True equilibrium measurement of transcription factor-DNA binding affinities using automated polarization microscopy

Jung, Christophe; Bandilla, Peter; Reutern, Marc von; Schnepf, Max; Rieder, Susanne; Unnerstall, Ulrich; Gaul, Ulrike

doi:10.1038/s41467-018-03977-4

Cited by 32 publications

(37 citation statements)

References 46 publications

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“…, corresponding to dissociation constants of ~1-10 ! nM and consistent with values measured for transcription factors (Gebhardt et al 2013;Jung et al 2018). We simulated the models using the Gillespie algorithm (Gillespie 1977).…”

Section: Stochastic Simulations Identify Stable Cell Fate Maintenancesupporting

confidence: 61%

Mechanism of life-long maintenance of neuron identity despite molecular fluctuations

Traets

Burght

Jansen

et al. 2020

Preprint

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Cell fate is maintained over long timescales, yet molecular fluctuations can lead to spontaneous loss of this differentiated state. We uncovered a mechanism that explains lifelong maintenance of ASE neuron fate in C. elegans by the terminal selector transcription factor CHE-1. Fluctuations in CHE-1 level are buffered by the reservoir of CHE-1 bound at its target promoters, which ensure continued che-1 expression by preferentially binding the che-1 promoter. We validated this mechanism by showing that che-1 expression was resilient to induced transient CHE-1 depletion, while both expression of CHE-1 targets and ASE function were lost. We identified a 130 bp che-1 promoter fragment responsible for this resilience, with deletion of a homeodomain binding site in this fragment causing stochastic loss of ASE identity long after its determination. Because network architectures that support this mechanism are highly conserved in cell differentiation, it may explain stable cell fate maintenance in many systems. KeywordsNeuronal cell fate, bistability, gene regulatory network, stochastic gene expression, molecular fluctuations, homeodomain proteins, chemotaxis, C. elegans, terminal selector CHE-1, a transcription factor whose expression is transiently induced by the nuclear hormone receptor NHR-67 at the time of determination (Sarin et al. 2009). CHE-1 induces the expression of 500-1000 ASE-specific target genes, such as chemosensory receptors, ion-channels, and neuropeptides, by binding ASE motifs within their promoters (Etchberger et al. 2007). Its continued presence is required for expression of target genes after subtype determination (Etchberger et al. 2009). CHE-1 also upregulates its own expression. This positive feedback loop is necessary for sustaining che-1 expression and ASE cell fate directly after cell determination (Etchberger et al. 2007; Leyva-Diaz and Hobert 2019). Yet, it is unknown whether this positive feedback loop is sufficient for long-term maintenance of ASE fate. The impact of molecular noise, such as variability in CHE-1 protein copy number, on ASE fate maintenance has not been studied. Overall, it is an open question whether a reversible, bistable switch based on positive CHE-1 autoregulation would be sufficiently stable to maintain ASE fate for the animal's lifetime, or if additional mechanisms are necessary to ensure that, once ASE fate is determined, che-1 expression becomes independent of CHE-1 itself and can no longer spontaneously switch off.We show that sufficiently long, transiently induced depletion of CHE-1 caused permanent loss of ASE fate, indicating that it is controlled by a switch that remains reversible long after specification. This raises the question how the switch is protected against molecular noise, which could cause it to spontaneously lose ASE fate. Combining experimental measurements of the key parameters that control the magnitude of noise, i.e. the copy numbers and lifetimes of che-1 mRNA and protein, with stochastic models of the che-1 genetic network, revealed a novel...

