Refereed Preprints

Review Commons provides authors with a Refereed Preprint, which includes the authors’ manuscript, reports from a single round of peer review and the authors’ response. These Refereed Preprints are transferred on the author’s behalf to bioRxiv. The most recently-completed Review Commons peer-reviews are listed below, with the most recently posted reviews at the top.

Latest Refereed Preprints

Cancer Biology | 25 Jan 2021
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Convergent organization of aberrant MYB complex controls oncogenic gene expression in acute myeloid leukemia

Sumiko Takao, Lauren Forbes, Masahiro Uni, Shuyuan Cheng, Jose Mario Bello Pineda, Yusuke Tarumoto, Paolo Cifani, Gerard Minuesa, Celine Chen, Michael G Kharas, Robert K Bradley, Christopher R Vakoc, Richard P Koche, Alex Kentsis

Dysregulated gene expression contributes to most prevalent features in human cancers. Here, we show that most subtypes of acute myeloid leukemia (AML) depend on the aberrant assembly of MYB transcriptional co-activator complex. By rapid and selective peptidomimetic interference with the binding of CBP/P300 to MYB, but not CREB or MLL1, we find that the leukemic functions of MYB are mediated by CBP/P300 co-activation of a distinct set of transcription factor complexes. These MYB complexes assemble aberrantly with LYL1, E2A, C/EBP family members, LMO2 and SATB1. They are organized convergently in genetically diverse subtypes of AML, and are at least in part associated with inappropriate transcription factor co-expression. Peptidomimetic remodeling of oncogenic MYB complexes is accompanied by specific proteolysis and dynamic redistribution of CBP/P300 with alternative transcription factors such as RUNX1 to induce myeloid differentiation and apoptosis. Thus, aberrant assembly and sequestration of MYB:CBP/P300 complexes provide a unifying mechanism of oncogenic gene expression in AML. This work establishes a compelling strategy for their pharmacologic reprogramming and therapeutic targeting for diverse leukemias and possibly other human cancers caused by dysregulated gene control.
Biophysics | 30 Sep 2020
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The genotype-phenotype landscape of an allosteric protein

Drew S. Tack, Peter D. Tonner, Abe Pressman, Nathanael D. Olson, Sasha F. Levy, Eugenia F. Romantseva, Nina Alperovich, Olga Vasilyeva, David Ross

Allostery is a fundamental biophysical mechanism that underlies cellular sensing, signaling, and metabolism. Yet a quantitative understanding of allosteric genotype-phenotype relationships remains elusive. Here we report the large-scale measurement of the genotype-phenotype landscape for an allosteric protein: the lac repressor from Escherichia coli, LacI. Using a method that combines long-read and short-read DNA sequencing, we quantitatively measure the dose-response curves for nearly 105 variants of the LacI genetic sensor. The resulting data provide a quantitative map of the effect of amino acid substitutions on LacI allostery and reveal systematic sequence-structure-function relationships. We find that in many cases, allosteric phenotypes can be quantitatively predicted with additive or neural-network models, but unpredictable changes also occur. For example, we were surprised to discover a new band-stop phenotype that challenges conventional models of allostery and that emerges from combinations of nearly silent amino acid substitutions.
Immunology | 14 Oct 2020
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IL-13 deficiency exacerbates lung damage and impairs epithelial-derived type 2 molecules during nematode infection

AL Chenery, S Rosini, JE Parkinson, JA Herrera, Craig Lawless, BHK Chan, P Loke, AS MacDonald, KE Kadler, TE Sutherland, JE Allen

