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Single-cell transcriptomics identifies Gadd45b as a regulator of herpesvirus-reactivating neurons

Hu, Hui-Lan; Srinivas, Kalanghad P; Wang, Shuoshuo; Chao, Moses V; Lionnet, Timothee; Mohr, Ian; Wilson, Angus C; Depledge, Daniel P; Huang, Tony T
Single-cell RNA sequencing (scRNA-seq) is a powerful technique for dissecting the complexity of normal and diseased tissues, enabling characterization of cell diversity and heterogeneous phenotypic states in unprecedented detail. However, this technology has been underutilized for exploring the interactions between the host cell and viral pathogens in latently infected cells. Herein, we use scRNA-seq and single-molecule sensitivity fluorescent in situ hybridization (smFISH) technologies to investigate host single-cell transcriptome changes upon the reactivation of a human neurotropic virus, herpes simplex virus-1 (HSV-1). We identify the stress sensor growth arrest and DNA damage-inducible 45 beta (Gadd45b) as a critical antiviral host factor that regulates HSV-1 reactivation events in a subpopulation of latently infected primary neurons. We show that distinct subcellular localization of Gadd45b correlates with the viral late gene expression program, as well as the expression of the viral transcription factor, ICP4. We propose that a hallmark of a "successful" or "aborted" HSV-1 reactivation state in primary neurons is determined by a unique subcellular localization signature of the stress sensor Gadd45b.
PMID: 34842321
ISSN: 1469-3178
CID: 5065412

Transcription Factor Dynamics

Lu, Feiyue; Lionnet, Timothée
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.
PMID: 34001530
ISSN: 1943-0264
CID: 4902872

Single-molecule imaging of chromatin remodelers reveals role of ATPase in promoting fast kinetics of target search and dissociation from chromatin

Kim, Jee Min; Visanpattanasin, Pat; Jou, Vivian; Liu, Sheng; Tang, Xiaona; Zheng, Qinsi; Li, Kai Yu; Snedeker, Jonathan; Lavis, Luke D; Lionnet, Timothee; Wu, Carl
Conserved ATP-dependent chromatin remodelers establish and maintain genome-wide chromatin architectures of regulatory DNA during cellular lifespan, but the temporal interactions between remodelers and chromatin targets have been obscure. We performed live-cell single-molecule tracking for RSC, SWI/SNF, CHD1, ISW1, ISW2, and INO80 remodeling complexes in budding yeast and detected hyperkinetic behaviors for chromatin-bound molecules that frequently transition to the free state for all complexes. Chromatin-bound remodelers display notably higher diffusion than nucleosomal histones, and strikingly fast dissociation kinetics with 4-7 s mean residence times. These enhanced dynamics require ATP binding or hydrolysis by the catalytic ATPase, uncovering an additional function to its established role in nucleosome remodeling. Kinetic simulations show that multiple remodelers can repeatedly occupy the same promoter region on a timescale of minutes, implicating an unending 'tug-of-war' that controls a temporally shifting window of accessibility for the transcription initiation machinery.
PMID: 34313223
ISSN: 2050-084x
CID: 4949212

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

Lionnet, Timothée; Wu, Carl
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.
PMID: 33422933
ISSN: 1879-0380
CID: 4925212

Live-cell single particle imaging reveals the role of RNA polymerase II in histone H2A.Z eviction

Ranjan, Anand; Nguyen, Vu Q; Liu, Sheng; Wisniewski, Jan; Kim, Jee Min; Tang, Xiaona; Mizuguchi, Gaku; Elalaoui, Ejlal; Nickels, Timothy J; Jou, Vivian; English, Brian P; Zheng, Qinsi; Luk, Ed; Lavis, Luke D; Lionnet, Timothee; Wu, Carl
The H2A.Z histone variant, a genome-wide hallmark of permissive chromatin, is enriched near transcription start sites in all eukaryotes. H2A.Z is deposited by the SWR1 chromatin remodeler and evicted by unclear mechanisms. We tracked H2A.Z in living yeast at single-molecule resolution, and found that H2A.Z eviction is dependent on RNA Polymerase II (Pol II) and the Kin28/Cdk7 kinase, which phosphorylates Serine 5 of heptapeptide repeats on the carboxy-terminal domain of the largest Pol II subunit Rpb1. These findings link H2A.Z eviction to transcription initiation, promoter escape and early elongation activities of Pol II. Because passage of Pol II through +1 nucleosomes genome-wide would obligate H2A.Z turnover, we propose that global transcription at yeast promoters is responsible for eviction of H2A.Z. Such usage of yeast Pol II suggests a general mechanism coupling eukaryotic transcription to erasure of the H2A.Z epigenetic signal.
PMID: 32338606
ISSN: 2050-084x
CID: 4411862

