Here are some of our publications!

for a complete list: Google Scholar | Pubmed

Thanks to progress in genomics, we know a lot about where Transcription Factors (TFs) bind, but what they do once bound remains poorly understood. Specifically, we wondered how TFs tune the dynamic properties of transcription. In collaboration with the labs of Mikko Taipale and Alex Holehouse, we discovered that TFs are kinetically specialized: some turn their target gene on, while others regulate how fast the gene is able to fire transcripts when on. Mining public protein-protein interaction databases, we developed a model that classifies arbitrary human TFs into kinetic families based on their known partners.

The Kinetic Landscape of Human Transcription Factors

Mamrak NE, Alerasool N, Griffith D, Holehouse AS, Taipale M & Lionnet T.* (2022)

* : corresponding author

Biorxiv

The labs of Marcus Noyes and Phil Kim, combined screening of billion of protein-DNA interactions with deep learning networks to build models able to design Zinc Fingers binding arbitrary sequences. Zinc Fingers are 8 times smaller than Cas9, less likely to trigger genetic instabilities, and less immunogenic, making them prime candidates for genome editing, epigenome editing, and fluorescent genome labeling (stay tuned!).

A universal deep-learning model for zinc finger design enables transcription factor reprogramming

Ichikawa DM, Abdin O, Alerasool N, Kogenaru M, Mueller AL, Wen H, Giganti DO, Goldberg GW, Adams S, Spencer JM, Razavi R, Nim S, Zheng H, Gionco C, Clark FT, Strokach A, Hughes TR, Lionnet T, Taipale M, Kim PM* &amp Noyes MB* (2023)

* : corresponding author

Nature Biotechnology

Several models for drug resistance have been proposed, including the selection of pre-existing resistant clones or therapy-induced resistance through genetic and non-genetic mechanisms. In collaboration with the lab of Itai Yanai , we provide compelling evidence that the life history of the cancer cells – in terms of the treatment duration and the dose exposure – are crucial determinants in eliciting cancer resistance. We show that cancer cells adapt according to the selective pressures imposed by the therapy along an evolutionary trajectory that we call the ‘resistance continuum’, with distinct physiological responses unfolding over time, including both genetic and epigenetic contributions.

Drug-induced adaptation along a resistance continuum in cancer cells

Franca G, Baron M, Pour M, King BR, Rao A, Misirlioglu S, Barkley D, Dolgalev I, Tang KH, Avital G, Kuperwaser F, Patel A, Levine DA, Lionnet T* & Yanai I* (2022)

* : equal last authors

Biorxiv

Despite an ever-expanding catalog of noncoding elements that are implicated in the control of mammalian gene expression, how the regulatory input from multiple elements is integrated across a genomic neighborhood has remained largely unclear. This challenge is exemplified at Hox clusters (~100 to 200 kb), which contain genes that specify positional identity along the anterior-posterior axis of the developing embryo. Taking inspiration from the bottom-up approaches of synthetic biology and biochemical reconstitution, the Mazzoni and Boeke labs synthesized large DNA constructs (>100 kb) that enable probing regulation at the scale of a native genomic neighborhood. The data suggest that the active gene and chromatin boundary specification at Hoxa in response to the developmental morphogen Retinoic Acid is primarily driven by internal transcription factor binding sites. Distal enhancers are dispensable for the specification of active genes but synergize with intracluster activator binding to boost the amount of transcription.

Synthetic regulatory reconstitution reveals principles of mammalian Hox cluster regulation

Pinglay S, Bulajic M, Rahe BP, Huang E, Brosh R, Mamrak NE, King BR, German S, Cadley JA, Rieber L, Easo N, Lionnet T, Mahony S, Maurano MT, Holt LJ, Mazzoni EO, Boeke JD (2022)

Science

We have developed a software for 3D spatial transcriptomics that enables detection of individual mRNA molecules in tissue volume robustly and rapidly (300x faster than popular alternative!). We share it as a user-friendly plugin for the open-source image analysis software FIJI. Collaboration with the Preibisch and Harrington labs.