IL-13 plays a key role during protective type 2 immune responses at mucosal sites, such as during infection with nematodes. However, dysregulation of IL-13 can also contribute to the pathogenesis of atopic and fibrotic diseases such as allergic asthma. Matrix remodelling is an important component of repair processes in the lung but also a hallmark of chronic conditions involving fibrosis. Hence, understanding the role of IL-13 in tissue remodelling has important clinical implications. Since IL-13 shares receptors and signalling pathways with IL-4, disentangling the relative contributions of these type 2 cytokines has been challenging. Additionally, little is known about the singular role of IL-13 following acute tissue injury. In this study, we used Nippostrongylus brasiliensis infection as a model of acute lung tissue damage comparing responses between WT and IL-13-deficient mice, in which IL-4 signalling is intact. Importantly, we found that IL-13 played a critical role in limiting tissue injury and haemorrhaging in the lung following infection. Through proteomic and transcriptomic profiling, we identified IL-13-dependent changes in matrix and associated regulators. We further showed that IL-13 is required for the induction of epithelial-derived type 2 effector molecules such as RELM-α and surfactant protein D. Pathway analyses predicted that IL-13 was heavily involved in the induction of cellular stress responses and regulation of lung epithelial cell differentiation by suppression of Foxa2 pathways. Thus, we propose that IL-13 has tissue-protective functions during lung injury and regulates epithelial cell responses during type 2 immunity in this acute setting.
Developmental Biology | 02 Oct 2020
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During development, gene expression regulates cell mechanics and shape to sculpt tissues. Epithelial folding proceeds through distinct cell shape changes that occur in different regions of a tissue. Here, using quantitative imaging in Drosophila melanogaster, we investigate how patterned cell shape changes promote tissue bending during early embryogenesis. We find that the transcription factors Twist and Snail combinatorially regulate a unique multicellular pattern of junctional F-actin density, which corresponds to whether cells apically constrict, stretch, or maintain their shape. Part of this pattern is a gradient in junctional F-actin and apical myosin-2, and the width of this gradient regulates tissue curvature. The actomyosin gradient results from a gradient in RhoA activation that is refined by a balance between RhoGEF2 and the RhoGAP C-GAP. Thus, cell behavior in the ventral furrow is choreographed by the interplay of distinct gene expression patterns and this coordination regulates tissue shape.
Genetics | 14 Sep 2020
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Q-STARZ: Quantitative Spatial and Temporal Assessment of Regulatory element activity in Zebrafish

Shipra Bhatia, Wendy Bickmore, Dirk Jan Kleinjan, Kirsty Uttley, Anita Mann, Nefeli Dellepiane

Noncoding regions of the genome harbouring cis-regulatory elements (CREs) or enhancers drive spatial and temporal gene expression. Mutations or single nucleotide polymorphisms (SNPs) in enhancers have been widely implicated in human diseases and disease-predispositions. However, our ability to assay the regulatory potential of genetic variants in enhancers is currently very limited, in part because of the need to assay these elements in an appropriate biological context. Here, we describe a method for simultaneous quantitative assessment of the spatial and temporal activity of wild-type (Wt) and disease-associated, mutant (Mut) human CRE alleles using live imaging in zebrafish embryonic development. We generated transgenic lines harbouring a dual-CRE dual-reporter cassette in a pre-defined neutral docking site in the zebrafish genome. Using this single transgenic cassette, the functional activity of each CRE allele is reported via expression of a specific fluorescent reporter, allowing the simultaneous visualisation of the activity of both alleles. This can reveal where and when in embryonic development the wild-type allele is active and how this activity is altered by the disease-associated mutation.
Bioinformatics | 07 Apr 2020
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Generating functional protein variants with variational autoencoders

Alex Hawkins-Hooker, Florence Depardieu, Sebastien Baur, Guillaume Couairon, Arthur Chen, David Bikard

