Chemically induced proximity in biology and medicine Stanton, Benjamin Z; Chory, Emma J; Crabtree, Gerald R
Science (American Association for the Advancement of Science),
03/2018, Letnik:
359, Številka:
6380
Journal Article
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Proximity, or the physical closeness of molecules, is a pervasive regulatory mechanism in biology. For example, most posttranslational modifications such as phosphorylation, methylation, and ...acetylation promote proximity of molecules to play deterministic roles in cellular processes. To understand the role of proximity in biologic mechanisms, chemical inducers of proximity (CIPs) were developed to synthetically model biologically regulated recruitment. Chemically induced proximity allows for precise temporal control of transcription, signaling cascades, chromatin regulation, protein folding, localization, and degradation, as well as a host of other biologic processes. A systematic analysis of CIPs in basic research, coupled with recent technological advances utilizing CRISPR, distinguishes roles of causality from coincidence and allows for mathematical modeling in synthetic biology. Recently, induced proximity has provided new avenues of gene therapy and emerging advances in cancer treatment.
Due to the rapid emergence of antibiotic-resistant bacteria, there is a growing need to discover new antibiotics. To address this challenge, we trained a deep neural network capable of predicting ...molecules with antibacterial activity. We performed predictions on multiple chemical libraries and discovered a molecule from the Drug Repurposing Hub—halicin—that is structurally divergent from conventional antibiotics and displays bactericidal activity against a wide phylogenetic spectrum of pathogens including Mycobacterium tuberculosis and carbapenem-resistant Enterobacteriaceae. Halicin also effectively treated Clostridioides difficile and pan-resistant Acinetobacter baumannii infections in murine models. Additionally, from a discrete set of 23 empirically tested predictions from >107 million molecules curated from the ZINC15 database, our model identified eight antibacterial compounds that are structurally distant from known antibiotics. This work highlights the utility of deep learning approaches to expand our antibiotic arsenal through the discovery of structurally distinct antibacterial molecules.
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•A deep learning model is trained to predict antibiotics based on structure•Halicin is predicted as an antibacterial molecule from the Drug Repurposing Hub•Halicin shows broad-spectrum antibiotic activities in mice•More antibiotics with distinct structures are predicted from the ZINC15 database
A trained deep neural network predicts antibiotic activity in molecules that are structurally different from known antibiotics, among which Halicin exhibits efficacy against broad-spectrum bacterial infections in mice.
Evolution occurs when selective pressures from the environment shape inherited variation over time. Within the laboratory, evolution is commonly used to engineer proteins and RNA, but experimental ...constraints have limited the ability to reproducibly and reliably explore factors such as population diversity, the timing of environmental changes and chance on outcomes. We developed a robotic system termed phage- and robotics-assisted near-continuous evolution (PRANCE) to comprehensively explore biomolecular evolution by performing phage-assisted continuous evolution in high-throughput. PRANCE implements an automated feedback control system that adjusts the stringency of selection in response to real-time measurements of each molecular activity. In evolving three distinct types of biomolecule, we find that evolution is reproducibly altered by both random chance and the historical pattern of environmental changes. This work improves the reliability of protein engineering and enables the systematic analysis of the historical, environmental and random factors governing biomolecular evolution.
Enhanced ascorbate peroxidase 2 (APEX2) is a protein generated with directed evolution by Lam et al. that has transformed our understanding of subcellular entities and phenomena. The rapid kinetics ...of this engineered protein highlights the power of directed evolution to expand the molecular toolkit for biologists.
The opposition between Polycomb repressive complexes (PRCs) and BAF (mSWI/SNF) complexes has a critical role in both development and disease. Mutations in the genes encoding BAF subunits contribute ...to more than 20% of human malignancies, yet the underlying mechanisms remain unclear, owing largely to a lack of assays to assess BAF function in living cells. To address this, we have developed a widely applicable recruitment assay system through which we find that BAF opposes PRC by rapid, ATP-dependent eviction, leading to the formation of accessible chromatin. The reversal of this process results in reassembly of facultative heterochromatin. Surprisingly, BAF-mediated PRC eviction occurs in the absence of RNA polymerase II (Pol II) occupancy, transcription, and replication. Further, we find that tumor-suppressor and oncogenic mutant BAF complexes have different effects on PRC eviction. The results of these studies define a mechanistic sequence underlying the resolution and formation of facultative heterochromatin, and they demonstrate that BAF opposes PRC on a minute-by-minute basis to provide epigenetic plasticity.
