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10 Janelia Publications

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    12/22/14 | Whole-cell, multicolor superresolution imaging using volumetric multifocus microscopy.
    Hajj B, Wisniewski J, El Beheiry M, Chen J, Revyakin A, Wu C, Dahan M
    Proceedings of the National Academy of Sciences of the United States of America. 2014 Dec 9;111(49):17480-5. doi: 10.1073/pnas.1412396111

    Single molecule-based superresolution imaging has become an essential tool in modern cell biology. Because of the limited depth of field of optical imaging systems, one of the major challenges in superresolution imaging resides in capturing the 3D nanoscale morphology of the whole cell. Despite many previous attempts to extend the application of photo-activated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM) techniques into three dimensions, effective localization depths do not typically exceed 1.2 µm. Thus, 3D imaging of whole cells (or even large organelles) still demands sequential acquisition at different axial positions and, therefore, suffers from the combined effects of out-of-focus molecule activation (increased background) and bleaching (loss of detections). Here, we present the use of multifocus microscopy for volumetric multicolor superresolution imaging. By simultaneously imaging nine different focal planes, the multifocus microscope instantaneously captures the distribution of single molecules (either fluorescent proteins or synthetic dyes) throughout an ∼4-µm-deep volume, with lateral and axial localization precisions of ∼20 and 50 nm, respectively. The capabilities of multifocus microscopy to rapidly image the 3D organization of intracellular structures are illustrated by superresolution imaging of the mammalian mitochondrial network and yeast microtubules during cell division.

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    12/11/14 | Regulation of RNA polymerase II activation by histone acetylation in single living cells.
    Stasevich TJ, Hayashi-Takanaka Y, Sato Y, Maehara K, Ohkawa Y, Sakata-Sogawa K, Tokunaga M, Nagase T, Nozaki N, McNally JG, Kimura H
    Nature. 2014 Dec 11;516(7530):272-5. doi: 10.1038/nature13714

    In eukaryotic cells, post-translational histone modifications have an important role in gene regulation. Starting with early work on histone acetylation, a variety of residue-specific modifications have now been linked to RNA polymerase II (RNAP2) activity, but it remains unclear if these markers are active regulators of transcription or just passive byproducts. This is because studies have traditionally relied on fixed cell populations, meaning temporal resolution is limited to minutes at best, and correlated factors may not actually be present in the same cell at the same time. Complementary approaches are therefore needed to probe the dynamic interplay of histone modifications and RNAP2 with higher temporal resolution in single living cells. Here we address this problem by developing a system to track residue-specific histone modifications and RNAP2 phosphorylation in living cells by fluorescence microscopy. This increases temporal resolution to the tens-of-seconds range. Our single-cell analysis reveals histone H3 lysine-27 acetylation at a gene locus can alter downstream transcription kinetics by as much as 50%, affecting two temporally separate events. First acetylation enhances the search kinetics of transcriptional activators, and later the acetylation accelerates the transition of RNAP2 from initiation to elongation. Signatures of the latter can be found genome-wide using chromatin immunoprecipitation followed by sequencing. We argue that this regulation leads to a robust and potentially tunable transcriptional response.

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    The hemoglobinopathies, such as β-thalassemia and sickle cell anemia (SCA), are characterized by mutations of the β-globin gene resulting in either decreased or functionally abnormal hemoglobin (Hb) production. As bone marrow transplant is the only curative option for these patients, there is a strong need for new therapeutic approaches. Both β-thalassemia and SCA represent ideal targets for gene therapy since introduction of a normal β-globin gene can ameliorate the phenotype, as we and others have shown previously. Overcoming the developmental silencing of the fetal γ-globin gene represents an additional approach for the treatment of hemoglobinopathies. Here, we directly compare a recently established approach to activate the γ-globin gene using forced chromatin looping with pharmacologic approaches to raise γ-globin expression.

    The β-type globin genes are activated through dynamic interactions with a distal upstream enhancer, the locus control region (LCR). The LCR physically contacts the developmental stage appropriate globin gene via chromatin looping, a process partially dependent on the protein Ldb1. Previously, we have shown that tethering Ldb1 to the murine β-globin promoter with a custom designed zinc finger protein (ZF-Ldb1) can induce loop formation and β-globin transcription in an erythroid cell line (Deng et al., 2012). Further work showed that forced chromatin looping can be exploited to potently reactivate fetal globin gene expression in adult human erythroid cells (Deng et al., 2014). Here we compared the efficacy and toxicity of ZF-Ldb1 to pharmacologic compounds that induce HbF in cultured hematopoietic stem progenitor cell-derived erythroid cultures from normal and SCA donors.

