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17 Publications

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    Tjian LabSinger LabTranscription Imaging
    08/31/15 | CASFISH: CRISPR/Cas9-mediated in situ labeling of genomic loci in fixed cells.
    Deng W, Shi X, Tjian R, Lionnet T, Singer RH
    Proceedings of the National Academy of Sciences of the United States of America. 2015 Aug 31;112(38):11870-5. doi: 10.1073/pnas.1515692112

    Direct visualization of genomic loci in the 3D nucleus is important for understanding the spatial organization of the genome and its association with gene expression. Various DNA FISH methods have been developed in the past decades, all involving denaturing dsDNA and hybridizing fluorescent nucleic acid probes. Here we report a novel approach that uses in vitro constituted nuclease-deficient clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated caspase 9 (Cas9) complexes as probes to label sequence-specific genomic loci fluorescently without global DNA denaturation (Cas9-mediated fluorescence in situ hybridization, CASFISH). Using fluorescently labeled nuclease-deficient Cas9 (dCas9) protein assembled with various single-guide RNA (sgRNA), we demonstrated rapid and robust labeling of repetitive DNA elements in pericentromere, centromere, G-rich telomere, and coding gene loci. Assembling dCas9 with an array of sgRNAs tiling arbitrary target loci, we were able to visualize nonrepetitive genomic sequences. The dCas9/sgRNA binary complex is stable and binds its target DNA with high affinity, allowing sequential or simultaneous probing of multiple targets. CASFISH assays using differently colored dCas9/sgRNA complexes allow multicolor labeling of target loci in cells. In addition, the CASFISH assay is remarkably rapid under optimal conditions and is applicable for detection in primary tissue sections. This rapid, robust, less disruptive, and cost-effective technology adds a valuable tool for basic research and genetic diagnosis.

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    08/28/15 | Chemical perturbation of an intrinsically disordered region of TFIID distinguishes two modes of transcription initiation.
    Zhang Z, Boskovic Z, Hussain MM, Hu W, Inouye C, Kim H, Abole AK, Doud MK, Lewis TA, Koehler AN, Schreiber SL, Tjian R
    eLife. 2015 Aug 28;4:. doi: 10.7554/eLife.07777

    Intrinsically disordered protein regions (IDRs) are peptide segments that fail to form stable 3-dimensional structures in the absence of partner proteins. They are abundant in eukaryotic proteomes and are often associated with human diseases, but their biological functions have been elusive to study. Here we report the identification of a tin(IV) oxochloride-derived cluster that binds an evolutionarily conserved IDR within the metazoan TFIID transcription complex. Binding arrests an isomerization of promoter-bound TFIID that is required for the engagement of Pol II during the first (de novo) round of transcription initiation. However, the specific chemical probe does not affect reinitiation, which requires the re-entry of Pol II, thus, mechanistically distinguishing these two modes of transcription initiation. This work also suggests a new avenue for targeting the elusive IDRs by harnessing certain features of metal-based complexes for mechanistic studies, and for the development of novel pharmaceutical interventions.

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    Grigorieff Lab
    08/28/15 | Evaluation of super-resolution performance of the K2 electron-counting camera using 2D crystals of aquaporin-0.
    Chiu P, Li X, Li Z, Beckett B, Brilot AF, Grigorieff N, Agard DA, Cheng Y, Walz T
    Journal of Structural Biology. 2015 Aug 28:. doi: 10.1016/j.jsb.2015.08.015

    The K2 Summit camera was initially the only commercially available direct electron detection camera that was optimized for high-speed counting of primary electrons and was also the only one that implemented centroiding so that the resolution of the camera can be extended beyond the Nyquist limit set by the physical pixel size. In this study, we used well-characterized two-dimensional crystals of the membrane protein aquaporin-0 to characterize the performance of the camera below and beyond the physical Nyquist limit and to measure the influence of electron dose rate on image amplitudes and phases.

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    08/28/15 | Extended-resolution structured illumination imaging of endocytic and cytoskeletal dynamics.
    Li D, Shao L, Chen B, Zhang X, Zhang M, Moses B, Milkie DE, Beach JR, Hammer JA, Pasham M, Kirchhausen T, Baird MA, Davidson MW, Xu P, Betzig E
    Science (New York, N.Y.). 2015 Aug 28;349(6251):. doi: 10.1126/science.aab3500

    Super-resolution fluorescence microscopy is distinct among nanoscale imaging tools in its ability to image protein dynamics in living cells. Structured illumination microscopy (SIM) stands out in this regard because of its high speed and low illumination intensities, but typically offers only a twofold resolution gain. We extended the resolution of live-cell SIM through two approaches: ultrahigh numerical aperture SIM at 84-nanometer lateral resolution for more than 100 multicolor frames, and nonlinear SIM with patterned activation at 45- to 62-nanometer resolution for approximately 20 to 40 frames. We applied these approaches to image dynamics near the plasma membrane of spatially resolved assemblies of clathrin and caveolin, Rab5a in early endosomes, and α-actinin, often in relationship to cortical actin. In addition, we examined mitochondria, actin, and the Golgi apparatus dynamics in three dimensions.

