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8 Publications

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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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    01/16/16 | Imaging transcription: past, present, and future.
    Coleman RA, Liu Z, Darzacq X, Tjian R, Singer RH, Lionnet T
    Cold Spring Harbor Symposia on Quantitative Biology. 2015;80:1-8. doi: 10.1101/sqb.2015.80.027201

    Transcription, the first step of gene expression, is exquisitely regulated in higher eukaryotes to ensure correct development and homeostasis. Traditional biochemical, genetic, and genomic approaches have proved successful at identifying factors, regulatory sequences, and potential pathways that modulate transcription. However, they typically only provide snapshots or population averages of the highly dynamic, stochastic biochemical processes involved in transcriptional regulation. Single-molecule live-cell imaging has, therefore, emerged as a complementary approach capable of circumventing these limitations. By observing sequences of molecular events in real time as they occur in their native context, imaging has the power to derive cause-and-effect relationships and quantitative kinetics to build predictive models of transcription. Ongoing progress in fluorescence imaging technology has brought new microscopes and labeling technologies that now make it possible to visualize and quantify the transcription process with single-molecule resolution in living cells and animals. Here we provide an overview of the evolution and current state of transcription imaging technologies. We discuss some of the important concepts they uncovered and present possible future developments that might solve long-standing questions in transcriptional regulation.

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    11/13/15 | Dynamics of CRISPR-Cas9 genome interrogation in living cells.
    Knight SC, Xie L, Deng W, Guglielmi B, Witkowsky LB, Bosanac L, Zhang ET, El Beheiry M, Masson J, Dahan M, Liu Z, Doudna JA, Tjian R
    Science (New York, N.Y.). 2015 Nov 13;350(6262):823-6. doi: 10.1126/science.aac6572

    The RNA-guided CRISPR-associated protein Cas9 is used for genome editing, transcriptional modulation, and live-cell imaging. Cas9-guide RNA complexes recognize and cleave double-stranded DNA sequences on the basis of 20-nucleotide RNA-DNA complementarity, but the mechanism of target searching in mammalian cells is unknown. Here, we use single-particle tracking to visualize diffusion and chromatin binding of Cas9 in living cells. We show that three-dimensional diffusion dominates Cas9 searching in vivo, and off-target binding events are, on average, short-lived (<1 second). Searching is dependent on the local chromatin environment, with less sampling and slower movement within heterochromatin. These results reveal how the bacterial Cas9 protein interrogates mammalian genomes and navigates eukaryotic chromatin structure.

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    09/22/15 | A specific E3 ligase/deubiquitinase pair modulates TBP protein levels during muscle differentiation.
    Li L, Martinez SS, Hu W, Liu Z, Tjian R
    eLife. 2015;4:. doi: 10.7554/eLife.08536

    TFIID-a complex of TATA-binding protein (TBP) and TBP-associated factors (TAFs)-is a central component of the Pol II promoter recognition apparatus. Recent studies have revealed significant downregulation of TFIID subunits in terminally differentiated myocytes, hepatocytes and adipocytes. Here, we report that TBP protein levels are tightly regulated by the ubiquitin-proteasome system. Using an in vitro ubiquitination assay coupled with biochemical fractionation, we identified Huwe1 as an E3 ligase targeting TBP for K48-linked ubiquitination and proteasome-mediated degradation. Upregulation of Huwe1 expression during myogenesis induces TBP degradation and myotube differentiation. We found that Huwe1 activity on TBP is antagonized by the deubiquitinase USP10, which protects TBP from degradation. Thus, modulating the levels of both Huwe1 and USP10 appears to fine-tune the requisite degradation of TBP during myogenesis. Together, our study unmasks a previously unknown interplay between an E3 ligase and a deubiquitinating enzyme regulating TBP levels during cellular differentiation.

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    12/24/14 | 3D imaging of Sox2 enhancer clusters in embryonic stem cells.
    Liu Z, Legant WR, Chen B, Li L, Grimm JB, Lavis LD, Betzig E, Tjian R
    eLife. 2014 Dec 24;3:. doi: 10.7554/eLife.04236

    Combinatorial cis-regulatory networks encoded in animal genomes represent the foundational gene expression mechanism for directing cell-fate commitment and maintenance of cell identity by transcription factors (TFs). However, the 3D spatial organization of cis-elements and how such sub-nuclear structures influence TF activity remain poorly understood. Here, we combine lattice light-sheet imaging, single-molecule tracking, numerical simulations, and ChIP-exo mapping to localize and functionally probe Sox2 enhancer-organization in living embryonic stem cells. Sox2 enhancers form 3D-clusters that are segregated from heterochromatin but overlap with a subset of Pol II enriched regions. Sox2 searches for specific binding targets via a 3D-diffusion dominant mode when shuttling long-distances between clusters while chromatin-bound states predominate within individual clusters. Thus, enhancer clustering may reduce global search efficiency but enables rapid local fine-tuning of TF search parameters. Our results suggest an integrated model linking cis-element 3D spatial distribution to local-versus-global target search modalities essential for regulating eukaryotic gene transcription.

