2024

Duyen Huynh, Philipp Hoffmeister, Tobias Friedrich, Kefan Zhang, Marek Bartkuhn, Francesca Ferrante, Benedetto Daniele Giaimo, Rhett Kovall, Tilman Borggrefe, Franz Oswald, J. Christof M. Gebhardt
Effective in vivo binding energy landscape illustrates kinetic stability of RBPJ-DNA binding
Transcription factors (TFs) such as the central DNA-binding hub in Notch signal transduction, RBPJ, bind to specific DNA sequences to regulate gene transcription. How the efficiency of gene regulation depends on the TF-DNA binding kinetics and cofactor interactions is mostly unknown. We determined the DNA binding kinetics and the transcriptional activity of RBPJ and several mutant variants by live-cell single-molecule tracking and reporter assays, and measured their genome-wide chromatin occupation by ChIP-Seq. We observed that cofactor binding, in addition to DNA binding, was required for target site specificity. Importantly, the target site search time of RBPJ was longer than its residence time, indicating kinetic rather than thermodynamic binding stability. Impaired DNA binding, e.g. by mutation K195E related to Adams-Oliver-Syndrome, modulated not only dissociation, but also association to target sites. Impaired cofactor binding mainly altered the rates of unspecific binding and target site association. For other TFs, we also observed longer search than residence times, indicating that kinetic rather than thermodynamic stability of DNA binding might be a general feature of TFs in vivo. We propose that an effective in vivo binding energy landscape of TF-DNA interactions constitutes an instructive visualization of TF-DNA binding kinetics and the changes upon mutations.
https://doi.org/10.1101/2023.12.19.572376

2023

Jonas Coßmann, Pavel I. Kos, Vassiliki Varamogianni-Mamatsi, Devin Assenheimer, Tobias Bischof, Timo Kuhn, Thomas Vomhof, Argyris Papantonis, Luca Giorgetti, J. Christof M. Gebhardt
Increasingly efficient chromatin binding of cohesin and CTCF supports chromatin architecture formation during zebrafish embryogenesis
The three-dimensional folding of chromosomes is essential for nuclear functions such as DNA replication and gene regulation. The emergence of chromatin architecture is thus an important process during embryogenesis. To shed light on the molecular and kinetic underpinnings of chromatin architecture formation, we characterized biophysical properties of cohesin and CTCF binding to chromatin and their changes upon cofactor depletion using single-molecule imaging in live developing zebrafish embryos. We found that chromatin-bound fractions of both cohesin and CTCF increased significantly between the 1000-cell and shield stages, which we could explain through changes in both their association and dissociation rates. Moreover, increasing binding of cohesin restricted chromatin motion, potentially via loop extrusion, and showed distinct stage-dependent nuclear distribution. Polymer simulations with experimentally derived parameters recapitulated the experimentally observed gradual emergence of chromatin architecture. Our findings suggest a kinetic framework of chromatin architecture formation during zebrafish embryogenesis.
https://doi.org/10.1101/2023.12.08.570809

International authors including Anja Reisser and J. Christof M. Gebhardt
Transcriptional reprogramming by mutated IRF4 in lymphoma
Disease-causing mutations in genes encoding transcription factors (TFs) can affect TF interactions with their cognate DNA-binding motifs. Whether and how TF mutations impact upon the binding to TF composite elements (CE) and the interaction with other TFs is unclear. Here, we report a distinct mechanism of TF alteration in human lymphomas with perturbed B cell identity, in particular classic Hodgkin lymphoma. It is caused by a recurrent somatic missense mutation c.295 T > C (p.Cys99Arg; p.C99R) targeting the center of the DNA-binding domain of Interferon Regulatory Factor 4 (IRF4), a key TF in immune cells. IRF4-C99R fundamentally alters IRF4 DNA-binding, with loss-of-binding to canonical IRF motifs and neomorphic gain-of-binding to canonical and non-canonical IRF CEs. IRF4-C99R thoroughly modifies IRF4 function by blocking IRF4-dependent plasma cell induction, and up-regulates disease-specific genes in a non-canonical Activator Protein-1 (AP-1)-IRF-CE (AICE)-dependent manner. Our data explain how a single mutation causes a complex switch of TF specificity and gene regulation and open the perspective to specifically block the neomorphic DNA-binding activities of a mutant TF.
https://doi.org/10.1038/s41467-023-41954-8

IRF4 International Consortium including Anja Reisser and J. Christof M. Gebhardt
A multimorphic mutation in IRF4 causes human autosomal dominant combined immunodeficiency
Interferon regulatory factor 4 (IRF4) is a transcription factor (TF) and key regulator of immune cell development and function. We report a recurrent heterozygous mutation in IRF4, p.T95R, causing an autosomal dominant combined immunodeficiency (CID) in seven patients from six unrelated families. The patients exhibited profound susceptibility to opportunistic infections, notably Pneumocystis jirovecii, and presented with agammaglobulinemia. Patients’ B cells showed impaired maturation, decreased immunoglobulin isotype switching, and defective plasma cell differentiation, whereas their T cells contained reduced T_H17 and T_FH populations and exhibited decreased cytokine production. A knock-in mouse model of heterozygous T95R showed a severe defect in antibody production both at the steady state and after immunization with different types of antigens, consistent with the CID observed in these patients. The IRF4^T95R variant maps to the TF’s DNA binding domain, alters its canonical DNA binding specificities, and results in a simultaneous multimorphic combination of loss, gain, and new functions for IRF4. IRF4^T95R behaved as a gain-of-function hypermorph by binding to DNA with higher affinity than IRF4^WT. Despite this increased affinity for DNA, the transcriptional activity on IRF4 canonical genes was reduced, showcasing a hypomorphic activity of IRF4^T95R. Simultaneously, IRF4^T95R functions as a neomorph by binding to noncanonical DNA sites to alter the gene expression profile, including the transcription of genes exclusively induced by IRF4^T95R but not by IRF4^WT. This previously undescribed multimorphic IRF4 pathophysiology disrupts normal lymphocyte biology, causing human disease.
https://doi.org/10.1126/sciimmunol.ade7953