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    08/28/15 | The transgenic RNAi project at Harvard Medical School: resources and validation.
    Perkins LA, Holderbaum L, Tao R, Hu Y, Sopko R, McCall K, Yang-Zhou D, Flockhart I, Binari R, Shim H, Miller A, Housden A, Foos M, Randkelv S, Kelley C, Namgyal P, Villalta C, Liu L, Jiang X, Huan-Huan Q, Xia W, Fujiyama A, Toyoda A, Ayers K, Blum A, Czech B, Neumuller R, Yan D, Cavallaro A, Hibbard K, Hall D, Cooley L, Hannon GJ, Lehmann R, Parks A, Mohr SE, Ueda R, Kondo S, Ni J, Perrimon N
    Genetics. 2015 Aug 28;201(3):843-52. doi: 10.1534/genetics.115.180208

    To facilitate large scale functional studies in Drosophila, the Drosophila Transgenic RNAi Project (TRiP) at Harvard Medical School (HMS) was established along with several goals: developing efficient vectors for RNAi that work in all tissues, generating a genome scale collection of RNAi stocks with input from the community, distributing the lines as they are generated through existing stock centers, validating as many lines as possible using RT-qPCR and phenotypic analyses, and developing tools and web resources for identifying RNAi lines and retrieving existing information on their quality. With these goals in mind, here we describe in detail the various tools we developed and the status of the collection, which is currently comprised of 11,491 lines and covering 71% of Drosophila genes. Data on the characterization of the lines either by RT-qPCR or phenotype is available on a dedicated web site, the RNAi Stock Validation and Phenotypes Project (RSVP; www.flyrnai.org/RSVP.html), and stocks are available from three stock centers, the Bloomington Drosophila Stock Center (USA), National Institute of Genetics (Japan), and TsingHua Fly Center (China).

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    Sternson Lab
    08/26/15 | Optogenetics: 10 years after ChR2 in neurons-views from the community.
    Adamantidis A, Arber S, Bains JS, Bamberg E, Bonci A, Buzsáki G, Cardin JA, Costa RM, Dan Y, Goda Y, Graybiel AM, Häusser M, Hegemann P, Huguenard JR, Insel TR, Janak PH, Johnston D, Josselyn SA, Koch C, Kreitzer AC, Lüscher C, Malenka RC, Miesenböck G, Nagel G, Roska B, Schnitzer MJ, Shenoy KV, Soltesz I, Sternson SM, Tsien RW, Tsien RY, Turrigiano GG, Tye KM, Wilson RI
    Nature Neuroscience. 2015 Aug 26;18(9):1202-12. doi: 10.1038/nn.4106
    Tjian Lab
    08/25/15 | Genome-wide errant targeting by Hairy.
    Kok K, Ay A, Li LM, Arnosti DN
    eLife. 2015 Aug 25;4:. doi: 10.7554/eLife.06394

    Metazoan transcriptional repressors regulate chromatin through diverse histone modifications. Contributions of individual factors to the chromatin landscape in development is difficult to establish, as global surveys reflect multiple changes in regulators. Therefore, we studied the conserved Hairy/Enhancer of Split family repressor Hairy, analyzing histone marks and gene expression in Drosophila embryos. This long-range repressor mediates histone acetylation and methylation in large blocks, with highly context-specific effects on target genes. Most strikingly, Hairy exhibits biochemical activity on many loci that are uncoupled to changes in gene expression. Rather than representing inert binding sites, as suggested for many eukaryotic factors, many regions are targeted errantly by Hairy to modify the chromatin landscape. Our findings emphasize that identification of active cis-regulatory elements must extend beyond the survey of prototypical chromatin marks. We speculate that this errant activity may provide a path for creation of new regulatory elements, facilitating the evolution of novel transcriptional circuits.

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    08/22/15 | C/D box sRNA-guided 2'-O-methylation patterns of archaeal rRNA molecules.
    Dennis PP, Tripp V, Lui L, Lowe T, Randau L
    BMC Genomics. 2015 Aug 22;16(1):632. doi: 10.1186/s12864-015-1839-z

    BACKGROUND: In archaea and eukaryotes, ribonucleoprotein complexes containing small C/D box s(no)RNAs use base pair complementarity to target specific sites within ribosomal RNA for 2'-O-ribose methylation. These modifications aid in the folding and stabilization of nascent rRNA molecules and their assembly into ribosomal particles. The genomes of hyperthermophilic archaea encode large numbers of C/D box sRNA genes, suggesting an increased necessity for rRNA stabilization at extreme growth temperatures.

