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  1. Article ; Online: Transcription Factor Dynamics.

    Lu, Feiyue / Lionnet, Timothée

    Cold Spring Harbor perspectives in biology

    2021  Volume 13, Issue 11

    Abstract: To predict transcription, one needs a mechanistic understanding of how the numerous required transcription factors (TFs) explore the nuclear space to find their target genes, assemble, cooperate, and compete with one another. Advances in fluorescence ... ...

    Abstract To predict transcription, one needs a mechanistic understanding of how the numerous required transcription factors (TFs) explore the nuclear space to find their target genes, assemble, cooperate, and compete with one another. Advances in fluorescence microscopy have made it possible to visualize real-time TF dynamics in living cells, leading to two intriguing observations: first, most TFs contact chromatin only transiently; and second, TFs can assemble into clusters through their intrinsically disordered regions. These findings suggest that highly dynamic events and spatially structured nuclear microenvironments might play key roles in transcription regulation that are not yet fully understood. The emerging model is that while some promoters directly convert TF-binding events into on/off cycles of transcription, many others apply complex regulatory layers that ultimately lead to diverse phenotypic outputs. Cracking this kinetic code is an ongoing and challenging task that is made possible by combining innovative imaging approaches with biophysical models.
    MeSH term(s) Chromatin/metabolism ; Gene Expression Regulation ; Microscopy, Fluorescence ; Protein Aggregates ; Transcription Factors/metabolism ; Transcription, Genetic
    Chemical Substances Chromatin ; Protein Aggregates ; Transcription Factors
    Language English
    Publishing date 2021-11-01
    Publishing country United States
    Document type Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't ; Review
    ISSN 1943-0264
    ISSN (online) 1943-0264
    DOI 10.1101/cshperspect.a040949
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  2. Article ; Online: Single-molecule tracking of transcription protein dynamics in living cells: seeing is believing, but what are we seeing?

    Lionnet, Timothée / Wu, Carl

    Current opinion in genetics & development

    2021  Volume 67, Page(s) 94–102

    Abstract: A universe of transcription factors (TFs), cofactors, as well as chromatin remodeling and modifying enzymes combine or compete on chromatin to control transcription. Measuring quantitatively how these proteins dynamically interact is required in order to ...

    Abstract A universe of transcription factors (TFs), cofactors, as well as chromatin remodeling and modifying enzymes combine or compete on chromatin to control transcription. Measuring quantitatively how these proteins dynamically interact is required in order to formulate models with predictive ability to elucidate transcription control mechanisms. Single molecule tracking (SMT) provides a powerful tool towards this goal: it is a fluorescence microscopy approach that measures the location and mobility of individual TF molecules, as well as their rates of association with and dissociation from chromatin in the physiological context of the living cell. Here we review SMT principles, and discuss key TF properties uncovered by live-cell SMT, such as fast turnover (seconds), and formation of clusters that locally increase activity.
    MeSH term(s) Chromatin/genetics ; Chromatin/ultrastructure ; Chromatin Assembly and Disassembly/genetics ; Chromosomes/genetics ; Chromosomes/ultrastructure ; Gene Expression Regulation/genetics ; Humans ; Protein Binding/genetics ; Single Molecule Imaging ; Transcription Factors/genetics ; Transcription, Genetic
    Chemical Substances Chromatin ; Transcription Factors
    Language English
    Publishing date 2021-01-07
    Publishing country England
    Document type Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't ; Review
    ZDB-ID 1077312-5
    ISSN 1879-0380 ; 0959-437X
    ISSN (online) 1879-0380
    ISSN 0959-437X
    DOI 10.1016/j.gde.2020.12.001
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  3. Article: Connecting Chromatin Structures to Gene Regulation Using Dynamic Polymer Simulations.

    Fu, Yi / Clark, Finnegan / Nomikou, Sofia / Tsirigos, Aristotelis / Lionnet, Timothee

    bioRxiv : the preprint server for biology

    2023  

    Abstract: The transfer of regulatory information between distal loci on chromatin is thought to involve physical proximity, but key biophysical features of these contacts remain unclear. For instance, it is unknown how close and for how long two loci need to be in ...