2022

Lisa Streit, Timo Kuhn, Thomas Vomhof, Verena Bopp, Albert C. Ludolph, Jochen H. Weishaupt, Christof M. Gebhardt, Jens Michaelis & Karin M. Danzer
Stress induced TDP-43 mobility loss independent of stress granules
TAR DNA binding protein 43 (TDP-43) is closely related to the pathogenesis of amyotrophic lateral sclerosis (ALS) and translocates to stress granules (SGs). The role of SGs as aggregation-promoting “crucibles” for TDP-43, however, is still under debate. We analyzed TDP-43 mobility and localization under different stress and recovery conditions using live cell single-molecule tracking and super-resolution microscopy. Besides reduced mobility within SGs, a stress induced decrease of TDP-43 mobility in the cytoplasm and the nucleus was observed. Stress removal led to a recovery of TDP-43 mobility, which strongly depended on the stress duration. ‘Stimulated-emission depletion microscopy’ (STED) and ‘tracking and localization microscopy’ (TALM) revealed not only TDP-43 substructures within stress granules but also numerous patches of slow TDP-43 species throughout the cytoplasm. This work provides insights into the aggregation of TDP-43 in living cells and provide evidence suggesting that TDP-43 oligomerization and aggregation takes place in the cytoplasm separate from SGs.
https://doi.org/10.1038/s41467-022-32939-0

Raphael Nold, Charles Babin, Joel Schmidt, Tobias Linkewitz, María T. Pérez Zaballos, Rainer Stöhr, Roman Kolesov, Vadim Vorobyov, Daniil M. Lukin, Rüdiger Boppert, Stefanie Barz, Jelena Vučković, J. Christof M. Gebhardt, Florian Kaiser, and Jörg Wrachtrup
Quantum Optical Microphone in the Audio Band
The ability to perform high-precision optical measurements is paramount in science and engineering. Laser interferometry enables interaction-free sensing with a precision ultimately limited by shot noise. Quantum optical sensors can surpass this limit but single- or multiphoton schemes are challenged by low experimental sampling rates, while squeezed-light approaches require complex optical setups and sophisticated time gating. Here, we introduce a simple method that infers optical phase shifts through standard intensity measurements while still maintaining the quantum advantage in the measurement precision. Capitalizing on the robustness and high sampling rates of our device, we implement a quantum optical microphone in the audio band. Its performance is benchmarked against a classical laser microphone in a standardized medically approved speech-recognition test on 45 subjects. We find that quantum-recorded words improve the speech-recognition threshold by $-0.57{\mathrm{dB}}_{\mathrm{SPL}}$, thus making the quantum advantage audible. Not only do these results open the door toward applications in quantum nonlinear interferometry but they also show that quantum phenomena can be experienced by humans.
https://doi.org/10.1103/PRXQuantum.3.020358

Oliver Kuchler, Jule Gerlach, Thomas Vomhof, Johannes Hettich, Julia Steinmetz, J. Christof M. Gebhardt, Jens Michaelis and Bernd Knöll
Single-molecule tracking (SMT) and localization of SRF and MRTF transcription factors during neuronal stimulation and differentiation
In cells, proteins encoded by the same gene do not all behave uniformly but engage in functional subpopulations induced by spatial or temporal segregation. While conventional microscopy has limitations in revealing such spatial and temporal diversity, single-molecule tracking (SMT) microscopy circumvented this problem and allows for high-resolution imaging and quantification of dynamic single-molecule properties. Particularly in the nucleus, SMT has identified specific DNA residence times of transcription factors (TFs), DNA-bound TF fractions and positions of transcriptional hot-spots upon cell stimulation. By contrast to cell stimulation, SMT has not been employed to follow dynamic TF changes along stages of cell differentiation. Herein, we analysed the serum response factor (SRF), a TF involved in the differentiation of many cell types to study nuclear single-molecule dynamics in neuronal differentiation. Our data in living mouse hippocampal neurons show dynamic changes in SRF DNA residence time and SRF DNA-bound fraction between the stages of adhesion, neurite growth and neurite differentiation in axon and dendrites. Using TALM (tracking and localization microscopy), we identified nuclear positions of SRF clusters and observed changes in their numbers and size during differentiation. Furthermore, we show that the SRF cofactor MRTF-A (myocardin-related TF or MKL1) responds to cell activation by enhancing the long-bound DNA fraction. Finally, a first SMT colocalization study of two proteins was performed in living cells showing enhanced SRF/MRTF-A colocalization upon stimulation. In summary, SMT revealed modulation of dynamic TF properties during cell stimulation and differentiation.
https://doi.org/10.1098/rsob.210383

Timo Kuhn, Amit N. Landge, David Mörsdorf, Jonas Coßmann, Johanna Gerstenecker, Daniel Čapek, Patrick Müller and J. Christof M. Gebhardt
Single-molecule tracking of Nodal and Lefty in live zebrafish embryos supports hindered diffusion model
The hindered diffusion model postulates that the movement of a signaling molecule through an embryo is affected by tissue geometry and binding-mediated hindrance, but these effects have not been directly demonstrated in vivo. Here, we visualize extracellular movement and binding of individual molecules of the activator-inhibitor signaling pair Nodal and Lefty in live developing zebrafish embryos using reflected light-sheet microscopy. We observe that diffusion coefficients of molecules are high in extracellular cavities, whereas mobility is reduced and bound fractions are high within cell-cell interfaces. Counterintuitively, molecules nevertheless accumulate in cavities, which we attribute to the geometry of the extracellular space by agent-based simulations. We further find that Nodal has a larger bound fraction than Lefty and shows a binding time of tens of seconds. Together, our measurements and simulations provide direct support for the hindered diffusion model and yield insights into the nanometer-to-micrometer-scale mechanisms that lead to macroscopic signal dispersal.
https://doi.org/10.1038/s41467-022-33704-z

Yukti Hari-Gupta , Natalia Fili, Ália dos Santos, Alexander W. Cook, Rosemarie E. Gough,
Hannah C. W. Reed, Lin Wang, Jesse Aaron, Tomas Venit, Eric Wait,
Andreas Grosse-Berkenbusch, J. Christof M. Gebhardt, Piergiorgio Percipalle, Teng-Leong Chew,
Marisa Martin-Fernandez & Christopher P. Toseland
Myosin VI regulates the spatial organisation of mammalian transcription initiation
During transcription, RNA Polymerase II (RNAPII) is spatially organised within the nucleus into clusters that correlate with transcription activity. While this is a hallmark of genome regulation in mammalian cells, the mechanisms concerning the assembly, organisation and stability remain unknown. Here, we have used combination of single molecule imaging and genomic approaches to explore the role of nuclear myosin VI (MVI) in the nanoscale organisation of RNAPII. We reveal that MVI in the nucleus acts as the molecular anchor that holds RNAPII in high density clusters. Perturbation of MVI leads to the disruption of RNAPII localisation, chromatin organisation and subsequently a decrease in gene expression. Overall, we uncover the fundamental role of MVI in the spatial regulation of gene expression.
https://doi.org/10.1038/s41467-022-28962-w

