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Search indexed bioRxiv preprints in genomics, neuroscience, cell biology and bioinformatics. Read source abstracts and check manuscript versions; preprints are not peer reviewed.

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bioRxiv2016

A systematic comparison reveals substantial differences in chromosomal versus episomal encoding of enhancer activity

Candidate enhancers can be identified on the basis of chromatin modifications, the binding of chromatin modifiers and transcription factors and cofactors, or chromatin accessibility. However, validating such candidates as bona fide enhancers requires functional characterization, typically achieved through reporter assays that test whether a sequence can drive expression of a transcriptional reporter via a minimal promoter. A longstanding concern is that reporter assays are mainly implemented on episomes, which are thought to lack physiological chromatin. However, the magnitude and determinants of differences in cis-regulation for regulatory sequences residing in episomes versus chromosomes remain almost completely unknown. To address this question in a systematic manner, we developed and applied a novel lentivirus-based massively parallel reporter assay (lentiMPRA) to directly compare the functional activities of 2,236 candidate liver enhancers in an episomal versus a chromosomally integrated context. We find that the activities of chromosomally integrated sequences are substantially different from the activities of the identical sequences assayed on episomes, and furthermore are correlated with different subsets of ENCODE annotations. The results of chromosomally-based reporter assays are also more reproducible and more strongly predictable by both ENCODE annotations and sequence-based models. With a linear model that combines chromatin annotations and sequence information, we achieve a Pearsons R2 of 0.347 for predicting the results of chromosomally integrated reporter assays. This level of prediction is better than with either chromatin annotations or sequence information alone and also outperforms predictive models of episomal assays. Our results have broad implications for how cis-regulatory elements are identified, prioritized and functionally validated.

Genomics↗

bioRxiv2016

HydDB: A web tool for hydrogenase classification and analysis

H2 metabolism is proposed to be the most ancient and diverse mechanism of energy-conservation. The metalloenzymes mediating this metabolism, hydrogenases, are encoded by over 60 microbial phyla and are present in all major ecosystems. We developed a classification system and web tool, HydDB, for the structural and functional analysis of these enzymes. We show that hydrogenase function can be predicted by primary sequence alone using an expanded classification scheme (comprising 29 [NiFe], 8 [FeFe], and 1 [Fe] hydrogenase classes) that defines 11 new classes with distinct biological functions. Using this scheme, we built a web tool that rapidly and reliably classifies hydrogenase primary sequences using a combination of k-nearest neighbors algorithms and CDD referencing. Demonstrating its capacity, the tool reliably predicted hydrogenase content and function in 12 newly-sequenced bacteria, archaea, and eukaryotes. HydDB provides the capacity to browse the amino acid sequences of 3248 annotated hydrogenase catalytic subunits and also contains a detailed repository of physiological, biochemical, and structural information about the 38 hydrogenase classes defined here. The database and classifier are freely and publicly available at http://services.birc.au.dk/hyddb/

Bioinformatics↗

bioRxiv2016

Foxc1 Controls Cell Fate Decisions During Transforming Growth Factor β Induced Epithelial to Mesenchymal Transition Through the Regulation of Fibroblast Growth Factor Receptor 1 Expression.

Epithelial to mesenchymal transition (EMT) is an important physiological process that drives tissue formation during development but also contributes to disease pathogenesis including fibrosis and cancer metastasis. The forkhead box transcription factor gene FOXC1 is an important developmental regulator in the generation of mesenchymal cells necessary in the formation of the anterior segment of the eye, the craniofacial skeleton and the meninges. Recently elevated expression of FOXC1 has been detected in several metastatic cancers that have undergone EMT events. We sought to determine the role of FOXC1 in the initiation of EMT events using NMuMG cells treated with TFG{beta}1. We found that although Foxc1 expression was increased following TFG{beta}1 induced EMT, Foxc1 was not required for this induction. Instead we propose that Foxc1 is required for the specification of the mesenchyme cell type, promoting an activated fibroblast phenotype over a myofibroblast phenotype following the initiation of EMT. This cells type specification was achieved through the regulation of Fibroblast growth factor (Fgfr) 1 expression. Using an RNA sequencing approach, we determined that levels of Fgfr1 normally activated upon TFG{beta}1 treatment were reduced in Foxc1-knockdown cells. Through chromatin immunoprecipitation experiments we determined that FOXC1 could bind to an Fgfr1 upstream regulatory region. Furthermore, expression of the myofibroblast marker -smooth muscle actin (SMA) was elevated in Foxc1 knockdown cells. Finally we determined that FGF2 mediated three dimensional migratory ability was greatly impaired in Foxc1-knockdown cells. Together these results define a role for Foxc1 in specifying a mesenchymal cell type following TFG{beta}1 mediated EMT events.