Three-dimensional chromatin landscapes in T cell acute lymphoblastic leukemia

Kloetgen, Andreas; Thandapani, Palaniraja; Ntziachristos, Panagiotis; Ghebrechristos, Yohana; Nomikou, Sofia; Lazaris, Charalampos; Chen, Xufeng; Hu, Hai; Bakogianni, Sofia; Wang, Jingjing; Fu, Yi; Boccalatte, Francesco; Zhong, Hua; Paietta, Elisabeth; Trimarchi, Thomas; Zhu, Yixing; Van Vlierberghe, Pieter; Inghirami, Giorgio G; Lionnet, Timothee; Aifantis, Iannis; Tsirigos, Aristotelis
Differences in three-dimensional (3D) chromatin architecture can influence the integrity of topologically associating domains (TADs) and rewire specific enhancer-promoter interactions, impacting gene expression and leading to human disease. Here we investigate the 3D chromatin architecture in T cell acute lymphoblastic leukemia (T-ALL) by using primary human leukemia specimens and examine the dynamic responses of this architecture to pharmacological agents. Systematic integration of matched in situ Hi-C, RNA-seq and CTCF ChIP-seq datasets revealed widespread differences in intra-TAD chromatin interactions and TAD boundary insulation in T-ALL. Our studies identify and focus on a TAD 'fusion' event associated with absence of CTCF-mediated insulation, enabling direct interactions between the MYC promoter and a distal super-enhancer. Moreover, our data also demonstrate that small-molecule inhibitors targeting either oncogenic signal transduction or epigenetic regulation can alter specific 3D interactions found in leukemia. Overall, our study highlights the impact, complexity and dynamic nature of 3D chromatin architecture in human acute leukemia.
PMID: 32203470
ISSN: 1546-1718
CID: 4357602

Histone H3K27 acetylation precedes active transcription during zebrafish zygotic genome activation as revealed by live-cell analysis

Sato, Yuko; Hilbert, Lennart; Oda, Haruka; Wan, Yinan; Heddleston, John M; Chew, Teng-Leong; Zaburdaev, Vasily; Keller, Philipp; Lionnet, Timothee; Vastenhouw, Nadine; Kimura, Hiroshi
Histone post-translational modifications are key gene expression regulators, but their rapid dynamics during development remain difficult to capture. We applied a Fab-based live endogenous modification labeling technique to monitor the changes in histone modification levels during zygotic genome activation (ZGA) in living zebrafish embryos. Among various histone modifications, H3 Lys27 acetylation (H3K27ac) exhibited most drastic changes, accumulating in two nuclear foci in the 64- to 1k-cell-stage embryos. The elongating form of RNA polymerase II, which is phosphorylated at Ser2 in heptad repeats within the C-terminal domain (RNAP2 Ser2ph), and miR-430 transcripts were also concentrated in foci closely associated with H3K27ac. When treated with α-amanitin to inhibit transcription or JQ-1 to inhibit binding of acetyl-reader proteins, H3K27ac foci still appeared but RNAP2 Ser2ph and miR-430 morpholino were not concentrated in foci, suggesting that H3K27ac precedes active transcription during ZGA. We anticipate that the method presented here could be applied to a variety of developmental processes in any model and non-model organisms.
PMID: 31570370
ISSN: 1477-9129
CID: 4116112

Imaging the Life and Death of mRNAs in Single Cells

Chao, Jeffrey A; Lionnet, Timothée
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.
PMID: 30510061
ISSN: 1943-0264
CID: 4310162

Single-Molecule Sensitivity RNA FISH Analysis of Influenza Virus Genome Trafficking

Chou, Yi-Ying; Lionnet, Timothée
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.
PMID: 30151575
ISSN: 1940-6029
CID: 3834622

Quantitative mRNA imaging throughout the entire Drosophila brain

Long, Xi; Colonell, Jennifer; Wong, Allan M; Singer, Robert H; Lionnet, Timothee
We describe a fluorescence in situ hybridization method that permits detection of the localization and abundance of single mRNAs (smFISH) in cleared whole-mount adult Drosophila brains. The approach is rapid and multiplexable and does not require molecular amplification; it allows facile quantification of mRNA expression with subcellular resolution on a standard confocal microscope. We further demonstrate single-mRNA detection across the entire brain using a custom Bessel beam structured illumination microscope (BB-SIM).
PMID: 28581495
ISSN: 1548-7105
CID: 2592002