Johannes Hettich and Christof M. Gebhardt
Periodic synchronization of isolated network elements facilitates simulating and inferring gene regulatory networks including stochastic molecular kinetics
The temporal progression of many fundamental processes in cells and organisms, including homeostasis, differentiation and development, are governed by gene regulatory networks (GRNs). GRNs balance fluctuations in the output of their genes, which trace back to the stochasticity of molecular interactions. Although highly desirable to understand life processes, predicting the temporal progression of gene products within a GRN is challenging when considering stochastic events such as transcription factor–DNA interactions or protein production and degradation.
We report a method to simulate and infer GRNs including genes and biochemical reactions at molecular detail. In our approach, we consider each network element to be isolated from other elements during small time intervals, after which we synchronize molecule numbers across all network elements. Thereby, the temporal behaviour of network elements is decoupled and can be treated by local stochastic or deterministic solutions. We demonstrate the working principle of this modular approach with a repressive gene cascade comprising four genes. By considering a deterministic time evolution within each time interval for all elements, our method approaches the solution of the system of deterministic differential equations associated with the GRN. By allowing genes to stochastically switch between on and off states or by considering stochastic production of gene outputs, we are able to include increasing levels of stochastic detail and approximate the solution of a Gillespie simulation. Thereby, CaiNet is able to reproduce noise-induced bi-stability and oscillations in dynamically complex GRNs. Notably, our modular approach further allows for a simple consideration of deterministic delays. We further infer relevant regulatory connections and steady-state parameters of a GRN of up to ten genes from steady-state measurements by identifying each gene of the network with a single perceptron in an artificial neuronal network and using a gradient decent method originally designed to train recurrent neural networks. To facilitate setting up GRNs and using our simulation and inference method, we provide a fast computer-aided interactive network simulation environment, CaiNet.
We developed a method to simulate GRNs at molecular detail and to infer the topology and steady-state parameters of GRNs. Our method and associated user-friendly framework CaiNet should prove helpful to analyze or predict the temporal progression of reaction networks or GRNs in cellular and organismic biology.
https://doi.org/10.1186/s12859-021-04541-6

2021

Philipp Hoffmeister, Aleksandra Turkiewicz, N. N. Duyen Huynh, Andreas Große-Berkenbusch, Uwe Knippschild, J. Christof M. Gebhardt, Bernd Baumann, Tilman Borggrefe, Franz Oswald
Transcription Factor RBPJL Is Able to Repress Notch Target Gene Expression but Is Non-Responsive to Notch Activation
The Notch signaling pathway is an evolutionary conserved signal transduction cascade present in almost all tissues and is required for embryonic and postnatal development, as well as for stem cell maintenance, but it is also implicated in tumorigenesis including pancreatic cancer and leukemia. The transcription factor RBPJ forms a coactivator complex in the presence of a Notch signal, whereas it represses Notch target genes in the absence of a Notch stimulus. In the pancreas, a specific paralog of RBPJ, called RBPJL, is expressed and found as part of the heterotrimeric PTF1-complex. However, the function of RBPJL in Notch signaling remains elusive. Using molecular modeling, biochemical and functional assays, as well as single-molecule time-lapse imaging, we show that RBPJL and RBPJ, despite limited sequence homology, possess a high degree of structural similarity. RBPJL is specifically expressed in the exocrine pancreas, whereas it is mostly undetectable in pancreatic tumour cell lines. Importantly, RBPJL is not able to interact with Notch−1 to −4 and it does not support Notch-mediated transactivation. However, RBPJL can bind to canonical RBPJ DNA elements and shows migration dynamics comparable to that of RBPJ in the nuclei of living cells. Importantly, RBPJL is able to interact with SHARP/SPEN, the central corepressor of the Notch pathway. In line with this, RBPJL is able to fully reconstitute transcriptional repression at Notch target genes in cells lacking RBPJ. Together, RBPJL can act as an antagonist of RBPJ, which renders cells unresponsive to the activation of Notch.
https://www.mdpi.com/2072-6694/13/19/5027

Sabine Vettorazzi, Denis Nalbantoglu, J. Christof M. Gebhardt, Jan Tuckermann
A guide to changing paradigms of glucocorticoid receptor function—a model system for genome regulation and physiology
The glucocorticoid receptor (GR) is a bona fide ligand-regulated transcription factor. Cloned in the 80s, the GR has become one of the best-studied and clinically most relevant members of the nuclear receptor superfamily. Cooperative activity of GR with other transcription factors and a plethora of coregulators contribute to the tissue- and context-specific response toward the endogenous and pharmacological glucocorticoids (GCs). Furthermore, nontranscriptional activities in the cytoplasm are emerging as an additional function of GR. Over the past 40 years, the concepts of GR mechanisms of action had been constantly changing. Different methodologies in the pregenomic and genomic era of molecular biological research and recent cutting-edge technology in single-cell and single-molecule analysis are steadily evolving the views, how the GR in particular and transcriptional regulation in general act in physiological and pathological processes. In addition to the development of technologies for GR analysis, the use of model organisms provides insights how the GR in vivo executes GC action in tissue homeostasis, inflammation, and energy metabolism. The model organisms, namely the mouse, but also rats, zebrafish, and recently fruit flies carrying mutations of the GR became a major driving force to analyze the molecular function of GR in disease models. This guide provides an overview of the exciting research and paradigm shifts in the GR field from past to present with a focus on GR transcription factor networks, GR DNA-binding and single-cell analysis, and model systems.
https://doi.org/10.1111/febs.16100

