrated by exquisitely timed cell lineage commitment, divisions, migration, and morphological changes at the single-cell level. In the past decade, extensive genetic, stem cell, and genomic studies provided crucial insights into molecular underpinnings and the functional importance of genetic pathways governing various cellular differentiation processes. However, it is still largely unknown how the precise coordination of these pathways is achieved at the whole-organism level and how the highly regulated spatiotemporal choreography of development is established in turn. Here, we discuss the latest technological advances in imaging and single-cell genomics that hold great promise for advancing our understanding of this intricate process. We propose an integrated approach that combines such methods to quantitatively decipher in vivo cellular dynamic behaviors and their underlying molecular mechanisms at the systems level with single-cell, single-molecule resolution.

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03/21/16 | Emerging imaging and genomic tools for developmental systems biology.
Liu Z, Keller PJ
Developmental Cell. 2016 Mar 21;36(6):597-610. doi: 10.1016/j.devcel.2016.02.016

Animal development is a complex and dynamic process orchestrated by exquisitely timed cell lineage commitment, divisions, migration, and morphological changes at the single-cell level. In the past decade, extensive genetic, stem cell, and genomic studies provided crucial insights into molecular underpinnings and the functional importance of genetic pathways governing various cellular differentiation processes. However, it is still largely unknown how the precise coordination of these pathways is achieved at the whole-organism level and how the highly regulated spatiotemporal choreography of development is established in turn. Here, we discuss the latest technological advances in imaging and single-cell genomics that hold great promise for advancing our understanding of this intricate process. We propose an integrated approach that combines such methods to quantitatively decipher in vivo cellular dynamic behaviors and their underlying molecular mechanisms at the systems level with single-cell, single-molecule resolution.

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