The design of novel proteins with specified function and controllable biochemical properties is a longstanding goal in bio-engineering with potential applications across medicine and nanotechnology. The vast expansion of protein sequence databases over the last decades provides an opportunity for new approaches which seek to learn the sequence-function relationship directly from natural sequence variation. Advances in deep generative models have led to the successful modelling of diverse kinds of high-dimensional data, from images to molecules, allowing the generation of novel, realistic samples. While deep models trained on protein sequence data have been shown to learn biologically meaningful representations helpful for a variety of downstream tasks, their potential for direct use in protein engineering remains largely unexplored. Here we show that variational autoencoders trained on a dataset of almost 70000 luciferase-like oxidoreductases can be used to generate novel, functional variants of the luxA bacterial luciferase. We propose separate VAE models to work with aligned sequence input (MSA VAE) and raw sequence input (AR-VAE), and offer evidence that while both are able to reproduce patterns of amino acid usage characteristic of the family, the MSA VAE is better able to capture long-distance dependencies reflecting the influence of 3D structure. To validate the practical utility of the models, we used them to generate variants of luxA whose function was tested experimentally. As further evidence of the practicality of these methods for design, we showed that conditional variants of both models could be used to increase the solubility of luxA without disrupting function. Altogether 18/24 of the variants generated using the AR-VAE and 21/23 variants generated using the MSA VAE retained some luminescence activity, despite containing as many as 35 differences relative to any training set sequence. These results demonstrate the feasibility of using deep generative models to explore the space of possible protein sequences and generate useful variants, providing a method complementary to rational design and directed evolution approaches.
Cell Biology | 09 Dec 2020
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During normal development and response to environmental stress, fungi must coordinate synthesis of the cell wall and plasma membrane. Septins, small cytoskeletal GTPases, colocalize with membrane sterol-rich regions and facilitate recruitment of cell wall synthases during dynamic wall remodeling. In this study we show that null mutants missing an Aspergillus nidulans core septin present in hexamers and octamers (ΔaspAcdc11, ΔaspBcdc3, or ΔaspCcdc12) are sensitive to multiple cell wall-disturbing agents known to activate the cell wall integrity MAPK pathway and that this sensitivity can be remediated by osmotic support. The null mutant missing the octamer-exclusive core septin (ΔaspDcdc10) showed similar osmotic-remedial sensitivity, but only to a single cell wall-disturbing agent and the null mutant missing the noncore septin (ΔaspE) showed very mild osmotic-remedial sensitivity to a different single agent. Representative core septin null mutants showed changes in cell wall polysaccharide composition, organization, and chitin synthase localization. Double mutant analysis with ΔmpkA suggested core septins interact with the cell wall integrity pathway. Null mutants missing any of the five septins were resistant to ergosterol-disrupting agents. The ΔaspAcdc11, ΔaspBcdc3, and ΔaspCcdc12 mutants showed increased sensitivity to sphingolipid-disrupting agents that was remediated by addition of exogenous phytosphingosine. Representative core septins were mislocalized after treatment with sphingolipid-disrupting agents, but not after treatment with ergosterol-disrupting agents. When challenged with both sphingolipid-disturbing and cell wall-disturbing agents in combination, remediation of the lipid defect restored proper growth to ΔaspAcdc11, ΔaspBcdc3, and ΔaspCcdc12, but remediation of the cell wall defect did not. Our data suggest that the core hexamer and octamer septins are involved in cell wall integrity signaling with the noncore septin playing a minor role; that all five septins are involved in monitoring ergosterol metabolism; that the hexamer septins are required for sphingolipid metabolism; and that septins require sphingolipids to coordinate the cell wall integrity response.
Cell Biology | 18 Nov 2020
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The multi-scale architecture of mammalian sperm flagella and implications for ciliary motility

Miguel Ricardo Leung, Marc C. Roelofs, Ravi Teja Ravi, Paula Maitan, Min Zhang, Heiko Henning, Elizabeth G. Bromfield, Stuart C. Howes, Bart M. Gadella, Hermes Bloomfield-Gadêlha, Tzviya Zeev-Ben-Mordehai