Eberle Julia, Wiehe Rahel Stefanie, Gole Boris, Mattis Liska Jule, Palmer Anja, Ständker Ludger, Forssmann Wolf-Georg, Münch Jan, Gebhardt J. Christof M., Wiesmüller Lisa
A Fibrinogen Alpha Fragment Mitigates Chemotherapy-Induced MLL Rearrangements
Rearrangements in the Mixed Lineage Leukemia breakpoint cluster region (MLLbcr) are frequently involved in therapy-induced leukemia, a severe side effect of anti-cancer therapies. Previous work unraveled Endonuclease G as the critical nuclease causing initial breakage in the MLLbcr in response to different types of chemotherapeutic treatment. To identify peptides protecting against therapy-induced leukemia, we screened a hemofiltrate-derived peptide library by use of an enhanced green fluorescent protein (EGFP)-based chromosomal reporter of MLLbcr rearrangements. Chromatographic purification of one active fraction and subsequent mass spectrometry allowed to isolate a C-terminal 27-mer of fibrinogen α encompassing amino acids 603 to 629. The chemically synthesized peptide, termed Fα27, inhibited MLLbcr rearrangements in immortalized hematopoietic cells following treatment with the cytostatics etoposide or doxorubicin. We also provide evidence for protection of primary human hematopoietic stem and progenitor cells from therapy-induced MLLbcr breakage. Of note, fibrinogen has been described to activate toll-like receptor 4 (TLR4). Dissecting the Fα27 mode-of action revealed association of the peptide with TLR4 in an antagonistic fashion affecting downstream NFκB signaling and pro-inflammatory cytokine production. In conclusion, we identified a hemofiltrate-derived peptide inhibitor of the genome destabilizing events causing secondary leukemia in patients undergoing chemotherapy.
https://www.frontiersin.org/article/10.3389/fonc.2021.689063

Timo Kuhn, Johannes Hettich, Rubina Davtyan, J. Christof M. Gebhardt
Single molecule tracking and analysis framework including theory-predicted parameter settings
Imaging, tracking and analyzing individual biomolecules in living systems is a powerful technology to obtain quantitative kinetic and spatial information such as reaction rates, diffusion coefficients and localization maps. Common tracking tools often operate on single movies and require additional manual steps to analyze whole data sets or to compare different experimental conditions. We report a fast and comprehensive single molecule tracking and analysis framework (TrackIt) to simultaneously process several multi-movie data sets. A user-friendly GUI offers convenient tracking visualization, multiple state-of-the-art analysis procedures, display of results, and data im- and export at different levels to utilize external software tools. We applied our framework to quantify dissociation rates of a transcription factor in the nucleus and found that tracking errors, similar to fluorophore photobleaching, have to be considered for reliable analysis. Accordingly, we developed an algorithm, which accounts for both tracking losses and suggests optimized tracking parameters when evaluating reaction rates. Our versatile and extensible framework facilitates quantitative analysis of single molecule experiments at different experimental conditions.
https://doi.org/10.1038/s41598-021-88802-7

Achim P. Popp, Johannes Hettich, J. Christof M. Gebhardt
Altering transcription factor binding reveals comprehensive transcriptional kinetics of a basic gene
Transcription is a vital process activated by transcription factor (TF) binding. The active gene releases a burst of transcripts before turning inactive again. While the basic course of transcription is well understood, it is unclear how binding of a TF affects the frequency, duration and size of a transcriptional burst. We systematically varied the residence time and concentration of a synthetic TF and characterized the transcription of a synthetic reporter gene by combining single molecule imaging, single molecule RNA-FISH, live transcript visualisation and analysis with a novel algorithm, Burst Inference from mRNA Distributions (BIRD). For this well-defined system, we found that TF binding solely affected burst frequency and variations in TF residence time had a stronger influence than variations in concentration. This enabled us to device a model of gene transcription, in which TF binding triggers multiple successive steps before the gene transits to the active state and actual mRNA synthesis is decoupled from TF presence. We quantified all transition times of the TF and the gene, including the TF search time and the delay between TF binding and the onset of transcription. Our quantitative measurements and analysis revealed detailed kinetic insight, which may serve as basis for a bottom-up understanding of gene regulation.
https://doi.org/10.1093/nar/gkab443

2020

Andreas Große-Berkenbusch, Johannes Hettich, Timo Kuhn, Natalia Fili, Alexander W. Cook, Yukti Hari-Gupta, Anja Palmer, Lisa Streit, Peter J.I. Ellis, Christopher P. Toseland and J. Christof M. Gebhardt
Myosin VI moves on nuclear actin filaments and supports long-range chromatin rearrangements
Nuclear myosin VI (MVI) enhances RNA polymerase II – dependent transcription, but the molecular mechanism is unclear. We used live cell single molecule tracking to follow individual MVI molecules inside the nucleus and observed micrometer-long motion of the motor. Besides static chromatin interactions lasting for tens of seconds, ATPase-dependent directed motion occurred with a velocity of 2 µm/s. The movement was frequently interrupted by short periods of slow restricted diffusion and increased in frequency upon stimulation of transcription. Mutagenesis and perturbation experiments demonstrated that nuclear MVI motion is independent of dimerization and occurs on nuclear actin filaments, which we also observed by two-color imaging. Using chromosome paint to quantify distances between chromosomes, we found that MVI is required for transcription-dependent long-range chromatin rearrangements. Our measurements reveal a transcription-coupled function of MVI in the nucleus, where it actively undergoes directed movement along nuclear actin filaments. Motion is potentially mediated by cooperating monomeric motors and might assist in enhancing transcription by supporting long-range chromatin rearrangements.
bioRxiv
https://doi.org/10.1101/2020.04.03.023614

Matthias Reisser, Johannes Hettich, Timo Kuhn, Achim P. Popp, Andreas Große-Berkenbusch & J. Christof M. Gebhardt
Inferring quantity and qualities of superimposed reaction rates from single molecule survival time distributions
Actions of molecular species, for example binding of transcription factors to chromatin, may comprise several superimposed reaction pathways. The number and the rate constants of such superimposed reactions can in principle be resolved by inverse Laplace transformation of the corresponding distribution of reaction lifetimes. However, current approaches to solve this transformation are challenged by photobleaching-prone fluorescence measurements of lifetime distributions. Here, we present a genuine rate identification method (GRID), which infers the quantity, rates and amplitudes of dissociation processes from fluorescence lifetime distributions using a dense grid of possible decay rates. In contrast to common multi-exponential analysis of lifetime distributions, GRID is able to distinguish between broad and narrow clusters of decay rates. We validate GRID by simulations and apply it to CDX2-chromatin interactions measured by live cell single molecule fluorescence microscopy. GRID reveals well-separated narrow decay rate clusters of CDX2, in part overlooked by multi-exponential analysis. We discuss the amplitudes of the decay rate spectrum in terms of frequency of observed events and occupation probability of reaction states. We further demonstrate that a narrow decay rate cluster is compatible with a common model of TF sliding on DNA.
Scientific Reports volume 10, Article number: 1758 (2020)
https://www.nature.com/articles/s41598-020-58634-y