Cell Biology↗

bioRxiv2016

Ancient coexistence of norepinephrine, tyramine, and octopamine signaling in bilaterians

Norepinephrine/noradrenaline is a neurotransmitter implicated in arousal and other aspects of vertebrate behavior and physiology. In invertebrates, adrenergic signaling is considered absent and analogous functions are performed by the biogenic amines octopamine and its precursor tyramine. These chemically similar transmitters signal by related families of GPCR in vertebrates and invertebrates, suggesting that octopamine/tyramine are the invertebrate equivalents of vertebrate norepinephrine. However, the evolutionary relationships and origin of these transmitter systems remain unclear. Using phylogenetic analysis and receptor pharmacology, here we establish that norepinephrine, octopamine, and tyramine receptors coexist in some marine invertebrates. In the protostomes Platynereis dumerilii (an annelid) and Priapulus caudatus (a priapulid) we identified and pharmacologically characterized adrenergic 1 and 2 receptors that coexist with octopamine , octopamine {beta}, tyramine type 1, and tyramine 2 receptors. These receptors represent the first examples of adrenergic receptors in protostomes. In the deuterostome Saccoglossus kowalewskii (a hemichordate), we identified and characterized octopamine , octopamine {beta}, tyramine type 1, and tyramine 2 receptors, representing the first example of these receptors in deuterostomes. S. kowalewskii also has adrenergic 1 and 2 receptors, indicating that all three signaling systems coexist in this animal. In phylogenetic analysis, we also identified adrenergic and tyramine receptor orthologs in xenacoelomorphs. Our results clarify the history of monoamine signaling in bilaterians. Since all six receptor families (two each for octopamine and tyramine and three for norepinephrine) can be found in representatives of the two major clades of Bilateria, the protostomes and the deuterostomes, all six receptors coexisted in the protostome-deuterostome last common ancestor. Adrenergic receptors were lost from most insects and nematodes and tyramine and octopamine receptors were lost from most deuterostomes. This complex scenario of differential losses cautions that octopamine signaling in protostomes is not a good model for adrenergic signaling in deuterostomes, and that the studies of marine animals where all three transmitter systems coexist will be needed for a better understanding of the origin and ancestral functions of these transmitters.

Evolutionary Biology↗

bioRxiv2016

Resource ratios determine nutrient limitation of primary productivity in the ocean

What is the ultimate limiting nutrient in the ocean? The dominant theory, which was first proposed by Redfield and later formalized by Tyrrell[1, 2], states that despite the scarcity of fixed nitrogen (N) in the surface ocean, phosphorus (P) availability ultimately determines primary productivity. Two recent findings directly challenge the assumptions of the Redfield-Tyrrell paradigm: the discovery of systematic variations of phytoplankton cellular N:P:Fe and widespread iron-limitation of phytoplankton. Here we use a simple model of nitrogen, phosphorus, and iron (Fe) cycling to show how the resource demand ratios and biogeography of phytoplankton interact with external resource supply ratios to govern nutrient cycling and primary productivity. We find that all three nutrients can limit global primary productivity, and that the ratio of geochemical supply to biological demand of each nutrient in each ocean region determines the limiting nutrients, with nitrogen N fixation providing a mechanism for the cycles to interact. These results have important consequences for our understanding of biogeochemical cycles, ocean-atmosphere interactions, marine ecology, and the response of ocean ecosystems to climate change. Our work demonstrates the importance of resource ratios and suggests that future studies of the physiological and geochemical regulation of these ratios are indispensable to building accurate theories and future predictions of nutrient cycling and primary productivity.

Ecology↗

bioRxiv2016

Beyond APs and VPs: evidences of chaos and self-organized criticality in electrical signals in plants.

Studies on plant electrophysiology are mostly focused on specific traits of action potentials (APs) and/or variation potentials (VPs), often in single cells. Inspired by the complexity of the signaling network in plants and by analogies with some traits of neurons in human brains, we have sought for evidences of high complexity in the electrical dynamics of plant signaling, beyond APs and VPs responses. Thus, from EEG-like data analyses of soybean plants, we showed consistent evidences of chaotic dynamics in the electrical time series. Furthermore, we have found that the dynamic complexity of electrical signals is affected by the plant physiological conditions, decreasing when plant was stressed. Surprisingly, but not unlikely, we have observed that, after stimuli, electrical spikes arise following a power law distribution, which is indicative of self-organized criticality (SOC). Since, as far as we know, these were the first evidences of chaos and SOC in plant electrophysiology, we have asked follow-up questions and we have proposed new hypotheses, seeking for improving our understanding about these findings.