Motile cilia are molecular machines used by a myriad of eukaryotic cells to swim through fluid environments. However, available molecular structures represent only a handful of cell types, limiting our understanding of how cilia are modified to support motility in diverse media. Here, we use cryo-focused ion beam milling-enabled cryo-electron tomography to image sperm flagella from three mammalian species. We resolve in-cell structures of centrioles, axonemal doublets, central pair apparatus, and endpiece singlets, revealing novel protofilament-bridging microtubule inner proteins throughout the flagellum. We present native structures of the flagellar base, which is crucial for shaping the flagellar beat. We show that outer dense fibers are directly coupled to microtubule doublets in the principal piece but not in the midpiece. Thus, mammalian sperm flagella are ornamented across scales, from protofilament-bracing structures rein-forcing microtubules at the nano-scale to accessory structures that impose micron-scale asymmetries on the entire assembly. Our structures provide vital foundations for linking molecular structure to ciliary motility and evolution.
Cell Biology | 20 Sep 2020
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Loading of the MCM replicative helicase onto origins of replication is a highly regulated process that precedes DNA replication in all eukaryotes. The number of MCM loaded on origins has been proposed to be a key determinant of when those origins initiate replication during S phase. Nevertheless, the genome-wide characteristics of MCM loading and their direct effect on replication timing remain unclear. In order to probe MCM loading dynamics and its effect on replication timing, we perturbed MCM levels in budding yeast cells and, for the first time, directly measured MCM levels and replication timing in the same experiment. Reduction of MCM levels through degradation of Mcm4, one of the six obligate components of the MCM complex, slowed progression through S phase and increased sensitivity to replication stress. Reduction of MCM levels also led to differential loading at origins during G1, revealing origins that are sensitive to reductions in MCM and others that are not. Sensitive origins loaded less MCM under normal conditions and correlated with a weak ability to recruit the origin recognition complex (ORC). Moreover, reduction of MCM loading at specific origins of replication led to a delay in their initiation during S phase. In contrast, overexpression of MCM had no effects on cell cycle progression, relative MCM levels at origins, or replication timing, suggesting that, under optimal growth conditions, cellular MCM levels not limiting for MCM loading. Our results support a model in which the loading activity of origins, controlled by their ability to recruit ORC and compete for MCM, determines the number of helicases loaded, which in turn affects replication timing.
Cell Biology | 05 Oct 2020
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Lumen extension in intracellular tubes can occur by the directed fusion of vesicles with an invading apical membrane domain. Within the C. elegans excretory cell, which contains an intracellular tube, the exocyst vesicle-tethering complex is enriched at the lumenal membrane domain and is required for tube formation, suggesting that it targets vesicles needed for lumen extension. Here, we identify a polarity pathway that promotes intracellular tube formation by enriching the exocyst at the lumenal membrane. We show that the PAR polarity proteins PAR-6 and PKC-3/aPKC localize to the lumenal membrane domain and function within the excretory cell to promote lumen extension, similar to exocyst component SEC-5 and exocyst regulator RAL-1. Using acute protein depletion, we find that PAR-6 is required to recruit the exocyst to the lumenal membrane domain, whereas PAR-3, which functions as an exocyst receptor in mammalian cells, appears to be dispensable for exocyst localization and lumen extension. Finally, we show that the Rho GTPase CDC-42 and the RhoGEF EXC-5/FGD act as upstream regulators of lumen formation by recruiting PAR-6 and PKC-3 to the lumenal membrane. Our findings reveal a molecular pathway that connects Rho GTPase signaling, cell polarity, and vesicle-tethering proteins to promote lumen extension in intracellular tubes.
Cell Biology | 29 Sep 2020
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Trim39 regulates neuronal apoptosis by acting as a SUMO-targeted E3 ubiquitin-ligase for the transcription factor NFATc3

Meenakshi Basu Shrivastava, Barbara Mojsa, Stéphan Mora, Ian Robbins, Guillaume Bossis, Iréna Lassot, Solange Desagher