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    03/13/14 | Single-molecule dynamics of enhanceosome assembly in embryonic stem cells.
    Chen J, Zhang Z, Li Li , Chen B, Revyakin A, Hajj B, Legant W, Dahan M, Lionnet T, Betzig E, Tjian R, Liu Z
    Cell. 2014 Mar 13;156:1274-85. doi: 10.1016/j.cell.2014.01.062

    Enhancer-binding pluripotency regulators (Sox2 and Oct4) play a seminal role in embryonic stem (ES) cell-specific gene regulation. Here, we combine in vivo and in vitro single-molecule imaging, transcription factor (TF) mutagenesis, and ChIP-exo mapping to determine how TFs dynamically search for and assemble on their cognate DNA target sites. We find that enhanceosome assembly is hierarchically ordered with kinetically favored Sox2 engaging the target DNA first, followed by assisted binding of Oct4. Sox2/Oct4 follow a trial-and-error sampling mechanism involving 84-97 events of 3D diffusion (3.3-3.7 s) interspersed with brief nonspecific collisions (0.75-0.9 s) before acquiring and dwelling at specific target DNA (12.0-14.6 s). Sox2 employs a 3D diffusion-dominated search mode facilitated by 1D sliding along open DNA to efficiently locate targets. Our findings also reveal fundamental aspects of gene and developmental regulation by fine-tuning TF dynamics and influence of the epigenome on target search parameters.

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    Tjian LabLiu (Zhe) Lab
    03/10/14 | Charting Brachyury-mediated developmental pathways during early mouse embryogenesis.
    Lolas M, Valenzuela PD, Tjian R, Liu Z
    Proceedings of the National Academy of Sciences of the United States of America. 2014 Mar 10;111(12):4478-83. doi: 10.1073/pnas.1402612111

    To gain insights into coordinated lineage-specification and morphogenetic processes during early embryogenesis, here we report a systematic identification of transcriptional programs mediated by a key developmental regulator-Brachyury. High-resolution chromosomal localization mapping of Brachyury by ChIP sequencing and ChIP-exonuclease revealed distinct sequence signatures enriched in Brachyury-bound enhancers. A combination of genome-wide in vitro and in vivo perturbation analysis and cross-species evolutionary comparison unveiled a detailed Brachyury-dependent gene-regulatory network that directly links the function of Brachyury to diverse developmental pathways and cellular housekeeping programs. We also show that Brachyury functions primarily as a transcriptional activator genome-wide and that an unexpected gene-regulatory feedback loop consisting of Brachyury, Foxa2, and Sox17 directs proper stem-cell lineage commitment during streak formation. Target gene and mRNA-sequencing correlation analysis of the T(c) mouse model supports a crucial role of Brachyury in up-regulating multiple key hematopoietic and muscle-fate regulators. Our results thus chart a comprehensive map of the Brachyury-mediated gene-regulatory network and how it influences in vivo developmental homeostasis and coordination.

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    Tjian LabLiu (Zhe) Lab
    09/02/11 | Control of embryonic stem cell lineage commitment by core promoter factor, TAF3.
    Liu Z, Scannell DR, Eisen MB, Tjian R
    Cell. 2011 Sep 2;146(5):720-31. doi: 10.1016/j.cell.2011.08.005

    Deciphering the molecular basis of pluripotency is fundamental to our understanding of development and embryonic stem cell function. Here, we report that TAF3, a TBP-associated core promoter factor, is highly enriched in ES cells. In this context, TAF3 is required for endoderm lineage differentiation and prevents premature specification of neuroectoderm and mesoderm. In addition to its role in the core promoter recognition complex TFIID, genome-wide binding studies reveal that TAF3 localizes to a subset of chromosomal regions bound by CTCF/cohesin that are selectively associated with genes upregulated by TAF3. Notably, CTCF directly recruits TAF3 to promoter distal sites and TAF3-dependent DNA looping is observed between the promoter distal sites and core promoters occupied by TAF3/CTCF/cohesin. Together, our findings support a new role of TAF3 in mediating long-range chromatin regulatory interactions that safeguard the finely-balanced transcriptional programs underlying pluripotency.

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