    Abstract The transfer of regulatory information between distal loci on chromatin is thought to involve physical proximity, but key biophysical features of these contacts remain unclear. For instance, it is unknown how close and for how long two loci need to be in order to productively interact. The main challenge is that it is currently impossible to measure chromatin dynamics with high spatiotemporal resolution at scale. Polymer simulations provide an accessible and rigorous way to test biophysical models of chromatin regulation, yet there is a lack of simple and general methods for extracting the values of model parameters. Here we adapt the Nelder-Mead simplex optimization algorithm to select the best polymer model matching a given Hi-C dataset, using the
    Language English
    Publishing date 2023-11-10
    Publishing country United States
    Document type Preprint
    DOI 10.1101/2023.11.07.566032
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  4. Article: Integrator-mediated clustering of poised RNA polymerase II synchronizes histone transcription.

    Lu, Feiyue / Park, Brandon J / Fujiwara, Rina / Wilusz, Jeremy E / Gilmour, David S / Lehmann, Ruth / Lionnet, Timothée

    bioRxiv : the preprint server for biology

    2024  

    Abstract: Numerous components of the transcription machinery, including RNA polymerase II (Pol II), accumulate in regions of high local concentration known as clusters, which are thought to facilitate transcription. Using the histone locus of : One sentence ... ...

    Abstract Numerous components of the transcription machinery, including RNA polymerase II (Pol II), accumulate in regions of high local concentration known as clusters, which are thought to facilitate transcription. Using the histone locus of
    One sentence summary: Using the
    Language English
    Publishing date 2024-01-04
    Publishing country United States
    Document type Preprint
    DOI 10.1101/2023.10.07.561364
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  5. Article: A zebrafish system for identifying genetic dependencies in melanocytes and melanoma.

    Perlee, Sarah / Ma, Yilun / Hunter, Miranda V / Swanson, Jacob B / Ming, Zhitao / Xia, Julia / Lionnet, Timothee / McGrail, Maura / White, Richard

    bioRxiv : the preprint server for biology

    2024  

    Abstract: The advent of large-scale sequencing in both development and disease has identified large numbers of candidate genes that may be linked to important phenotypes. Validating the function of these candidates in vivo is challenging, due to low efficiency and ...

    Abstract The advent of large-scale sequencing in both development and disease has identified large numbers of candidate genes that may be linked to important phenotypes. Validating the function of these candidates in vivo is challenging, due to low efficiency and low throughput of most model systems. This is especially the case in skin cells such as melanocytes, where the background mutation rate is high. We have developed a rapid and scalable system for assessing the role of candidate genes in a melanocyte specific manner using zebrafish. We generated transgenic zebrafish in which Cas9 was knocked-in to the endogenous mitfa locus, a master transcription factor of the melanocyte lineage. By introducing single guide RNA expression cassettes into mitfaCas9 embryos, we were able to achieve highly efficient melanocyte-specific mutation of genes important for melanocyte patterning and survival. These animals can be used to screen for dominant or recessive pigment defects in both the F0 generation (3 days) and F1 generation (3 months). We also utilized the mitfaCas9 line to study the role of melanoma genetic dependencies such as SOX10, demonstrating that loss of SOX10 reduces melanoma initiation yet promotes tumor progression by a switch to a SOX9hi state. This SOX10 to SOX9 switch has previously been observed in human patients, indicating that our system can be used to rapidly uncover biological states with relevance to human disease. Our high efficiency genetic approach can be readily applied to other cell lineages, with relevance to both development and disease.
    Language English
    Publishing date 2024-03-28
    Publishing country United States
    Document type Preprint
    DOI 10.1101/2024.03.22.586101
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  6. Article ; Online: Imaging the transcriptome.