RS-FISH: Precise, interactive and scalable smFISH spot detection using Radial Symmetry

Bahry E#, Breimann L#, Epstein L#, Kolyvanov K, Harrington KIS*, Lionnet T*, Preibisch S*. (2021)

* : corresponding authors | # : equal contributions

BiorXiv | video walk through | Github | Nature Methods (in press)

Transcription factors contact chromatin very briefly (seconds!), but can form large clusters. How do these unique dynamics regulate transcription? In this review, we present the tools that enable these observations and discuss possible models. This piece is part of the upcoming “The Nucleus” textbook (2nd edition, editors Tom Misteli, Ana Pombo & Martin Hetzer).

Transcription Factor Dynamics

Lu F & Lionnet T. (2021)

Cold Spring Harbor Perspectives in Biology | pdf

Nucleosomes help package the DNA to a manageable size into the nucleus. Chromatin remodelers rearrange nucleosomes in order to maintain active genome regions open for business, and ensure that silenced genome regions remain tightly compacted. Here we show in collaboration with the Wu lab that chromatin remodelers perform their tasks during very brief time intervals (seconds). Combined with genomics data, these results suggest that the open chromatin regions in the genome emerge from brief and frequent interactions with multiple chromatin remodelers.

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

Kim JM, Visanpattanasin P, Jou V, Liu S, Tang X, Zheng Q, Li KY, Snedeker J, Lavis LD, Lionnet T, Wu C (2021)

Biorxiv | eLife

This review introduces live-cell single-molecule imaging technologies to a broad audience, and discusses what these new approaches – some of them developed by the lab – can teach us about transcription regulation.

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

Lionnet T, Wu C (2021)

Current Opinion in Genetics and Development

The transcription preinitiation complex is a giant machine (100 proteins!). How fast does it assemble and turn over? Here are some answers thanks to a heroic effort led by Vu Nguyen in Carl Wu’s lab. Combining live cell single-molecule tracking and acute perturbations, Vu shows that PIC assembly is hierarchical, and likely highly efficient. Once assembled, the PIC is dynamic – disassembles in seconds upon transcription initiation. One really cool result is that Mediator and Pol II guide the nuclear exploration of other complex components, likely by generating local environments where those factors undergo subdiffusive behavior!

Spatio-Temporal Coordination of Transcription Preinitiation Complex Assembly in Live Cells

Nguyen VQ, Ranjan A, Liu S, Tang X, Ling YH, Wisniewski J, Mizuguchi G, Li KY, Jou V, Zheng Q, Lavis LD, Lionnet T, Wu C. (2020)

BioRxiv | Molecular Cell

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 of noncoding RNAs prior to premature termination, in addition to transcription of mRNAs, are 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.

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

Ranjan A, Nguyen VQ, Liu S, Wisniewski J, Kim JM, Tang X, Mizuguchi G, Elalaoui E, Nickels TJ, Jou V, English BP, Zheng Q, Luk E, Lavis LD, Lionnet T, Wu C. (2020)

Biorxiv | Elife

The spatial organization of the genome inside the nucleus is known to impact gene expression, for instance through physical contacts between the promoter of a gene and a regulatory DNA sequence localized far away along the chromosome. How DNA folding impacts the expression of key cancer driving genes is unclear. Here, we characterize the interplay between DNA organization and gene expression in T cell acute lymphoblastic leukemia (T-ALL) cells, demonstrating a change in physical organization linked with cancer cells at the Myc locus, correlated with expression of the oncogene.