Our understanding of complex living systems is limited by our capacity to perform experiments in high throughput. While robotic systems have automated many traditional hand‐pipetting protocols, ...software limitations have precluded more advanced maneuvers required to manipulate, maintain, and monitor hundreds of experiments in parallel. Here, we present Pyhamilton, an open‐source Python platform that can execute complex pipetting patterns required for custom high‐throughput experiments such as the simulation of metapopulation dynamics. With an integrated plate reader, we maintain nearly 500 remotely monitored bacterial cultures in log‐phase growth for days without user intervention by taking regular density measurements to adjust the robotic method in real‐time. Using these capabilities, we systematically optimize bioreactor protein production by monitoring the fluorescent protein expression and growth rates of a hundred different continuous culture conditions in triplicate to comprehensively sample the carbon, nitrogen, and phosphorus fitness landscape. Our results demonstrate that flexible software can empower existing hardware to enable new types and scales of experiments, empowering areas from biomanufacturing to fundamental biology.
SYNOPSIS
An open‐source Python platform enables advanced liquid handling robots to perform a variety of complex high‐throughput experiments that could never be performed manually.
Bioautomation can benefit from flexible, easily shared protocols in a widely used language.
Custom techniques such as plaque assays can be readily automated.
480 bacterial turbidostats using 100 different media compositions areused to map the metabolic fitness landscape for recombinant protein production.
An open‐source Python platform enables advanced liquid handling robots to perform a variety of complex high‐throughput experiments that could never be performed manually.
Understanding the causal link between epigenetic marks and gene regulation remains a central question in chromatin biology. To edit the epigenome we developed the FIRE-Cas9 system for rapid and ...reversible recruitment of endogenous chromatin regulators to specific genomic loci. We enhanced the dCas9-MS2 anchor for genome targeting with Fkbp/Frb dimerizing fusion proteins to allow chemical-induced proximity of a desired chromatin regulator. We find that mSWI/SNF (BAF) complex recruitment is sufficient to oppose Polycomb within minutes, leading to activation of bivalent gene transcription in mouse embryonic stem cells. Furthermore, Hp1/Suv39h1 heterochromatin complex recruitment to active promoters deposits H3K9me3 domains, resulting in gene silencing that can be reversed upon washout of the chemical dimerizer. This inducible recruitment strategy provides precise kinetic information to model epigenetic memory and plasticity. It is broadly applicable to mechanistic studies of chromatin in mammalian cells and is particularly suited to the analysis of endogenous multi-subunit chromatin regulator complexes.Understanding the link between epigenetic marks and gene regulation requires the development of new tools to directly manipulate chromatin. Here the authors demonstrate a Cas9-based system to recruit chromatin remodelers to loci of interest, allowing rapid, reversible manipulation of epigenetic states.
The ability to control translation of endogenous or exogenous RNAs in eukaryotic cells would facilitate a variety of biotechnological applications. Current strategies are limited by low fold changes ...in transgene output and the size of trigger RNAs (trRNAs). Here we introduce eukaryotic toehold switches (eToeholds) as modular riboregulators. eToeholds contain internal ribosome entry site sequences and form inhibitory loops in the absence of a specific trRNA. When the trRNA is present, eToeholds anneal to it, disrupting the inhibitory loops and allowing translation. Through optimization of RNA annealing, we achieved up to 16-fold induction of transgene expression in mammalian cells. We demonstrate that eToeholds can discriminate among viral infection status, presence or absence of gene expression and cell types based on the presence of exogenous or endogenous RNA transcripts.
Recent studies have indicated that nucleosome turnover is rapid, occurring several times per cell cycle. To access the effect of nucleosome turnover on the epigenetic landscape, we investigated H3K79 ...methylation, which is produced by a single methyltransferase (Dot1l) with no known demethylase. Using chemical-induced proximity (CIP), we find that the valency of H3K79 methylation (mono-, di-, and tri-) is determined by nucleosome turnover rates. Furthermore, propagation of this mark is predicted by nucleosome turnover simulations over the genome and accounts for the asymmetric distribution of H3K79me toward the transcriptional unit. More broadly, a meta-analysis of other conserved histone modifications demonstrates that nucleosome turnover models predict both valency and chromosomal propagation of methylation marks. Based on data from worms, flies, and mice, we propose that the turnover of modified nucleosomes is a general means of propagation of epigenetic marks and a determinant of methylation valence.
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•Chemical-induced proximity of DOT1L leads to ordered addition of H3K79me1, me2, and me3•Methylation kinetics are loci specific and related to the rate of nucleosome turnover•Genome-wide analyses reveal quantitative relationship between turnover/demethylation•Simulations of turnover predict valence and topology of various histone modifications
Previous work has revealed that “writers” and “erasers” of histone modifications play a critical role in regulating gene expression. Now, studies by Chory et al. reveal that, in addition, the patterns, kinetics, and topology of histone modifications over the genome can be shaped by the rate of nucleosome turnover.
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