TY - JOUR AB - Gene expression is regulated by the set of transcription factors (TFs) that bind to the promoter. The ensuing regulating function is often represented as a combinational logic circuit, where output (gene expression) is determined by current input values (promoter bound TFs) only. However, the simultaneous arrival of TFs is a strong assumption, since transcription and translation of genes introduce intrinsic time delays and there is no global synchronisation among the arrival times of different molecular species at their targets. We present an experimentally implementable genetic circuit with two inputs and one output, which in the presence of small delays in input arrival, exhibits qualitatively distinct population-level phenotypes, over timescales that are longer than typical cell doubling times. From a dynamical systems point of view, these phenotypes represent long-lived transients: although they converge to the same value eventually, they do so after a very long time span. The key feature of this toy model genetic circuit is that, despite having only two inputs and one output, it is regulated by twenty-three distinct DNA-TF configurations, two of which are more stable than others (DNA looped states), one promoting and another blocking the expression of the output gene. Small delays in input arrival time result in a majority of cells in the population quickly reaching the stable state associated with the first input, while exiting of this stable state occurs at a slow timescale. In order to mechanistically model the behaviour of this genetic circuit, we used a rule-based modelling language, and implemented a grid-search to find parameter combinations giving rise to long-lived transients. Our analysis shows that in the absence of feedback, there exist path-dependent gene regulatory mechanisms based on the long timescale of transients. The behaviour of this toy model circuit suggests that gene regulatory networks can exploit event timing to create phenotypes, and it opens the possibility that they could use event timing to memorise events, without regulatory feedback. The model reveals the importance of (i) mechanistically modelling the transitions between the different DNA-TF states, and (ii) employing transient analysis thereof. AU - Petrov, Tatjana AU - Igler, Claudia AU - Sezgin, Ali AU - Henzinger, Thomas A AU - Guet, Calin C ID - 9647 JF - Theoretical Computer Science SN - 0304-3975 TI - Long lived transients in gene regulation VL - 893 ER - TY - JOUR AB - Attachment of adhesive molecules on cell culture surfaces to restrict cell adhesion to defined areas and shapes has been vital for the progress of in vitro research. In currently existing patterning methods, a combination of pattern properties such as stability, precision, specificity, high-throughput outcome, and spatiotemporal control is highly desirable but challenging to achieve. Here, we introduce a versatile and high-throughput covalent photoimmobilization technique, comprising a light-dose-dependent patterning step and a subsequent functionalization of the pattern via click chemistry. This two-step process is feasible on arbitrary surfaces and allows for generation of sustainable patterns and gradients. The method is validated in different biological systems by patterning adhesive ligands on cell-repellent surfaces, thereby constraining the growth and migration of cells to the designated areas. We then implement a sequential photopatterning approach by adding a second switchable patterning step, allowing for spatiotemporal control over two distinct surface patterns. As a proof of concept, we reconstruct the dynamics of the tip/stalk cell switch during angiogenesis. Our results show that the spatiotemporal control provided by our “sequential photopatterning” system is essential for mimicking dynamic biological processes and that our innovative approach has great potential for further applications in cell science. AU - Zisis, Themistoklis AU - Schwarz, Jan AU - Balles, Miriam AU - Kretschmer, Maibritt AU - Nemethova, Maria AU - Chait, Remy P AU - Hauschild, Robert AU - Lange, Janina AU - Guet, Calin C AU - Sixt, Michael K AU - Zahler, Stefan ID - 9822 IS - 30 JF - ACS Applied Materials and Interfaces SN - 19448244 TI - Sequential and switchable patterning for studying cellular processes under spatiotemporal control VL - 13 ER - TY - JOUR AB - Evolutionary adaptation is a major source of antibiotic resistance in bacterial pathogens. Evolution-informed therapy aims to constrain resistance by accounting for bacterial evolvability. Sequential treatments with antibiotics that target different bacterial processes were previously shown to limit adaptation through genetic resistance trade-offs and negative hysteresis. Treatment with homogeneous sets of antibiotics is generally viewed to be disadvantageous, as it should rapidly lead to cross-resistance. We here challenged this assumption by determining the evolutionary response of Pseudomonas aeruginosa to experimental sequential treatments involving both heterogenous and homogeneous antibiotic