Dynamic 3D chromosomal landscapes in acute leukemia

Kloetgen K*, Thandapani P*, Ntziachristos P*, Ghebrechristos Y, Nomikou S, Lazaris C, Chen X, Hu H, Bakogianni S, Wang J, Fu Y, Boccalatte F, Zhong H, Paietta E, Trimarchi T, Zhu Y, van Vlierberghe P, Inghirami G, Lionnet T, Aifantis I and Tsirigos A. (2019) BiorXiv | Nature Genetics

Embryos initially contain parental mRNA and do not transcribe their own genes. They remain silent until the Zygotic Genome Activation, when the first genes are transcribed. How this happens, and how chromatin changes to favor the emergence of transcription remains unclear. This work demonstrates how fluorescently labeled antibody fragments (Fabs) can be used to track the changes in chromatin modifications that pave the way for the onset of transcription.

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

Sato Y, Hilbert L, Oda H, Wan Y, Heddleston JM, Chew TL, Zaburdaev V, Keller P, Lionnet T, Vastenhouw N, Kimura H.
(2019) Development 146, dev179127 | BiorXiv

This protocol describes in detail the technique used in Trcek et al to visualize and quantify the spatial organization of RNAs in embryos of the fruit fly Drosophila. The enrichment of specific RNAs in the posterior region of the embryo gives rise to the germ line (the cells that will eventually become the reproductive cells of the animal).

mRNA quantification using single-molecule FISH in Drosophila embryos.
Trcek T, Lionnet T, Shroff H, Lehmann R.
(2017) Nature Protocols 12(7):1326-1348.

The fruit fly (Drosophila) is one of the most important animals used to understand brain circuits. This is because one can use extensive fly genetics tools to perturb specific neurons and understand their role in various behaviors. However, knowing which genes each neuron expresses in order to achieve the required tasks has remained difficult. We developed a technique to image mRNAs (the gene intermediate between DNA and protein) in entire brains of fruit flies that provides this missing tool. First, we process the fixed tissue so that fluorescent probes efficiently bind to mRNAs in the entire brain. These probes can then be imaged seamlessly thanks to a clearing treatment that makes the tissue optically transparent. Combined with a light sheet microscope capable of light sheet illumination, the technique allows counting individual molecules of mRNA to detect when, where and how many mRNAs are expressed throughout the brain.

Quantitative mRNA Imaging Throughout the Entire Drosophila Brain

Long X*, Colonell J, Wong AM, Singer RH, Lionnet T*, (2017) Nature Methods 14(7):703-706; Bioarxiv

*Corresponding author

We built on the Janelia Fluor™ dyes to design organic fluors that can be turned on with blue light. The dyes penetrate living cells and can be conjugated in vivo to target proteins (HaloTag). Because the fluors are bright and we can easily control their concentration using blue light, they are perfectly suited for single molecule tracking in vivo.

Bright photoactivatable fluorophores for single-molecule Imaging.

Grimm JB, English BP, Choi H, Muthusamy AK, Mehl BP, Dong P, Brown TA, Lippincott-Schwartz J, Liu Z, Lionnet T*, Lavis LD* (2016) Nature Methods doi:10.1038/nmeth.4034 Bioarxiv PMC

*Corresponding author

A new imaging technique that captures on camera the nascent peptides being produced by individual mRNA molecules in living cells. This technology opens the door to investigating when and where translation occurs.

Real-time Quantification of single RNA translation dynamics in living cells.

Morisaki T, Lyon K, DeLuca KF, DeLuca JG, English BP, Zhang Z, Lavis LD, Grimm JB, Viswanathan S, Looger LL, Lionnet T, Stasevich T. (2016) Science Jun 17; 352 (6292): 1425-9

We observed that the enzyme that synthesizes mRNA from DNA (Pol II) accumulates around genes when they are induced. Dozens of Pol II form these clusters for a few seconds before dissociating. The longer these clusters last, the more RNA is produced. The factors that trigger these elusive clusters remain to be discovered!

RNA Polymerase II cluster dynamics predicts mRNA ouput in living cells.

Cho WK, Jayanth N, English BP, Inoue T, Andrews JO, Conway W, Grimm JB, Spille JH, Lavis LD, Lionnet T*, Cisse II* (2016) eLife PMC

*Corresponding author

A novel design for the optics at the center of the Multifocus Microscope (MFM): a microscope capable of acquiring the full volume of a sample in a single camera shot.