2019

Matthias Reisser, Johannes Hettich, Timo Kuhn, View ORCID ProfileJ. Christof M. Gebhardt
Inferring quantity and qualities of superimposed reaction rates in single molecule survival time distributions
Actions of molecular species, for example binding of transcription factors to chromatin, are intrinsically stochastic and may comprise several mutually exclusive pathways. Inverse Laplace transformation in principle resolves the rate constants and frequencies of superimposed reaction processes, however current approaches are challenged by single molecule fluorescence time series prone to photobleaching. Here, we present a genuine rate identification method (GRID) that infers the quantity, rates and frequencies of dissociation processes from single molecule fluorescence survival time distributions using a dense grid of possible decay rates. In particular, GRID is able to resolve broad clusters of rate constants not accessible to common models of one to three exponential decay rates. We validate GRID by simulations and apply it to the problem of in-vivo TF-DNA dissociation, which recently gained interest due to novel single molecule imaging technologies. We consider dissociation of the transcription factor CDX2 from chromatin. GRID resolves distinct, decay rates and identifies residence time classes overlooked by other methods. We confirm that such sparsely distributed decay rates are compatible with common models of TF sliding on DNA.
bioRxiv, posted June 21, 2019, 1-12
https://www.nature.com/articles/s41598-020-58634-y

Mahé Raccaud, Elias T. Friman, Andrea B. Alber, Harsha Agarwa, Cédric Deluz, Timo Kuhn, J. Christof M. Gebhardt & David M. Suter
Mitotic chromosome binding predicts transcription factor properties in interphase
Mammalian transcription factors (TFs) differ broadly in their nuclear mobility and sequence- specific/non-specific DNA binding. How these properties affect their ability to occupy specific genomic sites and modify the epigenetic landscape is unclear. The association of TFs with mitotic chromosomes observed by fluorescence microscopy is largely mediated by non-specific DNA interactions and differs broadly between TFs. Here we combine quantitative measure- ments of mitotic chromosome binding (MCB) of 501 TFs, TF mobility measurements by fluorescence recovery after photobleaching, single molecule imaging of DNA binding, and mapping of TF binding and chromatin accessibility. TFs associating to mitotic chromosomes are enriched in DNA-rich compartments in interphase and display slower mobility in interphase and mitosis. Remarkably, MCB correlates with relative TF on-rates and genome-wide specific site occupancy, but not with TF residence times. This suggests that non-specific DNA binding properties of TFs regulate their search efficiency and occupancy of specific genomic sites.
Nat Commun. 2019 Jan 30;10(1):487
https://www.nature.com/articles/s41467-019-08417-5.pdf

2018

Lisa Hipp, Judith Beer, Oliver Kuchler, Matthias Reisser, Daniela Sinske, Jens Michaelis, J. Christof M. Gebhardt, and Bernd Knöll
Single-molecule imaging of the transcription factor SRF reveals prolonged chromatin-binding kinetics upon cell stimulation
Serum response factor (SRF) mediates immediate early gene (IEG) and cytoskeletal gene expression programs in almost any cell type. So far, SRF transcriptional dynamics have not been investigated at single-molecule resolution. We provide a study of single Halo-tagged SRF molecules in fibroblasts and primary neurons. In both cell types, individual binding events of SRF molecules segregated into three chromatin residence time regimes, short, intermediate, and long binding, indicating a cell type-independent SRF property. The chromatin residence time of the long bound fraction was up to 1 min in quiescent cells and significantly increased upon stimulation. Stimulation also enhanced the long bound SRF fraction at specific timepoints (20 and 60 min) in both cell types. These peaks correlated with activation of the SRF cofactors MRTF-A and MRTF-B (myocardin-related transcription factors). Interference with signaling pathways and cofactors demonstrated modulation of SRF chromatin occupancy by actin signaling, MAP kinases, and MRTFs.
PNAS January 15, 2019 116 (3) 880-889
doi.org/10.1073/pnas.1812734116

Matthias Reisser, Anja Palmer, Achim P. Popp, Christopher Jahn, Gilbert Weidinger & J. Christof M. Gebhardt
Single-molecule imaging correlates decreasing nuclear volume with increasing TF-chromatin associations during zebrafish development
Zygotic genome activation (ZGA), the onset of transcription after initial quiescence, is a major developmental step in many species, which occurs after ten cell divisions in zebrafish embryos. How transcription factor (TF)-chromatin interactions evolve during early development to support ZGA is largely unknown. We establish single molecule tracking in live developing zebrafish embryos using reflected light-sheet microscopy to visualize two fluorescently labeled TF species, mEos2-TBP and mEos2-Sox19b. We further develop a data acquisition and analysis scheme to extract quantitative information on binding kinetics and bound fractions during fast cell cycles. The chromatin-bound fraction of both TFs increases during early development, as expected from a physical model of TF-chromatin interactions including a decreasing nuclear volume and increasing DNA accessibility. For Sox19b, data suggests the increase is mainly due to the shrinking nucleus. Our single molecule approach provides quantitative insight into changes of TF-chromatin associations during the developmental period embracing ZGA.
Nature Communicationsvolume 9, Article number: 5218 (2018)
nature.com/articles/s41467-018-07731-8