    ZF-Ldb1 increased HbF synthesis in SCA erythroid cells (N=8) up to 86% and, concurrently, reduced sickle Hb (HbS) below 15%, consistent with previous studies of erythroid cells from normal probands. Preliminary results obtained from treating SCA specimens (N=3) show that the induction of HbF in cells treated with ZF-Ldb1 is twice as high (+35.55% ± 8.34%, at a dose of ~ one ZF-Ldb1 transgene copy per cell) as that observed using pomalidomide (+16.50% ± 14.57%, 20μM) and decitabine (+15.60% ± 12.36%, 0.5μM). Tranylcypromine and hydroxyurea showed the lowest HbF increase (+9.67% ± 3.26% and +5.06 ± 2.82%, 1.5μM and 150μM respectively).

    Importantly, decitabine and pomalidomide treatment lowered cell viability to 39% and 26%, respectively, while ZF-Ldb1 expressing cells retained normal viability similar to control populations.

    In related experiments, we are comparing the expression of a battery of genes known to regulate HbF levels (BCL11A, SOX6, KLF1 and C-Myb) in normal and SCA derived erythroid cells treated with ZF-Ldb1 or HbF inducers and compared to controls. Preliminary analyses indicate altered expression of KLF1 in SCA versus normal cells, consistent with a superior response of SCA cells to HbF induction.

    In conclusion, lentiviral-mediated ZF-Ldb1 gene transfer appears superior to pharmacologic compounds in terms of efficacy and cell viability further supporting suitability for the reactivation of HbF in SCA erythroid cells.

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    12/01/14 | Quantifying histone and RNA polymerase II post-translational modification dynamics in mother and daughter cells.
    Stasevich TJ, Sato Y, Nozaki N, Kimura H
    Methods. 2014 Dec;70(2-3):77-88. doi: 10.1016/j.ymeth.2014.08.002

    Post-translational histone modifications are highly correlated with transcriptional activity, but the relative timing of these marks and their dynamic interplay during gene regulation remains controversial. To shed light on this problem and clarify the connections between histone modifications and transcription, we demonstrate how FabLEM (Fab-based Live Endogenous Modification labeling) can be used to simultaneously track histone H3 Lysine 9 acetylation (H3K9ac) together with RNA polymerase II Serine 2 and Serine 5 phosphorylation (RNAP2 Ser2ph/Ser5ph) in single living cells and their progeny. We provide a detailed description of the FabLEM methodology, including helpful tips for preparing and loading fluorescently conjugated antigen binding fragments (Fab) into cells for optimal results. We also introduce simple procedures for analyzing and visualizing FabLEM data, including color-coded scatterplots to track correlations between modifications through the cell cycle and temporal cross-correlation analysis to dissect modification dynamics. Using these methods, we find significant correlations that span cell generations, with a relatively strong correlation between H3K9ac and Ser5ph that appears to peak a few hours before mitosis and may reflect the bookmarking of genes for efficient re-initiation following mitosis. The techniques we have developed are broadly applicable and should help clarify how histone modifications dynamically contribute to gene regulation.

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    08/14/14 | Reactivation of developmentally silenced globin genes by forced chromatin looping.
    Deng W, Rupon JW, Krivega I, Breda L, Motta I, Jahn KS, Reik A, Gregory PD, Rivella S, Dean A, Blobel GA
    Cell. 2014 Aug 14;158(4):849-60. doi: 10.1016/j.cell.2014.05.050

    Distal enhancers commonly contact target promoters via chromatin looping. In erythroid cells, the locus control region (LCR) contacts β-type globin genes in a developmental stage-specific manner to stimulate transcription. Previously, we induced LCR-promoter looping by tethering the self-association domain (SA) of Ldb1 to the β-globin promoter via artificial zinc fingers. Here, we show that targeting the SA to a developmentally silenced embryonic globin gene in adult murine erythroblasts triggers its transcriptional reactivation. This activity depends on the LCR, consistent with an LCR-promoter looping mechanism. Strikingly, targeting the SA to the fetal γ-globin promoter in primary adult human erythroblasts increases γ-globin promoter-LCR contacts, stimulating transcription to approximately 85% of total β-globin synthesis, with a reciprocal reduction in adult β-globin expression. Our findings demonstrate that forced chromatin looping can override a stringent developmental gene expression program and suggest a novel approach to control the balance of globin gene transcription for therapeutic applications.