Plant Biology↗

bioRxiv2016

Gene regulation mechanism in drought-responsive grapevine leaves as revealed by transcriptomic analysis

Grapevine is economically important and widely cultivated fruit crop, which is seriously hampered by drought worldwide. It is necessary to understand the impact of glitches incurred by the drought on grapevine genetic resources. Therefore, in the present study RNA-sequencing analysis was performed using cDNA libraries constructed from both drought-stress and control plants. Results yielded, a total of 12,451 differentially expressed genes (DEGs) out of which 8,022 genes were up-regulated and 4,430 were down-regulated. Further physiological and biochemical analyses were carried out to validate the various biological processes involved in the development of grapevine in response to drought stress. Results also showed that decrease in rate of stomatal conductance in-turn decrease the photosynthetic activity and CO2 assimilation rate in the grapevine leaves and most ROS detoxification systems, including stress enzymes, stress related proteins and secondary metabolites were strongly induced. Moreover, various hormones were known to be induced in the present study in response to drought. Overall the present study concludes that these DEGs play both positive and negative role in drought tolerance by regulating different biological pathways of grapevine. However our findings have provided valuable gene information for future studies of abiotic stress in grapevine and other fruit crops.

Molecular Biology↗

bioRxiv2016

Probabilistic adaptation in changing microbial environments

Microbes growing in animal host environments face fluctuations that have elements of both randomness and predictability. In the mammalian gut, fluctuations in nutrient levels and other physiological parameters are structured by the animal hosts behavior, diet, health and microbiota composition. Microbial cells that are able to anticipate these fluctuations by exploiting this structure would likely gain a fitness advantage, by adapting their internal state in advance. We propose that the problem of adaptive growth in these structured changing environments can be viewed as probabilistic inference. We analyze environments that are \"meta-changing\": where there are changes in the way the environment fluctuates, governed by a mechanism unobservable to cells. We develop a dynamic Bayesian model of these environments and show that a real-time inference algorithm (particle filtering) for this model can be used as a microbial growth strategy implementable in molecular circuits. The growth strategy suggested by our model outperforms heuristic strategies, and points to a class of algorithms that could support real-time probabilistic inference in natural or synthetic cellular circuits.

Systems Biology↗

bioRxiv2016

Mechanisms of self-assembly and fibrillization of the prion-like domains

The mechanism of the self-assembly and fibrillization of the prion-like domains lies at the heart of their physiology and pathology. Here with the same methods previously established, we aimed to further decipher the mechanism by characterizing two prion-like sequences with the electrostatic properties very different from that of the full-length TDP-43 prion-like domain with a very basic pI value: namely the C-half of the TDP-43 prion-like domain only abundant in Gly, Ser, Asn and Gln with a pI of ~6.3, and the FUS prion-like domain enriched with Gly, Ser, Gln and Tyr with a pI of ~3.5. Interestingly, the C-half with the TDP-43 unique hydrophobic region removed is no longer able to form insoluble aggregates/fibrils but still capable of self-assembling into the reversible hydrogel with cross-{beta} structures, despite being much slower than the full-length. On the other hand, the FUS prion-like domain rapidly self-assembles into the reversible hydrogel with cross-{beta} fibrillar structures in 1 mM phosphate buffer at pH 6.8 but its self-assembly becomes very slow in 50 mM MES buffer at pH 5.5. Our study reveals that despite having completely different electrostatic properties, the full-length and C-half of the TDP-43 prion-like domain, as well as FUS prion-like domain all have the similar pH-dependence in self-assembly as we previously reported (Lim et al., [2016] PLOS Biol 14:e1002338). This unambiguously indicates that the self-assembly of the prion-like domains is not generally governed by the electrostatic interaction. Rather, their self-assembly and fibrillization are specified by the sequences despite being highly polar and degenerative. Furthermore, our study provides the first evidence that the formation of reversible hydrogel with cross-{beta} structures is separable from fibrillization of the prion-like domain. Finally, our results also successfully reconcile the previous discrepancy about the conformation and mechanism of the self-assembly of the FUS prion-like domain.