NFATc3 is the predominant member of the NFAT family of transcription factor in neurons, where it plays a pro-apoptotic role. Mechanisms controlling NFAT protein stability are poorly understood. Here we identify Trim39 as an E3 ubiquitin-ligase of NFATc3. Indeed, Trim39 ubiquitinates NFATc3 in vitro and in cells, whereas silencing of endogenous Trim39 decreases NFATc3 ubiquitination. We also show that Trim17 inhibits Trim39-mediated ubiquitination of NFATc3 by reducing both the E3 ubiquitin-ligase activity of Trim39 and the NFATc3/Trim39 interaction. Moreover, mutation of SUMOylation sites in NFATc3 or SUMO-interacting motif in Trim39 reduces the NFATc3/Trim39 interaction and Trim39-induced ubiquitination of NFATc3. As a consequence, silencing of Trim39 increases the protein level and transcriptional activity of NFATc3, resulting in enhanced neuronal apoptosis. Likewise, a SUMOylation-deficient mutant of NFATc3 exhibits increased stability and pro-apoptotic activity. Taken together, these data indicate that Trim39 modulates neuronal apoptosis by acting as a SUMO-targeted E3 ubiquitin-ligase for NFATc3.
Plant Biology | 24 Sep 2020
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Individual plant cells possess a genetic network, the circadian clock, that times internal processes to the day-night cycle. Mathematical models of the clock network have driven a mechanistic understanding of the clock in plants. However, these models are typically either ‘whole plant’ models that ignore tissue or cell type specific clock behavior, or ‘phase only’ models that do not include clock network components explicitly. It is increasingly clear that in order to reveal the design principles of the plant circadian clock, clock network models must address spatial differences. This is because complex spatial behaviours have been observed in tissues and cells in plants, including period and phase differences between cells and spatial waves of gene expression between organs. Here, we implement an up to date clock network model on a spatial template of the plant. In our model, the sensitivity to light inputs varies across the plant, and cells communicate their clock timing locally via the levels of core clock mRNA levels by cell-to-cell coupling. We found that differences in sensitivities to environmental input in the model can explain the experimentally observed differences in clock periods in different organs, and we show using the model that a plausible coupling mechanism can generate the experimentally observed waves in clock gene expression across the plant. We then examined what features of the plant circadian system allow it to keep time under noisy light-dark (LD) cycles. We found that differences in sensitivity to light can allow regional flexibility in phase even under LD cycles, whilst local cell-to-cell coupling minimized variability in clock rhythms in neighboring cells. Thus, local sensitivity to environmental inputs combined with cell-to-cell coupling allows for flexible yet robust circadian timing under noisy environments.
Systems Biology | 27 Aug 2020
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The circadian oscillator analysed at the single-transcript level

Nicholas E. Phillips, Alice Hugues, Jake Yeung, Eric Durandau, Damien Nicolas, Felix Naef

The circadian clock is an endogenous and self-sustained oscillator that anticipates daily environmental cycles and coordinates physiology accordingly. While rhythmic gene expression of circadian genes is well-described in populations of cells, the single-cell mRNA dynamics of multiple core-clock genes remain largely unknown. Here we use single molecule fluorescence in-situ hybridization (smFISH) at multiple time points to measure pairs of core-clock transcripts, Rev-erbα (Nr1d1), Cry1 and Bmal1, in mouse fibroblasts at single-molecule resolution. The mean mRNA level oscillates over 24 hours for all three genes, but mRNA numbers show considerable spread between cells. While transcript number scales with cell size for all genes, gene-to-gene correlations of mRNA number depends on the gene pair. To account for these features of the data, we develop a probabilistic model for multivariate smFISH mRNA counts that quantifies changes in transcriptional bursting across genes and over circadian time. We identify a mixture model of negative binomials as the preferred model of the mRNA count distributions, which accounts for cell-to-cell heterogeneity, notably in cell size. The paired count data and modelling allows the decomposition of mRNA variability into distinct noise sources, showing that circadian clock time contributes only a small fraction of the total variability in mRNA number between cells. Thus, our results highlight the intrinsic biological challenges in estimating circadian phase from single-cell mRNA counts and suggest that circadian phase in single cells is encoded post-transcriptionally.
Developmental Biology | 23 Sep 2020
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Glial Hedgehog and lipid metabolism regulate neural stem cell proliferation in Drosophila

Qian Dong, Michael Zavortink, Francesca Froldi, Sofya Golenkina, Tammy Lam, Louise Y. Cheng

The final size and function of the adult central nervous system (CNS) is determined by neuronal lineages generated by neural stem cells (NSCs) in the developing brain. In Drosophila, NSCs called neuroblasts (NBs) reside within a specialised microenvironment called the glial niche. Here, we explore non-autonomous glial regulation of NB proliferation. We show that lipid droplets (LDs) which reside within the glial niche are closely associated with the signalling molecule Hedgehog (Hh). Under physiological conditions, cortex glial Hh is autonomously required to sustain niche chamber formation, and non-autonomously restrained to prevent ectopic Hh signalling in the NBs. In the context of cortex glial overgrowth, induced by Fibroblast Growth Factor (FGF) activation, Hh and lipid storage regulators Lsd-2 and Fasn1 were upregulated, resulting in activation of Hh signalling in the NBs; which in turn disrupted NB cell cycle progression and reduced neuronal production. We show that the LD regulator Lsd-2 modulates Hh’s ability to signal to NBs, and de novo lipogenesis gene Fasn1 regulates Hh post-translational modification via palmitoylation. Together, our data suggest that the glial niche non-autonomously regulates NB proliferation and neural lineage size via Hh signaling that is modulated by lipid metabolism genes.
Bioinformatics | 17 Dec 2020
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Predicting cell health phenotypes using image-based morphology profiling