    RESULTS: We have identified the complete sets of C/D box sRNAs from seven archaea using RNA-Seq methodology. In total, 489 C/D box sRNAs were identified, each containing two guide regions. A combination of computational and manual analyses predicts 719 guide interactions with 16S and 23S rRNA molecules. This first pan-archaeal description of guide sequences identifies (i) modified rRNA nucleotides that are frequently conserved between species and (ii) regions within rRNA that are hotspots for 2'-O-methylation. Gene duplication, rearrangement, mutational drift and convergent evolution of sRNA genes and guide sequences were observed. In addition, several C/D box sRNAs were identified that use their two guides to target locations distant in the rRNA sequence but close in the secondary and tertiary structure. We propose that they act as RNA chaperones and facilitate complex folding events between distant sequences.

    CONCLUSIONS: This pan-archaeal analysis of C/D box sRNA guide regions identified conserved patterns of rRNA 2'-O-methylation in archaea. The interaction between the sRNP complexes and the nascent rRNA facilitates proper folding and the methyl modifications stabilize higher order rRNA structure within the assembled ribosome.

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    Singer Lab
    08/21/15 | A three-camera imaging microscope for high-speed single-molecule tracking and super-resolution imaging in living cells.
    English BP, Singer RH
    Proceedings of SPIE. 2015 Aug 21;9550:955008 . doi: 10.1117/12.2190246

    Our aim is to develop quantitative single-molecule assays to study when and where molecules are interacting inside living cells and where enzymes are active. To this end we present a three-camera imaging microscope for fast tracking of multiple interacting molecules simultaneously, with high spatiotemporal resolution. The system was designed around an ASI RAMM frame using three separate tube lenses and custom multi-band dichroics to allow for enhanced detection efficiency. The frame times of the three Andor iXon Ultra EMCCD cameras are hardware synchronized to the laser excitation pulses of the three excitation lasers, such that the fluorophores are effectively immobilized during frame acquisitions and do not yield detections that are motion-blurred. Stroboscopic illumination allows robust detection from even rapidly moving molecules while minimizing bleaching, and since snapshots can be spaced out with varying time intervals, stroboscopic illumination enables a direct comparison to be made between fast and slow molecules under identical light dosage. We have developed algorithms that accurately track and co-localize multiple interacting biomolecules. The three-color microscope combined with our co-movement algorithms have made it possible for instance to simultaneously image and track how the chromosome environment affects diffusion kinetics or determine how mRNAs diffuse during translation. Such multiplexed single-molecule measurements at a high spatiotemporal resolution inside living cells will provide a major tool for testing models relating molecular architecture and biological dynamics.

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    08/19/15 | Novel behavioral paradigm reveals lower temporal limits on mouse olfactory decisions.
    Resulaj A, Rinberg D
    The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. 2015 Aug 19;35(33):11667-73. doi: 10.1523/JNEUROSCI.4693-14.2015

    UNLABELLED: Temporal limits on perceptual decisions set strict boundaries on the possible underlying neural computations. How odor information is encoded in the olfactory system is still poorly understood. Here, we sought to define the limit on the speed of olfactory processing. To achieve this, we trained mice to discriminate different odor concentrations in a novel behavioral setup with precise odor delivery synchronized to the sniffing cycle. Mice reported their choice by moving a horizontal treadmill with their front limbs. We found that mice reported discriminations of 75% accuracy in 70-90 ms after odor inhalation. For a low concentration and nontrigeminal odorant, this time was 90-140 ms, showing that mice process odor information rapidly even in the absence of trigeminal stimulation. These response times establish, after accounting for odor transduction and motor delays, that olfactory processing can take tens of milliseconds. This study puts a strong limit on the underlying neural computations and suggests that the action potentials forming the neural basis for these decisions are fired in a few tens of milliseconds.

    SIGNIFICANCE STATEMENT: Understanding how sensory information is processed requires different approaches that span multiple levels of investigation from genes to neurons to behavior. Limits on behavioral performance constrain the possible neural mechanisms responsible for specific computations. Using a novel behavioral paradigm, we established that mice can make decisions about odor intensity surprisingly fast. After accounting for sensory and motor delays, the limit on some olfactory neural computations can be as low as a few tens of milliseconds, which suggests that only the first action potentials across a population of neurons contribute to these computations.

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