Multifocus microscopy with precise color multi-phase diffractive optics applied in functional neuronal imaging.

Abrahamsson S, Ilic R, Wisniewski J, Mehl B, Yu L, Chen L, Davanco M, Oudjedi L, Fiche J-B, Hajj B, Jin X, Pulupa J, Cho C, Mir M, El Beheiry M, Darzacq X, Nollmann M, Dahan M, Wu C, Lionnet T, Liddle AJ, Bargmann CI (2016) Biomedical Optics Express 7 (3) 855 PMC

By co-tracking individual molecules of mRNAs and ribosomes (the molecules synthesizing proteins from mRNA), we were able to identify locations of the cell where RNA is made into proteins.

Mapping translation ‘hot spots’ in live cells by tracking single molecules of mRNA and ribosomes.

Katz ZB, English BP, Lionnet T, Yoon YJ, Monnier N, Ovryn B, Bathe M, Singer RH. (2016) eLife 10415 PMC

We harnessed the CRISPR system to label genomic loci in fixed cells. The CasFISH technique can provide results in as little as 15 minutes, a significant improvement on the traditional DNA-FISH based methods.

CASFISH : CRISPR/Cas9-mediated in situ labeling of genomic loci in fixed cells.

Deng W, Shi X, Tjian R, Lionnet T, Singer RH. (2015) Proc. Natl. Acad. Sci., 112 (38), 11870-11875 PMC

In Drosophila embryos, mRNA molecules from specific genes aggregate into granules at the posterior pole (the region of the embryo that will eventually give rise to gametes). Within each of these small small (~100 nanometers) granules, the positions of mRNAs is not random; rather, mRNAs from the same species accumulate together at preferred locations.

Drosophila germ granules are structured and contain homotypic mRNA clusters.

Treck T, Grosch M, York A, Shroff H, Lionnet T, Lehman R. (2015) Nature Communications, 6:7962 PMC

We used the response of the Actin gene to serum as a model for rapid transcription dynamics and interrogated the influence of different upstream factors on transcription kinetics and robustness.

Cellular Levels of Signaling Factors Are Sensed by β-actin Alleles to Modulate Transcriptional Pulse Intensity.

Kalo A, Kanter I, Shraga A, Sheinberger J, Tzemach H, Kinor N, Singer RH, Lionnet T, Shav-Tal Y. (2015) Cell Reports 11(3) 419-32 PMC

We labeled mRNAs with 2 colors which allowed us to distinguish whether each mRNA molecule in a cell had been translated (read by the protein synthesizing machinery).

An RNA biosensor for imaging the first round of translation from single cells to living animals.

Halstead JM*, Lionnet T*, Wilbertz JH*, Wippich F*, Ephrussi A, Singer RH, Chao JA. (2015) Science 347(6228) 1367-671 PMC

*Equal Contributions

We designed and characterized novel cell permeable dyes – Janelia Fluor™ : dyes that label proteins in living cells with fluorophores brighter and more stable than existing ones (or fluorescent proteins).

A general method to improve fluorophores for live-cell and single-molecule microscopy.

Grimm JB, English BP, Chen J, Slaughter JP, Zhang Z, Revyakin A, Patel R, Macklin JJ, Normanno D, Singer RH, Lionnet T*, Lavis LD.* (2015) Nature Methods 12(3) 244-50 PMC

*Corresponding author

We measured the dynamics of individual molecules of transcription factors to quantitatively address how these proteins find their preferred sequences within the billions of basepairs that constitute the genome. This helps understand how they can selectively activate their target genes.

Single-molecule dynamics of enhanceosome assembly in embryonic stem cells.

Chen J, Zhang Z, Li L, Chen BC, Revyakin A, Hajj B, Legant W, Dahan M, Lionnet T, Betzig E, Tjian R, Liu Z. (2014) Cell 156 (6) 1274-85 PMC

We used multiplex RNA-FISH to visualize where in the cell the RNA strands that form the influenza virus genome come together. This helped us uncover a new step in the influenza infection cycle.