Rahel Stefanie Wiehe, Boris Gole, Laurent Chatre, Paul Walther, Enrico Calzia, Anja Palmer, J. Christof M. Gebhardt, Miria Ricchetti and Lisa Wiesmüller
Endonuclease G promotes mitochondrial genome cleavage and replication
Endonuclease G (EndoG) is a nuclear-encoded endonuclease, mostly localised in mitochondria. In the nucleus EndoG participates in site-specific cleavage during replication stress and genome-wide DNA degradation during apoptosis. However, the impact of EndoG on mitochondrial DNA (mtDNA) metabolism is poorly understood. Here, we investigated whether EndoG is involved in the regulation of mtDNA replication and removal of aberrant copies. We applied the single-cell mitochondrial Transcription and Replication Imaging Protocol (mTRIP) and PCR-based strategies on human cells after knockdown/knockout and re-expression of EndoG. Our analysis revealed that EndoG stimulates both mtDNA replication initiation and mtDNA depletion, the two events being interlinked and dependent on EndoG’s nuclease activity. Stimulation of mtDNA replication by EndoG was independent of 7S DNA processing at the replication origin. Importantly, both mtDNA-directed activities of EndoG were promoted by oxidative stress. Inhibition of base excision repair (BER) that repairs oxidative stress- induced DNA damage unveiled a pronounced effect of EndoG on mtDNA removal, reminiscent of recently discovered links between EndoG and BER in the nucleus. Altogether with the downstream effects on mitochondrial transcription, protein expression, redox status and morphology, this study demonstrates that removal of damaged mtDNA by EndoG and compensatory replication play a critical role in mitochondria homeostasis.
Oncotarget. 2018 Apr 6;9(26):18309-18326
doi.org/10.18632/oncotarget.24822

J. Hettich J.C.M. Gebhardt
Transcription factor target site search and gene regulation in a background of unspecific binding sites
Response time and transcription level are vital parameters of gene regulation. They depend on how fast transcription factors (TFs) find and how efficient they occupy their specific target sites. It is well known that target site search is accelerated by TF binding to and sliding along unspecific DNA and that unspecific associations alter the occupation frequency of a gene. However, whether target site search time and occupation frequency can be optimized simultaneously is mostly unclear. We developed a transparent and intuitively accessible state-based formalism to calculate search times to target sites on and occupation frequencies of promoters of arbitrary state structure. Our formalism is based on dissociation rate constants experimentally accessible in live cell experiments. To demonstrate our approach, we consider promoters activated by a single TF, by two coactivators or in the presence of a competitive inhibitor. We find that target site search time and promoter occupancy differentially vary with the unspecific dissociation rate constant. Both parameters can be harmonized by adjusting the specific dissociation rate constant of the TF. However, while measured DNA residence times of various eukaryotic TFs correspond to a fast search time, the occupation frequencies of target sites are generally low. Cells might tolerate low target site occupancies as they enable timely gene regulation in response to a changing environment.
Journal of Theoretical Biology, Volume 454, 7 October 2018, Pages 91-101
https://doi.org/10.1016/j.jtbi.2018.05.037

2017

Achim P. Popp, Karen Clauß, Lena Schulze, Johannes Hettich, Matthias Reisser, Laura Escoter Torres, N. Henriette Uhlenhaut, J. Christof M. Gebhardt
DNA residence time is a regulatory factor of transcription repression
Transcription comprises a highly regulated sequence of intrinsically stochastic processes, resulting in bursts of transcription intermitted by quiescence. In transcription activation or repression, a transcription factor binds dynamically to DNA, with a residence time unique to each factor. Whether the DNA residence time is important in the transcription process is unclear. Here, we designed a series of transcription repressors differing in their DNA residence time by utilizing the modular DNA binding domain of transcription activator-like effectors (TALEs) and varying the number of nucleotide-recognizing repeat domains. We characterized the DNA residence times of our repressors in living cells using single molecule tracking. The residence times depended non-linearly on the number of repeat domains and differed by more than a factor of six. The factors provoked a residence time-dependent decrease in transcript level of the glucocorticoid receptor-activated gene SGK1. Down regulation of transcription was due to a lower burst frequency in the presence of long binding repressors and is in accordance with a model of competitive inhibition of endogenous activator binding. Our single molecule experiments reveal transcription factor DNA residence time as a regulatory factor controlling transcription repression and establish TALE-DNA binding domains as tools for the temporal dissection of transcription regulation.
Nucleic Acids Research, Volume 45, Issue 19, 2 November 2017, Pages 11121–11130
https://doi.org/10.1093/nar/gkx728

Harsha Agarwal, Matthias Reisser, Celina Wortmann, J. Christof M. Gebhardt
Direct Observation of Cell-Cycle-Dependent Interactions between CTCF and Chromatin
The three-dimensional arrangement of chromatin encodes regulatory traits important for nuclear processes such as transcription and replication. Chromatin topology is in part mediated by the architectural protein CCCTC-binding factor (CTCF) that binds to the boundaries of topologically associating domains. Whereas sites of CTCF interactions are well characterized, little is known on how long CTCF binds to chromatin and how binding evolves during the cell cycle. We monitored CTCF-chromatin interactions by live cell single molecule tracking in different phases of the cell cycle. In G1-, S-, and G2-phases, a majority of CTCF molecules was bound transiently (∼0.2 s) to chromatin, whereas minor fractions were bound dynamically (∼4 s) or stably (>15 min). During mitosis, CTCF was mostly excluded from chromatin. Our data suggest that CTCF scans DNA in search for two different subsets of specific target sites and provide information on the timescales over which topologically associating domains might be restructured. During S-phase, dynamic and stable interactions decreased considerably compared to G1-phase, but were resumed in G2-phase, indicating that specific interactions need to be dissolved for replication to proceed.
Biophysical Journal 112, 1-5, May 23, 2017
doi.org/10.1016/j.bpj.2017.04.018