Gregory P. Way, Maria Kost-Alimova, Tsukasa Shibue, William F. Harrington, Stanley Gill, Federica Piccioni, Tim Becker, Hamdah Shafqat-Abbasi, William C. Hahn, Anne E. Carpenter, Francisca Vazquez, Shantanu Singh

Genetic and chemical perturbations impact diverse cellular phenotypes, including multiple indicators of cell health. These readouts reveal toxicity and antitumorigenic effects relevant to drug discovery and personalized medicine. We developed two customized microscopy assays, one using four targeted reagents and the other three targeted reagents, to collectively measure 70 specific cell health phenotypes including proliferation, apoptosis, reactive oxygen species (ROS), DNA damage, and cell cycle stage. We then tested an approach to predict multiple cell health phenotypes using Cell Painting, an inexpensive and scalable image-based morphology assay. In matched CRISPR perturbations of three cancer cell lines, we collected both Cell Painting and cell health data. We found that simple machine learning algorithms can predict many cell health readouts directly from Cell Painting images, at less than half the cost. We hypothesized that these trained models can be applied to accurately predict cell health assay outcomes for any future or existing Cell Painting dataset. For Cell Painting images from a set of 1,500+ compound perturbations across multiple doses, we validated predictions by orthogonal assay readouts, and by confirming mitotic arrest, ROS, and DNA damage phenotypes via PLK, proteasome, and aurora kinase/tubulin inhibition, respectively. We provide an intuitive web app to browse all predictions athttp://broad.io/cell-health-app. Our approach can be used to add cell health annotations to Cell Painting perturbation datasets.
Infectious Diseases (except HIV/AIDS) | 30 Oct 2020
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A sensitive and affordable multiplex RT-qPCR assay for SARS-CoV-2 detection

Martin A.M. Reijns, Louise Thompson, Juan Carlos Acosta, Holly A. Black, Francisco J. Sanchez-Luque, Austin Diamond, David A. Parry, Alison Daniels, Marie O’Shea, Carolina Uggenti, Maria C. Sanchez, Alan O’Callaghan, Michelle L.L. McNab, Martyna Adamowicz, Elias T. Friman, Toby Hurd, Edward J. Jarman, Frederic Li Mow Chee, Jacqueline K. Rainger, Marion Walker, Camilla Drake, Dasa Longman, Christine Mordstein, Sophie J. Warlow, Stewart McKay, Louise Slater, Morad Ansari, Ian P.M. Tomlinson, David Moore, Nadine Wilkinson, Jill Shepherd, Kate Templeton, Ingolfur Johannessen, Christine Tait-Burkard, Jürgen G. Haas, Nick Gilbert, Ian R. Adams, Andrew P. Jackson

With the ongoing COVID-19 pandemic, caused by the novel coronavirus SARS-CoV-2, there is need for sensitive, specific and affordable diagnostic tests to identify infected individuals, not all of whom are symptomatic. The most sensitive test involves the detection of viral RNA using RT-qPCR, with many commercial kits now available for this purpose. However, these are expensive and supply of such kits in sufficient numbers cannot always be guaranteed. We therefore developed a multiplex assay using well-established SARS-CoV-2 targets alongside a human cellular control (RPP30) and a viral spike-in control (PhHV-1), which monitor sample quality and nucleic acid extraction efficiency respectively. Here, we establish that this test performs as well as widely used commercial assays, but at substantially reduced cost. Furthermore, we demonstrate >1,000-fold variability in material routinely collected by nose-and-throat swabbing, and establish a statistically significant correlation between the detected level of human and SARS-CoV-2 nucleic acids. The inclusion of the human control probe in our assay therefore provides a quantitative measure of sample quality that could help reduce false negative rates. We demonstrate feasibility of establishing a robust RT-qPCR assay at ∼10% of the cost of equivalent commercial assays, which could benefit low resource environments and make high volume testing more affordable.