TY - JOUR AB - In Gram negative bacteria, the multiple antibiotic resistance or mar operon, is known to control the expression of multi-drug efflux genes that protect bacteria from a wide range of drugs. As many different chemical compounds can induce this operon, identifying the parameters that govern the dynamics of its induction is crucial to better characterize the processes of tolerance and resistance. Most experiments have assumed that the properties of the mar transcriptional network can be inferred from population measurements. However, measurements from an asynchronous population of cells can mask underlying phenotypic variations of single cells. We monitored the activity of the mar promoter in single Escherichia coli cells in linear micro-colonies and established that the response to a steady level of inducer was most heterogeneous within individual colonies for an intermediate value of inducer. Specifically, sub-lineages defined by contiguous daughter-cells exhibited similar promoter activity, whereas activity was greatly variable between different sub-lineages. Specific sub-trees of uniform promoter activity persisted over several generations. Statistical analyses of the lineages suggest that the presence of these sub-trees is the signature of an inducible memory of the promoter state that is transmitted from mother to daughter cells. This single-cell study reveals that the degree of epigenetic inheritance changes as a function of inducer concentration, suggesting that phenotypic inheritance may be an inducible phenotype. AU - Guet, Calin C AU - Bruneaux, L AU - Oikonomou, P AU - Aldana, M AU - Cluzel, P ID - 12478 JF - Frontiers in Microbiology TI - Monitoring lineages of growing and dividing bacteria reveals an inducible memory of mar operon expression VL - 14 ER - TY - JOUR AB - Understanding and characterising biochemical processes inside single cells requires experimental platforms that allow one to perturb and observe the dynamics of such processes as well as computational methods to build and parameterise models from the collected data. Recent progress with experimental platforms and optogenetics has made it possible to expose each cell in an experiment to an individualised input and automatically record cellular responses over days with fine time resolution. However, methods to infer parameters of stochastic kinetic models from single-cell longitudinal data have generally been developed under the assumption that experimental data is sparse and that responses of cells to at most a few different input perturbations can be observed. Here, we investigate and compare different approaches for calculating parameter likelihoods of single-cell longitudinal data based on approximations of the chemical master equation (CME) with a particular focus on coupling the linear noise approximation (LNA) or moment closure methods to a Kalman filter. We show that, as long as cells are measured sufficiently frequently, coupling the LNA to a Kalman filter allows one to accurately approximate likelihoods and to infer model parameters from data even in cases where the LNA provides poor approximations of the CME. Furthermore, the computational cost of filtering-based iterative likelihood evaluation scales advantageously in the number of measurement times and different input perturbations and is thus ideally suited for data obtained from modern experimental platforms. To demonstrate the practical usefulness of these results, we perform an experiment in which single cells, equipped with an optogenetic gene expression system, are exposed to various different light-input sequences and measured at several hundred time points and use parameter inference based on iterative likelihood evaluation to parameterise a stochastic model of the system. AU - Davidović, Anđela AU - Chait, Remy P AU - Batt, Gregory AU - Ruess, Jakob ID - 10939 IS - 3 JF - PLoS Computational Biology SN - 1553-734X TI - Parameter inference for stochastic biochemical models from perturbation experiments parallelised at the single cell level VL - 18 ER - TY - JOUR AB - Objective: MazF is a sequence-specific endoribonuclease-toxin of the MazEF toxin–antitoxin system. MazF cleaves single-stranded ribonucleic acid (RNA) regions at adenine–cytosine–adenine (ACA) sequences in the bacterium Escherichia coli. The MazEF system has been used in various biotechnology and synthetic biology applications. In this study, we infer how ectopic mazF overexpression affects production of heterologous proteins. To this end, we quantified the levels of fluorescent proteins expressed in E. coli from reporters translated from the ACA-containing or ACA-less messenger RNAs (mRNAs). Additionally, we addressed the impact of the 5′-untranslated region of these reporter mRNAs under the same conditions by comparing expression from mRNAs that comprise (canonical mRNA) or lack this region (leaderless mRNA). Results: Flow cytometry analysis indicates