sets. To our surprise, we found that fast switching between only β-lactam antibiotics resulted in increased extinction of bacterial populations. We demonstrate that extinction is favored by low rates of spontaneous resistance emergence and low levels of spontaneous cross-resistance among the antibiotics in sequence. The uncovered principles may help to guide the optimized use of available antibiotics in highly potent, evolution-informed treatment designs. AU - Batra, Aditi AU - Römhild, Roderich AU - Rousseau, Emilie AU - Franzenburg, Sören AU - Niemann, Stefan AU - Schulenburg, Hinrich ID - 9746 JF - eLife TI - High potency of sequential therapy with only beta-lactam antibiotics VL - 10 ER - TY - JOUR AB - Erythropoietin enhances oxygen delivery and reduces hypoxia-induced cell death, but its pro-thrombotic activity is problematic for use of erythropoietin in treating hypoxia. We constructed a fusion protein that stimulates red blood cell production and neuroprotection without triggering platelet production, a marker for thrombosis. The protein consists of an anti-glycophorin A nanobody and an erythropoietin mutant (L108A). The mutation reduces activation of erythropoietin receptor homodimers that induce erythropoiesis and thrombosis, but maintains the tissue-protective signaling. The binding of the nanobody element to glycophorin A rescues homodimeric erythropoietin receptor activation on red blood cell precursors. In a cell proliferation assay, the fusion protein is active at 10−14 M, allowing an estimate of the number of receptor–ligand complexes needed for signaling. This fusion protein stimulates erythroid cell proliferation in vitro and in mice, and shows neuroprotective activity in vitro. Our erythropoietin fusion protein presents a novel molecule for treating hypoxia. AU - Lee, Jungmin AU - Vernet, Andyna AU - Gruber, Nathalie AU - Kready, Kasia M. AU - Burrill, Devin R. AU - Way, Jeffrey C. AU - Silver, Pamela A. ID - 10363 JF - Protein Engineering, Design and Selection SN - 1741-0126 TI - Rational engineering of an erythropoietin fusion protein to treat hypoxia VL - 34 ER - TY - JOUR AB - Gene expression levels are influenced by multiple coexisting molecular mechanisms. Some of these interactions such as those of transcription factors and promoters have been studied extensively. However, predicting phenotypes of gene regulatory networks (GRNs) remains a major challenge. Here, we use a well-defined synthetic GRN to study in Escherichia coli how network phenotypes depend on local genetic context, i.e. the genetic neighborhood of a transcription factor and its relative position. We show that one GRN with fixed topology can display not only quantitatively but also qualitatively different phenotypes, depending solely on the local genetic context of its components. Transcriptional read-through is the main molecular mechanism that places one transcriptional unit (TU) within two separate regulons without the need for complex regulatory sequences. We propose that relative order of individual TUs, with its potential for combinatorial complexity, plays an important role in shaping phenotypes of GRNs. AU - Nagy-Staron, Anna A AU - Tomasek, Kathrin AU - Caruso Carter, Caroline AU - Sonnleitner, Elisabeth AU - Kavcic, Bor AU - Paixão, Tiago AU - Guet, Calin C ID - 9283 JF - eLife KW - Genetics and Molecular Biology SN - 2050-084X TI - Local genetic context shapes the function of a gene regulatory network VL - 10 ER - TY - THES AB - Bacteria-host interactions represent a continuous trade-off between benefit and risk. Thus, the host immune response is faced with a non-trivial problem – accommodate beneficial commensals and remove harmful pathogens. This is especially difficult as molecular patterns, such as lipopolysaccharide or specific surface organelles such as pili, are conserved in both, commensal and pathogenic bacteria. Type 1 pili, tightly regulated by phase variation, are considered an important virulence factor of pathogenic bacteria as they facilitate invasion into host cells. While invasion represents a de facto passive mechanism for pathogens to escape the host immune response, we demonstrate a fundamental role of type 1 pili as active modulators of the innate and adaptive immune response. AU - Tomasek, Kathrin ID - 10307 SN - 2663-337X TI - Pathogenic Escherichia coli hijack the host immune response ER - TY - GEN AB - A key attribute of persistent or recurring bacterial infections is the ability of the pathogen to evade the host’s immune response. Many Enterobacteriaceae express type 1 pili, a pre-adapted virulence trait, to invade host epithelial cells and establish persistent infections. However, the molecular mechanisms and strategies by which bacteria actively circumvent the immune response of the host remain poorly understood. Here, we identified CD14, the major co-receptor for lipopolysaccharide detection, on dendritic cells as a previously undescribed binding partner of FimH, the protein located at the tip of the type 1 pilus of Escherichia coli. The FimH amino acids involved in CD14 binding are