Colocalization of different influenza viral RNA segments in the cytoplasm before viral budding as shown by single-molecule sensitivity FISH analysis.

Chou YY, Heaton NS, Gao Q, Palese P, Singer R, Lionnet T.* (2013) PLoS Pathogens, 9 (5) e1003358 PMC

Corresponding Author

A review of the phenomenon known as transcription bursting: the still poorly understood observation that transcription (the synthesis of mRNA from a gene’s DNA) occurs mostly in infrequent bursts of activity.

Transcription goes digital.

Lionnet T, Singer RH (2012). Embo Reports, 13(4):313-21 PMC

Identifying the RNA sequence recognized by the protein ZBP1 with biochemistry enables identifying the protein’s target genes.

Spatial arrangement of conserved recognition elements identifies RNA regulatory networks.

Patel VL, Mitra S, Harris R, Buxbaum AR, Lionnet T, Girvin M, Levy M, Almo SC, Brenowitz M, Singer RH, Chao JA. (2012) Genes & Development, 26 (1) 43-53; PMC

The first demonstration in a mammalian organism that the MS2 reporter (a technology that renders mRNAs fluorescent) can be used on endogenous genes without detrimental effects.

A transgenic mouse for in vivo detection of endogenous labeled mRNA.

Lionnet T, Czaplinski K, Darzacq X, Shav-Tal Y, Wells AL, Chao JA, Park HY, de Turris V, Lopez-Jones M, Singer RH (2011). Nature Methods, 8(2) 165-70 PMC

By counting the number of mRNAs in yeast cells, we concluded that transcription of so-called housekeeping genes (genes continuously expressed by the cell) was not coordinated, even within genes that shared the same biological function.

Transcription of functionally related genes is not coordinated.

Gandhi SJ, Zenklusen D, Lionnet T, Singer RH (2011). Nature Structural & Molecular Biology, 18 (1) 27-34 PMC

We measured how fast a helicase could unwind a DNA hairpin as a function of the magnitude of an assisting force pulling the DNA apart (helicases are proteins that unzip DNA). This helped us demonstrate that the helicase exploits natural opening fluctuations of the fork, rather than actively destabilizing the double helix.

Real-time observation of bacteriophage T4 gp41 helicase reveals an unwinding mechanism.
Lionnet T, Spiering MM, Benkovic SJ, Bensimon D, Croquette V (2007). Proc. Natl. Acad. Sci. 104 (50): 19790-5 PMC

We predicted the effect of the DNA sequence on the double helix twist-stretch coupling (the amount that the DNA lengthens upon twisting). Our results show that this mechanical property depends strongly one the DNA sequence, suggesting it would be possible to read the chemical sequence of the DNA by purely mechanical means.

Sequence dependent twist-stretch coupling in DNA.

Lionnet T, Lankas F (2007). Biophys. J. 92 (4): L30-32 PMC

We twisted a single strand of DNA and measured its length with nanometer accuracy. This allowed us to discover a counterintuitive property: the double helix unwinds when stretched, in contrast with our everyday intuition of a braided rope. This effect could have important implications in the way DNA binding proteins recognize their target sequence.

Wringing out DNA.

Lionnet T, Joubaud S, Lavery R, Bensimon D, Croquette V (2006). Phys. Rev. Lett., 96 (17): 178102 PMC

This is the first direct observation of the UvrD helicase at the single molecule level (helicases are proteins that unzip the DNA). We discovered that UvrD switches DNA strands, a property that might help it repair DNA more efficiently.

Single-molecule assay reveals strand switching and enhanced processivity of UvrD.

Dessinges MN, Lionnet T, Xi XG, Bensimon D, Croquette V (2004). Proc. Natl. Acad. Sci. 101 (17): 6439-44 PMC