that during mazF overexpression, fluorescent proteins are translated from the canonical as well as leaderless mRNAs. Our analysis further indicates that longer mazF overexpression generally increases the concentration of fluorescent proteins translated from ACA-less mRNAs, however it also substantially increases bacterial population heterogeneity. Finally, our results suggest that the strength and duration of mazF overexpression should be optimized for each experimental setup, to maximize the heterologous protein production and minimize the amount of phenotypic heterogeneity in bacterial populations, which is unfavorable in biotechnological processes. AU - Nikolic, Nela AU - Sauert, Martina AU - Albanese, Tanino G. AU - Moll, Isabella ID - 11713 JF - BMC Research Notes KW - General Biochemistry KW - Genetics and Molecular Biology KW - General Medicine SN - 1756-0500 TI - Quantifying heterologous gene expression during ectopic MazF production in Escherichia coli VL - 15 ER - TY - JOUR AB - Predicting function from sequence is a central problem of biology. Currently, this is possible only locally in a narrow mutational neighborhood around a wildtype sequence rather than globally from any sequence. Using random mutant libraries, we developed a biophysical model that accounts for multiple features of σ70 binding bacterial promoters to predict constitutive gene expression levels from any sequence. We experimentally and theoretically estimated that 10–20% of random sequences lead to expression and ~80% of non-expressing sequences are one mutation away from a functional promoter. The potential for generating expression from random sequences is so pervasive that selection acts against σ70-RNA polymerase binding sites even within inter-genic, promoter-containing regions. This pervasiveness of σ70-binding sites implies that emergence of promoters is not the limiting step in gene regulatory evolution. Ultimately, the inclusion of novel features of promoter function into a mechanistic model enabled not only more accurate predictions of gene expression levels, but also identified that promoters evolve more rapidly than previously thought. AU - Lagator, Mato AU - Sarikas, Srdjan AU - Steinrueck, Magdalena AU - Toledo-Aparicio, David AU - Bollback, Jonathan P AU - Guet, Calin C AU - Tkačik, Gašper ID - 10736 JF - eLife TI - Predicting bacterial promoter function and evolution from random sequences VL - 11 ER - TY - JOUR AB - Several promising strategies based on combining or cycling different antibiotics have been proposed to increase efficacy and counteract resistance evolution, but we still lack a deep understanding of the physiological responses and genetic mechanisms that underlie antibiotic interactions and the clinical applicability of these strategies. In antibiotic-exposed bacteria, the combined effects of physiological stress responses and emerging resistance mutations (occurring at different time scales) generate complex and often unpredictable dynamics. In this Review, we present our current understanding of bacterial cell physiology and genetics of responses to antibiotics. We emphasize recently discovered mechanisms of synergistic and antagonistic drug interactions, hysteresis in temporal interactions between antibiotics that arise from microbial physiology and interactions between antibiotics and resistance mutations that can cause collateral sensitivity or cross-resistance. We discuss possible connections between the different phenomena and indicate relevant research directions. A better and more unified understanding of drug and genetic interactions is likely to advance antibiotic therapy. AU - Römhild, Roderich AU - Bollenbach, Mark Tobias AU - Andersson, Dan I. ID - 10812 JF - Nature Reviews Microbiology KW - General Immunology and Microbiology KW - Microbiology KW - Infectious Diseases SN - 1740-1526 TI - The physiology and genetics of bacterial responses to antibiotic combinations VL - 20 ER - TY - JOUR AB - The interaction between a cell and its environment shapes fundamental intracellular processes such as cellular metabolism. In most cases growth rate is treated as a proximal metric for understanding the cellular metabolic status. However, changes in growth rate might not reflect metabolic variations in individuals responding to environmental fluctuations. Here we use single-cell microfluidics-microscopy combined with transcriptomics, proteomics and mathematical modelling to quantify the accumulation of glucose within Escherichia coli cells. In contrast to the current consensus, we reveal that environmental conditions which are comparatively unfavourable for growth, where both nutrients and salinity are depleted, increase glucose accumulation rates in individual bacteria and population subsets. We find that these changes in metabolic function are underpinned by variations at the translational and posttranslational level but not at the transcriptional level and are