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    07/16/14 | Accessing the third dimension in localization-based super-resolution microscopy.
    Hajj B, El Beheiry M, Izeddin I, Darzacq X, Dahan M
    Physical Chemistry Chemical Physics. 2014 Jul 16;16(31):16340-8. doi: 10.1039/c4cp01380h

    Only a few years after its inception, localization-based super-resolution microscopy has become widely employed in biological studies. Yet, it is primarily used in two-dimensional imaging and accessing the organization of cellular structures at the nanoscale in three dimensions (3D) still poses important challenges. Here, we review optical and computational techniques that enable the 3D localization of individual emitters and the reconstruction of 3D super-resolution images. These techniques are grouped into three main categories: PSF engineering, multiple plane imaging and interferometric approaches. We provide an overview of their technical implementation as well as commentary on their applicability. Finally, we discuss future trends in 3D localization-based super-resolution microscopy.

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    06/12/14 | Single-molecule tracking in live cells reveals distinct target-search strategies of transcription factors in the nucleus.
    Izeddin I, Récamier V, Bosanac L, Cisse II, Boudarene L, Dugast-Darzacq C, Proux F, Bénichou O, Voituriez R, Bensaude O, Dahan M, Darzacq X
    eLife. 2014 Jun 12:e02230. doi: 10.7554/eLife.02230

    Gene regulation relies on transcription factors (TFs) exploring the nucleus searching their targets. So far, most studies have focused on how fast TFs diffuse, underestimating the role of nuclear architecture. We implemented a single-molecule tracking assay to determine TFs dynamics. We found that c-Myc is a global explorer of the nucleus. In contrast, the positive transcription elongation factor P-TEFb is a local explorer that oversamples its environment. Consequently, each c-Myc molecule is equally available for all nuclear sites while P-TEFb reaches its targets in a position-dependent manner. Our observations are consistent with a model in which the exploration geometry of TFs is restrained by their interactions with nuclear structures and not by exclusion. The geometry-controlled kinetics of TFs target-search illustrates the influence of nuclear architecture on gene regulation, and has strong implications on how proteins react in the nucleus and how their function can be regulated in space and time.

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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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    01/28/14 | Single-molecule tracking of the transcription cycle by sub-second RNA detection.
    Zhang Z, Revyakin A, Grimm JB, Lavis LD, Tjian R
    eLife. 2014 Jan 28;3:e01775. doi: 10.7554/eLife.01775

    Transcription is an inherently stochastic, noisy, and multi-step process, in which fluctuations at every step can cause variations in RNA synthesis, and affect physiology and differentiation decisions in otherwise identical cells. However, it has been an experimental challenge to directly link the stochastic events at the promoter to transcript production. Here we established a fast fluorescence in situ hybridization (fastFISH) method that takes advantage of intrinsically unstructured nucleic acid sequences to achieve exceptionally fast rates of specific hybridization (\~{}10e7 M(-1)s(-1)), and allows deterministic detection of single nascent transcripts. Using a prototypical RNA polymerase, we demonstrated the use of fastFISH to measure the kinetic rates of promoter escape, elongation, and termination in one assay at the single-molecule level, at sub-second temporal resolution. The principles of fastFISH design can be used to study stochasticity in gene regulation, to select targets for gene silencing, and to design nucleic acid nanostructures. DOI:

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    01/24/14 | Visualization of dynamics of single endogenous mRNA labeled in live mouse.
    Park HY, Lim H, Yoon YJ, Follenzi A, Nwokafor C, Lopez-Jones M, Meng X, Singer RH
    Science. 2014 Jan 24;343(6169):422-4. doi: 10.1126/science.1239200

    The transcription and transport of messenger RNA (mRNA) are critical steps in regulating the spatial and temporal components of gene expression, but it has not been possible to observe the dynamics of endogenous mRNA in primary mammalian tissues. We have developed a transgenic mouse in which all β-actin mRNA is fluorescently labeled. We found that β-actin mRNA in primary fibroblasts localizes predominantly by diffusion and trapping as single mRNAs. In cultured neurons and acute brain slices, we found that multiple β-actin mRNAs can assemble together, travel by active transport, and disassemble upon depolarization by potassium chloride. Imaging of brain slices revealed immediate early induction of β-actin transcription after depolarization. Studying endogenous mRNA in live mouse tissues provides insight into its dynamic regulation within the context of the cellular and tissue microenvironment.

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