2014

Z.W. Zhao*, R. Roy*, J.C.M. Gebhardt*, D.M. Suter*, A.R. Chapman, and X.S. Xie
Spatial organization of RNA polymerase II inside a mammalian cell nucleus revealed by reflected light-sheet superresolution microscopy
Superresolution microscopy based on single-molecule centroid determination has been widely applied to cellular imaging in recent years. However, quantitative imaging of the mammalian nucleus has been challenging due to the lack of 3D optical sectioning methods for normal-sized cells, as well as the inability to accurately count the absolute copy numbers of biomolecules in highly dense structures. Here we report a reflected light-sheet superresolution microscopy method capable of imaging inside the mammalian nucleus with superior signal-to-background ratio as well as molecular counting with single-copy accuracy. Using reflected light-sheet superresolution microscopy, we probed the spatial organization of transcription by RNA polymerase II (RNAP II) molecules and quantified their global extent of clustering inside the mammalian nucleus. Spatiotemporal clustering analysis that leverages on the blinking photophysics of specific organic dyes showed that the majority (>70%) of the transcription foci originate from single RNAP II molecules, and no significant clustering between RNAP II molecules was detected within the length scale of the reported diameter of “transcription factories.” Colocalization measurements of RNAP II molecules equally labeled by two spectrally distinct dyes confirmed the primarily unclustered distribution, arguing against a prevalent existence of transcription factories in the mammalian nucleus as previously proposed. The methods developed in our study pave the way for quantitative mapping and stoichiometric characterization of key biomolecular species deep inside mammalian cells.
(* equal contribution)
Proc. Natl. Acad. Sci. (2014)
DOI: 10.1073/pnas.1318496111

2013

Z.W. Zhao, J.C.M. Gebhardt, D,M. Suter, and X.S. Xie
Reply to "Convergence of chromatin binding estimates in live cells"
Nat. methods 10 (2013) 692
DOI: 10.1038/nmeth.2574

J.C.M. Gebhardt*, D.M. Suter*, R. Roy, Z. Zhao, A. R. Chapman, S. Basu, T. Maniatis, and X.S. Xie
Single-molecule imaging of transcription factor binding to DNA in live mammalian cells
Imaging single fluorescent proteins in living mammalian cells is challenged by out-of-focus fluorescence excitation. To reduce out-of-focus fluorescence we developed reflected light-sheet microscopy (RLSM), a fluorescence microscopy method allowing selective plane illumination throughout the nuclei of living mammalian cells. A thin light sheet parallel to the imaging plane and close to the sample surface is generated by reflecting an elliptical laser beam incident from the top by 90° with a small mirror. The thin light sheet allows for an increased signal-to-background ratio superior to that in previous illumination schemes and enables imaging of single fluorescent proteins with up to 100 HZ time resolution. We demonstrated the single-molecule sensitivity of RLSM by measuring the DNA-bound fraction of glucocorticoid receptor (GR) and determining the residence times on DNA of various oligomerization states and mutants of GR and estrogen receptor-α (ER), which permitted us to resolve different modes of DNA binding of GR. We demonstrated two-color single-molecule imaging by observing the spatiotemporal colocalization of two different protein pairs. Our single- molecule measurements and statistical analysis revealed dynamic properties of transcriptionfactors.
(*equal contribution)
Nat. Methods 10 (2013) 421-426
doi: 10.1038/nmeth.2411

M. Hinczewski, J.C.M. Gebhardt, M. Rief, and D. Thirumalai
From mechanical folding trajectories to intrinsic energy landscapes of proteins
In single-molecule laser optical tweezer (LOT) pulling experiments, a protein or RNA is juxtaposed between DNA handles that are attached to beads in optical traps. The LOT generates folding trajectories under force in terms of time-dependent changes in the distance between the beads. How to construct the full intrinsic folding landscape (without the handles and beads) from the measured time series is a major unsolved problem. By using rigorous theoretical methods - which account for fluctuations of the DNA handles, rotation of the optical beads, variations in applied tension due to finite trap stiffness, as well as environmental noise and limited bandwidth of the apparatus - we provide a trac-table method to derive intrinsic free-energy profiles. We validate the method by showing that the exactly calculable intrinsic free-energy profile for a generalized Rouse model, which mimics the two-state behavior in nucleic acid hairpins, can be accurately extracted from simulated time series in a LOT setup regardless of the stiffness of the handles. We next apply the approach to trajectories from coarse-grained LOT molecular simulations of a coiled-coil protein based on the GCN4 leucine zipper and obtain a free-energy landscape that is in quantitative agreement with simulations performed without the beads and handles. Finally, we extract the intrinsic free-energy landscape from experimental LOT measurements for the leucine zipper.
Proc. Natl. Acad. Sci. 110 (2013) 4500-4505
doi: 10.1073/pnas.1214051110

2011

J. Stigler, F. Ziegler, A. Gieseke, J.C.M. Gebhardt, and M. Rief
The Complex Folding Network of Single Calmodulin Molecules
Direct observation of the detailed conformational fluctuations of a single protein molecule en route to its folded state has so far been realized only in silico. We have used single-molecule force spectroscopy to study the folding transitions of single calmodulin molecules. High-resolution optical tweezers assays in combination with hidden Markov analysis reveal a complex network of on- and off-pathway intermediates. Cooperative and anticooperative interactions across domain boundaries can be observed directly. The folding network involves four intermediates. Two off-pathway intermediates exhibit non-native interdomain interactions and compete with the ultrafast productive folding pathway.
Science 28 (2011) 512 – 516
doi: 10.1126/science.1207598

2010

M. Brunnbauer, F. Mueller-Planitz, S. Kösem, T.H. Ho, R. Dombi, J.C.M. Gehardt, M. Rief, and Z. Ökten
Regulation of a Heterodimeric Kinesin-2 Through an Unprocessive Motor Domain that is Turned Processive by its Partner
Cilia are microtubule-based protrusions of the plasma membrane found on most eukaryotic cells. Their assembly is mediated through the conserved intraflagellar transport mechanism. One class of motor proteins involved in intraflagellar transport, kinesin-2, is unique among kinesin motors in that some of its members are composed of two distinct polypeptides. However, the biological reason for heterodimerization has remained elusive. Here we provide several interdependent reasons for the heterodimerization of the kinesin-2 motor KLP11/KLP20 of Caenorhabditis elegans cilia. One motor domain is unprocessive as a homodimer, but hetero-dimerization with a processive partner generates processivity. The “unprocessive” subunit is kept in this partnership as it mediates an asymmetric autoregulation of the motor activity. Finally, heterodimerization is necessary to bind KAP1, the in vivo link between motor and cargo.
Proc. Natl. Acad. Sci. 107 (2010) 10460-10465
doi: 10.1073/pnas.1005177107