    Lionnet, Timothée

    Molecular systems biology

    2013  Volume 9, Page(s) 710

    MeSH term(s) Animals ; Automation, Laboratory ; Gene Expression Profiling ; Genetic Heterogeneity ; Humans ; In Situ Hybridization, Fluorescence/methods ; Molecular Imaging/methods ; Transcriptome
    Language English
    Publishing date 2013-11-26
    Publishing country England
    Document type News ; Research Support, Non-U.S. Gov't
    ISSN 1744-4292
    ISSN (online) 1744-4292
    DOI 10.1038/msb.2013.67
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  7. Article ; Online: Imaging the Life and Death of mRNAs in Single Cells.

    Chao, Jeffrey A / Lionnet, Timothée

    Cold Spring Harbor perspectives in biology

    2018  Volume 10, Issue 12

    Abstract: RNA plays a central role in gene expression from its transcription in the nucleus through translation and degradation in the cytoplasm. Technological advances in fluorescent microscopy and labeling methodologies have made it possible to detect single ... ...

    Abstract RNA plays a central role in gene expression from its transcription in the nucleus through translation and degradation in the cytoplasm. Technological advances in fluorescent microscopy and labeling methodologies have made it possible to detect single molecules of RNA in both fixed and living cells. Here, we focus on the recent developments in RNA imaging that have allowed quantitatively measuring the lives of individual transcripts from birth to death and all the events in between in single cells and tissues. Direct observation of RNAs within their native cellular environment has revealed a complex layer of spatial and temporal regulation that has profoundly impacted our understanding of RNA biology.
    MeSH term(s) Animals ; Gene Expression Regulation/physiology ; Humans ; RNA, Messenger/metabolism ; Single-Cell Analysis/methods
    Chemical Substances RNA, Messenger
    Language English
    Publishing date 2018-12-03
    Publishing country United States
    Document type Journal Article ; Research Support, Non-U.S. Gov't ; Review
    ISSN 1943-0264
    ISSN (online) 1943-0264
    DOI 10.1101/cshperspect.a032086
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  8. Article ; Online: Single-Molecule Sensitivity RNA FISH Analysis of Influenza Virus Genome Trafficking.

    Chou, Yi-Ying / Lionnet, Timothée

    Methods in molecular biology (Clifton, N.J.)

    2018  Volume 1836, Page(s) 195–211

    Abstract: Influenza A virus is an enveloped virus with a segmented genome consisting of eight negative-sense, single-stranded RNAs. Accumulating evidence has revealed that influenza viruses selectively package their genomes. However, less is known about how ... ...

    Abstract Influenza A virus is an enveloped virus with a segmented genome consisting of eight negative-sense, single-stranded RNAs. Accumulating evidence has revealed that influenza viruses selectively package their genomes. However, less is known about how different viral RNA segments are selected for incorporation into progeny virions. Understanding the trafficking routes and assembly process of various viral RNA segments during infection will shed light on the mechanisms of selective genome packaging for influenza A viruses. This chapter describes the single-molecule sensitivity RNA fluorescence in situ hybridization assay (smFISH) for influenza viral RNAs, a method used to analyze the distributions and trafficking of viral RNAs in infected cells with segment specificity. Hybridization using 20 or more short fluorescently labeled DNA probes allows the detection of viral RNAs with single-molecule sensitivity. The following imaging analyses provide information regarding quantitative measurements of vRNA abundance and the relative positions of the different viral RNA segments in cells. This chapter also includes a protocol for combining immunofluorescence techniques with smFISH, which is useful to analyze the positions of viral RNAs relative to viral/cellular proteins in infected cells.
    MeSH term(s) Animals ; Cell Line ; Fluorescent Dyes ; Genome, Viral ; Humans ; Image Processing, Computer-Assisted ; In Situ Hybridization, Fluorescence/methods ; Influenza A virus/genetics ; Influenza, Human/diagnosis ; Influenza, Human/virology ; RNA, Viral ; Single Molecule Imaging/methods
    Chemical Substances Fluorescent Dyes ; RNA, Viral
    Language English
    Publishing date 2018-08-24
    Publishing country United States
    Document type Journal Article
    ISSN 1940-6029
    ISSN (online) 1940-6029
    DOI 10.1007/978-1-4939-8678-1_10
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  9. Article ; Online: mRNA quantification using single-molecule FISH in Drosophila embryos.