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Section: Stochastic Simulations Identify Stable Cell Fate Maintenancesupporting

confidence: 61%

Mechanism of life-long maintenance of neuron identity despite molecular fluctuations

Traets

Burght

Jansen

et al. 2020

Preprint

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“…Commercial liquid handling equipment can also be used to scale up throughput. Binding energy landscapes can be generated by many approaches including PBMs (Bulyk et al 2001), MITOMI (Maerkl and Quake 2009), SELEX-seq (Zhao et al 2009), and HiP-FA (Jung et al 2018). While our binding energy landscapes are based on direct a nity measurements, it may be su cient to use PWMs from indirect measurements as found in other high-throughput techniques.…”

Section: Discussionmentioning

confidence: 99%

“…Although the development of new technologies is steadily enabling progress in this area, our understanding of GRNs remains limited as exemplified by our inability to predict in vivo gene expression levels in essentially any organism, and the di culty asso-ciated with de novo engineering of GRNs. Although methods exist for high-throughput in vitro characterization of transcription factor binding specificities(Bulyk et al 2001, Jung et al 2018, Maerkl and Quake 2007, Zhao et al 2009) and medium to high-throughput approaches are used to understand gene regulation in vivo(Barnes et al 2018, Mogno et al 2013, Rajkumar et al 2013, Sharon et al 2012 both approaches have limitations. Both an advantage and disadvantage of in vitro methods is that they generally include only the smallest number of components necessary, i.e.…”

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confidence: 99%

Cell-free gene regulatory network engineering with synthetic transcription factors

Swank

Laohakunakorn

Maerkl

2018

Preprint

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Gene regulatory networks are ubiquitous in nature and critical for bottom-up engineering of synthetic networks. Transcriptional repression is a fundamental function that can be tuned at the level of DNA, protein, and cooperative protein -protein interactions, necessitating high-throughput experimental approaches for in-depth characterization. Here we used a cell-free system in combination with a high-throughput microfluidic device to comprehensively study the di↵erent tuning mechanisms of a synthetic zinc-finger repressor library, whose a nity and cooperativity can be rationally engineered. The device is integrated into a comprehensive workflow that includes determination of transcription factor binding energy landscapes and mechanistic modeling, enabling us to generate a library of well-characterized synthetic transcription factors and corresponding promoters, which we then used to build gene regulatory networks de novo. The well-characterized synthetic parts and insights gained should be useful for rationally engineering gene regulatory networks and for studying the biophysics of transcriptional regulation. ⇤ equal first authors † Correspondence: sebastian.maerkl@epfl.ch

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“…Each TF binding site mimics a short (6-12bp) DNA sequence. Specific recognition of DNA motifs by TFs (Weirauch et al, 2014) is mediated by typical TF-DNA binding strengths corresponding to nanomolar dissociation equilibrium constants (Jung et al, 2018), which is the range of TF-DNA interaction energies that we have studied in our simulations (Methods). TFs and coactivators contain large IDRs that interact weakly with each other (Boija et al, 2018;Sabari et al, 2018).…”

Section: Development Of a Computational Modelmentioning

confidence: 99%

Enhancer features that drive formation of transcriptional condensates

Shrinivas

Sabari

Coffey

et al. 2018

Preprint

126

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Enhancers, DNA elements that regulate gene expression, contain transcription factor (TF) binding sites. TFs bind short sequence motifs that are present throughout the genome at much higher frequency than active enhancers, and so the features that define active enhancers are not well understood. We show that DNA elements with TF binding site valency, density, and binding affinity above sharply defined thresholds can recruit TFs and coactivators in condensates by the cooperative process of phase separation. We demonstrate that weak cooperative interactions between IDRs of TFs and coactivators in combination with specific TF-DNA interactions are required for forming such transcriptional condensates. IDR-IDR interactions are relatively nonspecific with the same molecular interactions shared by many TFs and coactivators, and phase separation is a universal cooperative mechanism. Therefore, whether a genomic locus is an enhancer that can assemble a transcriptional condensate is determined predominantly by its cognate TFs' binding site valency and density. helpful discussions. We acknowledge the support of Wendy Salmon and the W.M Keck Microscopy Facility at the Whitehead Institute.

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True equilibrium measurement of transcription factor-DNA binding affinities using automated polarization microscopy

Cited by 32 publications

References 46 publications

Mechanism of life-long maintenance of neuron identity despite molecular fluctuations

Mechanism of life-long maintenance of neuron identity despite molecular fluctuations

Cell-free gene regulatory network engineering with synthetic transcription factors

Enhancer features that drive formation of transcriptional condensates

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