J.C.M. Gebhardt, Z. Ökten, and M. Rief
The Lever Arm Effects a Mechanical Asymmetry of the Myosin-V – Actin Bond
Myosin-V is a two-headed molecular motor taking multiple ATP-dependent steps toward the plus end (forward) of actin filaments. At high mechanical loads, the motor processively steps toward the minus end (backward) even in the absence of ATP, whereas analogous forward steps cannot be induced. The detailed mechanism underlying this mechanical asymmetry is not known. We investigate the effect of force on individual single headed myosin-V constructs bound to actin in the absence of ATP. If pulled forward, the myosin-V head dissociates at forces twice as high than if pulled backward. Moreover, backward but not forward distances to the unbinding barrier are dependent on the lever arm length. This asymmetry of unbinding force distributions in a single headed myosin forms the basis of the two-headed asymmetry. Under load, the lever arm functions as a true lever in a mechanical sense.
Biophys J 98 (2010) 277-281
doi: 10.1016/j.bpj.2009.10.017

J.C.M. Gebhardt, T. Bornschlögl, and M. Rief
Full Distance-Resolved Folding Energy Landscape of one Single Protein Molecule
Kinetic bulk and single molecule folding experiments characterize barrier properties but the shape of folding landscapes between barrier top and native state is difficult to access. Here, we directly extract the full free energy landscape of a single molecule of the GCN4 leucine zipper using dual beam optical tweezers. To this end, we use deconvolution force spectroscopy to follow an individual molecule’s trajectory with high temporal and spatial resolution. We find a heterogeneous energy landscape of the GCN4 leucine zipper domain. The energy profile is divided into two stable C-terminal heptad repeats and two less stable repeats at the N-ter- minus. Energies and transition barrier positions were confirmed by single molecule kinetic analysis. We anticipate that deconvolution sampling is a powerful tool for the model-free investigation of protein energy landscapes.
Proc. Natl. Acad. Sci. USA 107 (2010) 2013-1018
doi: 10.1073/pnas.0909854107

2009

J.C.M. Gebhardt and M. Rief
Force Signaling in Biology
Many processes in our body, like muscle contraction, cell locomotion and division, or transport processes, need force-producing actuators such as molecular motors. In turn, biological systems can also sense mechanical forces. Examples are the sense of touch, hearing, and the strengthening of muscle tissues upon physical exercise. In these cases, force triggers a biochemical signal cascade, but the mechanisms by which forces affect biomolecular conformation and biochemical signaling have long remained elusive. The development of ultrasensitive instruments for nanomanipulation - such as atomic force microscopy and optical and magnetic tweezers - has allowed the effect of forces on protein conformation and function to be probed at the single-molecule level (1–4).
Science 324 (2009) 1278-1280
doi: 10.1126/science.1175874

T. Bornschlogl, J.C.M. Gebhardt, and M. Rief
Designing the Folding Mechanics of Coiled Coils
Naturally occurring coiled coils are often not homogeneous throughout their entire structure but rather interrupted by sequence discontinuities and non-coiled-coil-forming subsegments. We apply atomic force microscopy to locally probe the mechanical folding/unfolding process of a well-understood model coiled coil when unstructured subsegments with different sizes are added. We find that the refolding force decreases from 7.8 pN with increasing size of the added unstructured subsegment, while the unfolding properties of the model coiled coil remain unchanged. We show that this behavior results from the increased size of the nucleation seed which has to form before further coiled-coil folding can proceed. Since the nucleation seed size is linked to the width of the energetic folding barrier, we are able to directly measure the dependence of folding forces on the barrier width. Our results allow the design of coiled coils with designated refolding forces by simply adjusting the nucleation seed size.
Chemphyschem 10 (2009) 2800-2804
doi: 10.1002/cphc.200900575

2007

G. Cappello, P. Pierobon, C. Symonds, L. Busoni, J.C.M. Gebhardt, M. Rief, and J. Prost
Myosin-V Stepping Mechanism
We observe the myosin V stepping mechanism by traveling wave tracking. This technique, associated with optical tweezers, allows one to follow a scattering particle in a two-dimensional plane, with nanometer accuracy and a temporal resolution in the microsecond range. We have observed that, at the millisecond time scale, the myosin V combines longitudinal and vertical motions during the step. Because at this time scale the steps appear heterogeneous, we deduce their general features by aligning and averaging a large number of them. Our data show that the 36-nm step occurs in three main stages. First, the myosin center of mass moves forward 5 nm; the duration of this short prestep depends on the ATP concentration. Second, the motor performs a fast motion over 23 nm; this motion is associated to a vertical movement of the myosin center of mass, whose distance from the actin filament increases by 6 nm. Third, the myosin head freely diffuses toward the next binding site and the vertical position is recovered. We propose a simple model to describe the step mechanism of the dimeric myosin V.
Proc. Nat. Acad. Sci. 104 (2007) 15328-15333
doi: 10.1073/pnas.0706653104

2006

J.C.M. Gebhardt, A.E.-M. Clemen, J. Jaud, and M. Rief
Myosin-V is a Mechanical Ratchet
Myosin-V is a linear molecular motor that hydrolyzes ATP to move processively toward the plus end of actin filaments. Motion of this motor under low forces has been studied recently in various single-molecule assays. In this paper we show that myosin-V reacts to high forces as a mechanical ratchet. High backward loads can induce rapid and processive backward steps along the actin filament. This motion is completely independent of ATP binding and hydrolysis. In contrast, forward forces cannot induce ATP-independent forward steps. We can explain this pronounced mechanical asymmetry by a model in which the strength of actin binding of a motor head is modulated by the lever arm conformation. Knowledge of the complete force–velocity dependence of molecular motors is important to understand their function in the cellular environment.
Proc. Nat. Acad. Sci. 103 (2006) 8680-8685
doi: 10.1073/pnas.0510191103

2003

P. Persson, J.C.M. Gebhardt, and S. Lunell
The Smallest Possible Nanocrystals of Semiionic Oxides
General bonding principles are used to predict the structure of individual nanocrystals in nanocrystalline materials with semiionic bonding. The relationship between the general principles and actual nanocrystal structures is demonstrated using titanium dioxide in the anatase form. The proposed nanocrystals simultaneously fulfill strict criteria of stoichiometry, high coordination, and balanced charge distribution. The smallest such nanocrystals are remarkably simple, e.g., consisting of less than 100 atoms in anatase. According to computer simulations, these nanocrystals show strong quantum size effects, while other clusters of similar size instead show typical defect characteristics.
J. Phys. Chem. B 107 (2003) 3336-3339
doi: 10.1021/jp022036e