highly conserved across pathogenic and non-pathogenic strains. Binding of pathogenic bacteria to CD14 lead to reduced dendritic cell migration and blunted expression of co-stimulatory molecules, both rate-limiting factors of T cell activation. While defining an active molecular mechanism of immune evasion by pathogens, the interaction between FimH and CD14 represents a potential target to interfere with persistent and recurrent infections, such as urinary tract infections or Crohn’s disease. AU - Tomasek, Kathrin AU - Leithner, Alexander F AU - Glatzová, Ivana AU - Lukesch, Michael S. AU - Guet, Calin C AU - Sixt, Michael K ID - 10316 T2 - bioRxiv TI - Type 1 piliated uropathogenic Escherichia coli hijack the host immune response by binding to CD14 ER - TY - CHAP AB - Understanding how the activity of membrane receptors and cellular signaling pathways shapes cell behavior is of fundamental interest in basic and applied research. Reengineering receptors to react to light instead of their cognate ligands allows for generating defined signaling inputs with high spatial and temporal precision and facilitates the dissection of complex signaling networks. Here, we describe fundamental considerations in the design of light-regulated receptor tyrosine kinases (Opto-RTKs) and appropriate control experiments. We also introduce methods for transient receptor expression in HEK293 cells, quantitative assessment of signaling activity in reporter gene assays, semiquantitative assessment of (in)activation time courses through Western blot (WB) analysis, and easy to implement light stimulation hardware. AU - Kainrath, Stephanie AU - Janovjak, Harald L ED - Niopek, Dominik ID - 8173 T2 - Photoswitching Proteins TI - Design and application of light-regulated receptor tyrosine kinases VL - 2173 ER - TY - THES AB - In the thesis we focus on the interplay of the biophysics and evolution of gene regulation. We start by addressing how the type of prokaryotic gene regulation – activation and repression – affects spurious binding to DNA, also known as transcriptional crosstalk. We propose that regulatory interference caused by excess regulatory proteins in the dense cellular medium – global crosstalk – could be a factor in determining which type of gene regulatory network is evolutionarily preferred. Next,we use a normative approach in eukaryotic gene regulation to describe minimal non-equilibrium enhancer models that optimize so-called regulatory phenotypes. We find a class of models that differ from standard thermodynamic equilibrium models by a single parameter that notably increases the regulatory performance. Next chapter addresses the question of genotype-phenotype-fitness maps of higher dimensional phenotypes. We show that our biophysically realistic approach allows us to understand how the mechanisms of promoter function constrain genotypephenotype maps, and how they affect the evolutionary trajectories of promoters. In the last chapter we ask whether the intrinsic instability of gene duplication and amplification provides a generic alternative to canonical gene regulation. Using mathematical modeling, we show that amplifications can tune gene expression in many environments, including those where transcription factor-based schemes are hard to evolve or maintain. AU - Grah, Rok ID - 8155 SN - 2663-337X TI - Gene regulation across scales – how biophysical constraints shape evolution ER - TY - GEN AB - In prokaryotes, thermodynamic models of gene regulation provide a highly quantitative mapping from promoter sequences to gene expression levels that is compatible with in vivo and in vitro bio-physical measurements. Such concordance has not been achieved for models of enhancer function in eukaryotes. In equilibrium models, it is difficult to reconcile the reported short transcription factor (TF) residence times on the DNA with the high specificity of regulation. In non-equilibrium models, progress is difficult due to an explosion in the number of parameters. Here, we navigate this complexity by looking for minimal non-equilibrium enhancer models that yield desired regulatory phenotypes: low TF residence time, high specificity and tunable cooperativity. We find that a single extra parameter, interpretable as the “linking rate” by which bound TFs interact with Mediator components, enables our models to escape equilibrium bounds and access optimal regulatory phenotypes, while remaining consistent with the reported phenomenology and simple enough to be inferred from upcoming experiments. We further find that high specificity in non-equilibrium models is in a tradeoff with gene expression noise, predicting bursty dynamics — an experimentally-observed hallmark of eukaryotic transcription. By drastically reducing the vast parameter space to a much smaller subspace that optimally realizes biological function prior to inference from data, our normative approach holds promise for mathematical models in systems biology. AU - Grah, Rok AU - Zoller, Benjamin AU - Tkačik, Gašper ID - 7675 T2 - bioRxiv TI - Normative models of enhancer function ER -