not dictated by changes in cell size. The metabolic response-characteristics identified greatly advance our fundamental understanding of the interactions between bacteria and their environment and have important ramifications when investigating cellular processes where salinity plays an important role. AU - Glover, Georgina AU - Voliotis, Margaritis AU - Łapińska, Urszula AU - Invergo, Brandon M. AU - Soanes, Darren AU - O’Neill, Paul AU - Moore, Karen AU - Nikolic, Nela AU - Petrov, Peter AU - Milner, David S. AU - Roy, Sumita AU - Heesom, Kate AU - Richards, Thomas A. AU - Tsaneva-Atanasova, Krasimira AU - Pagliara, Stefano ID - 11339 JF - Communications Biology TI - Nutrient and salt depletion synergistically boosts glucose metabolism in individual Escherichia coli cells VL - 5 ER - TY - JOUR 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 mouse dendritic cells (DCs) as a 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 the pathogenic strain CFT073 to CD14 reduced DC migration by overactivation of integrins and blunted expression of co-stimulatory molecules by overactivating the NFAT (nuclear factor of activated T-cells) pathway, both rate-limiting factors of T cell activation. This response was binary at the single-cell level, but averaged in larger populations exposed to both piliated and non-piliated pathogens, presumably via the exchange of immunomodulatory cytokines. 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 - 11843 JF - eLife TI - Type 1 piliated uropathogenic Escherichia coli hijack the host immune response by binding to CD14 VL - 11 ER - TY - JOUR AB - Together, copy-number and point mutations form the basis for most evolutionary novelty, through the process of gene duplication and divergence. While a plethora of genomic data reveals the long-term fate of diverging coding sequences and their cis-regulatory elements, little is known about the early dynamics around the duplication event itself. In microorganisms, selection for increased gene expression often drives the expansion of gene copy-number mutations, which serves as a crude adaptation, prior to divergence through refining point mutations. Using a simple synthetic genetic reporter system that can distinguish between copy-number and point mutations, we study their early and transient adaptive dynamics in real time in Escherichia coli. We find two qualitatively different routes of adaptation, depending on the level of functional improvement needed. In conditions of high gene expression demand, the two mutation types occur as a combination. However, under low gene expression demand, copy-number and point mutations are mutually exclusive; here, owing to their higher frequency, adaptation is dominated by copy-number mutations, in a process we term amplification hindrance. Ultimately, due to high reversal rates and pleiotropic cost, copy-number mutations may not only serve as a crude and transient adaptation, but also constrain sequence divergence over evolutionary time scales. AU - Tomanek, Isabella AU - Guet, Calin C ID - 12333 JF - eLife TI - Adaptation dynamics between copynumber and point mutations VL - 11 ER - TY - GEN AB - Copy-number and point mutations form the basis for most evolutionary novelty through the process of gene duplication and divergence. While a plethora of genomic sequence data reveals the long-term fate of diverging coding sequences and their cis-regulatory elements, little is known about the early dynamics around the duplication event itself. In microorganisms, selection for increased gene expression often drives the expansion of gene copy-number mutations, which serves as a crude adaptation, prior to divergence through refining point mutations. Using a simple synthetic genetic system that allows us to distinguish copy-number and point mutations, we study their early and transient adaptive dynamics in real-time in Escherichia coli. We find two qualitatively different routes of adaptation depending on the level of functional improvement selected for: In conditions of high gene expression demand, the two types of mutations occur as a combination. Under low gene expression demand, negative epistasis between the two types of mutations renders them mutually exclusive. Thus, owing to their higher frequency, adaptation is dominated by copy-number mutations. Ultimately, due to high rates of reversal and pleiotropic cost, copy-number mutations may not only serve as a crude and transient adaptation but also constrain sequence divergence over evolutionary time scales. AU - Tomanek, Isabella AU - Guet, Calin C ID - 12339 TI - Flow cytometry YFP and CFP data and deep sequencing data of populations evolving in galactose ER - TY - JOUR AU - Römhild, Roderich AU - Andersson, Dan I. ID - 9046 IS - 1 JF - PLoS Pathogens SN - 15537366 TI - Mechanisms and therapeutic potential of collateral sensitivity to antibiotics VL - 17 ER - 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 -