    Trcek, Tatjana / Lionnet, Timothée / Shroff, Hari / Lehmann, Ruth

    Nature protocols

    2016  Volume 12, Issue 7, Page(s) 1326–1348

    Abstract: Spatial information is critical to the interrogation of developmental and tissue-level regulation of gene expression. However, this information is usually lost when global mRNA levels from tissues are measured using reverse transcriptase PCR, microarray ... ...

    Abstract Spatial information is critical to the interrogation of developmental and tissue-level regulation of gene expression. However, this information is usually lost when global mRNA levels from tissues are measured using reverse transcriptase PCR, microarray analysis or high-throughput sequencing. By contrast, single-molecule fluorescence in situ hybridization (smFISH) preserves the spatial information of the cellular mRNA content with subcellular resolution within tissues. Here we describe an smFISH protocol that allows for the quantification of single mRNAs in Drosophila embryos, using commercially available smFISH probes (e.g., short fluorescently labeled DNA oligonucleotides) in combination with wide-field epifluorescence, confocal or instant structured illumination microscopy (iSIM, a super-resolution imaging approach) and a spot-detection algorithm. Fixed Drosophila embryos are hybridized in solution with a mixture of smFISH probes, mounted onto coverslips and imaged in 3D. Individual fluorescently labeled mRNAs are then localized within tissues and counted using spot-detection software to generate quantitative, spatially resolved gene expression data sets. With minimum guidance, a graduate student can successfully implement this protocol. The smFISH procedure described here can be completed in 4-5 d.
    MeSH term(s) Animals ; Drosophila/embryology ; Gene Expression Regulation, Developmental ; In Situ Hybridization, Fluorescence/methods ; RNA, Messenger/analysis ; RNA, Messenger/genetics ; Spatio-Temporal Analysis
    Chemical Substances RNA, Messenger
    Language English
    Publishing date 2016-06-08
    Publishing country England
    Document type Journal Article
    ZDB-ID 2244966-8
    ISSN 1750-2799 ; 1754-2189
    ISSN (online) 1750-2799
    ISSN 1754-2189
    DOI 10.1038/nprot.2017.030
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  10. Article ; Online: Transcription goes digital.

    Lionnet, Timothée / Singer, Robert H

    EMBO reports

    2012  Volume 13, Issue 4, Page(s) 313–321

    Abstract: Transcription is a complex process that integrates the state of the cell and its environment to generate adequate responses for cell fitness and survival. Recent microscopy experiments have been able to monitor transcription from single genes in ... ...

    Abstract Transcription is a complex process that integrates the state of the cell and its environment to generate adequate responses for cell fitness and survival. Recent microscopy experiments have been able to monitor transcription from single genes in individual cells. These observations have revealed two striking features: transcriptional activity can vary markedly from one cell to another, and is subject to large changes over time, sometimes within minutes. How the chromatin structure, transcription machinery assembly and signalling networks generate such patterns is still unclear. In this review, we present the techniques used to investigate transcription from single genes, introduce quantitative modelling tools, and discuss transcription mechanisms and their implications for gene expression regulation.
    MeSH term(s) Animals ; Gene Expression Regulation ; Genes/genetics ; Humans ; Models, Genetic ; Stochastic Processes ; Transcription, Genetic
    Language English
    Publishing date 2012-04-02
    Publishing country England
    Document type Journal Article ; Research Support, N.I.H., Extramural ; Review
    ZDB-ID 2020896-0
    ISSN 1469-3178 ; 1469-221X
    ISSN (online) 1469-3178
    ISSN 1469-221X
    DOI 10.1038/embor.2012.31
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    In stock of ZB MED Cologne/Königswinter

    Zs.A 5433: Show issues Location:
    Je nach Verfügbarkeit (siehe Angabe bei Bestand)
    bis Jg. 1994: Bestellungen von Artikeln über das Online-Bestellformular
    Jg. 1995 - 2021: Lesesall (2.OG)
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    Zs.MG 41: Show issues

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