Biophysics↗

bioRxiv2016

Biophysical properties of human β-cardiac myosin with converter mutations that cause hypertrophic cardiomyopathy

Hypertrophic cardiomyopathy (HCM) affects 1 in 500 individuals and is an important cause of arrhythmias and heart failure. Clinically, HCM is characterized as causing hyper-contractility, and therapies are aimed toward controlling the hyperactive physiology. {beta}-cardiac myosin comprises ~40 percent of genetic mutations associated with HCM and the converter domain of myosin is a hot spot for HCM-causing mutations, but the underlying primary effects of these mutations on myosin's biomechanical function remain elusive. We hypothesize that these mutations affect the biomechanical properties of myosin, such as increasing its intrinsic force and/or its duty ratio and therefore the ensemble force of the sarcomere. Using recombinant human {beta}-cardiac myosin, we characterize the molecular effects of three severe HCM-causing converter domain mutations R719W, R723G and G741R. Contrary to our hypothesis, the intrinsic forces of R719W and R723G mutant myosins are decreased compared to wild type, and unchanged for G741R. Actin and regulated thin filament gliding velocities are ~15 percent faster for R719W and R723G myosin, while there is no change in velocity for G741R. ATPase activities and the load-dependent velocity change profiles of all three mutant proteins are very similar to wild type. These results indicate that the net biomechanical properties of human {beta}-cardiac myosin carrying these converter domain mutations are very similar to wild type or even slightly hypo-contractile, leading us to consider an alternative mechanism for the clinically observed hyper-contractility. Future work includes how these mutations affect protein interactions within the sarcomere that increase the availability of myosin heads participating in force production.\n\nOne Sentence Summary: Biophysical analysis of human {beta} -cardiac myosin with converter domain HCM mutations reveal minimal changes compared to wild type myosin to slightly hypo-contractile profiles in ensemble force production, in contrast to the clinically observed hyper-contractility, leading to a speculative alternative mechanism involving availability of myosin heads participating in the sarcomere.

Biophysics↗

bioRxiv2016

Mind over matter: A perceptual decision bias toward filled-in stimuli in the blind spot

Humans often evaluate sensory signals according to their reliability for optimal decision-making. However, how do we evaluate percepts generated in the absence of direct input that are, therefore, completely unreliable? Here, we utilize the phenomenon of filling-in occurring at the physiological blind-spots to compare partially inferred and veridical percepts. Subjects chose between stimuli that elicit filling-in, and perceptually equivalent ones presented outside the blind-spots, looking for a Gabor stimulus without a small orthogonal inset. In ambiguous conditions, when the stimuli were physically identical and the inset was absent in both, subjects behaved opposite to optimal, preferring the blind-spot stimulus as the better example of a collinear stimulus, even though no relevant veridical information was available. Thus, a percept that is partially inferred is paradoxically considered more reliable than a percept based on external input. In other words: Humans treat filled-in inferred percepts as more real than veridical ones.

Neuroscience↗

bioRxiv2016

Transient protein-protein interactions perturb E.coli metabolome and cause gene dosage toxicity

Several genes exhibit gene dosage toxicity yet its molecular underpinnings remain unknown. Here we demonstrate that overexpression of DHFR in E. coli causes toxic metabolic imbalance triggered by interactions with several enzymes involved in 1-carbon metabolism, in particular GlyA and PurH. DHFR overexpression partially inhibits activity of these enzymes, but at physiological concentrations, PurH-DHFR interaction enhances catalytic efficiency of DHFR, implying a functional interaction in vivo. Surprisingly, overexpression of orthologous DHFRs from other bacterial species caused minimal metabolic and fitness perturbations, despite pulling out more interacting partners than overexpressed endogenous DHFR. Orthologous DHFRs were less potent in inhibiting E. coli GlyA and PurH, or gaining a catalytic improvement upon interaction with PurH, indicating a partial loss of interaction specificity due to evolutionary divergence. This study shows how protein overexpression perturbs a dynamic network of weak yet potentially functional PPI with consequences for the metabolic state of cells and their fitness.

Systems Biology↗

bioRxiv2016

Toxoplasma gondii establishes an extensive filamentous network consisting of stable F-actin during replication

Apicomplexan actin is well conserved and clearly important during the parasite's life cycle. Several studies assert that its polymerization kinetics are unusual, permitting only short, unstable F-actin filaments. However, it has not been possible to study actin in vivo, so its physiological role has remained obscure. This has led to functional models which are mutually conflicting, incompatible with actin behavior from other eukaryotes, and cannot explain actin's importance during basic processes such as parasite replication and egress. Here we use a chromobody that specifically binds F-actin to demonstrate that Toxoplasma forms stable actin filaments in vivo. F-actin is not only important for parasite replication, but also forms an extensive network that connects individuals both within and between parasitophorous vacuoles, and allows vesicles to be exchanged between parasites within a vacuole. During host cell egress, prior to motility, this network collapses in a calcium-dependent manner. This study demonstrates unexpected roles of Toxoplasma actin during the asexual life cycle, and proves that formation of F-actin depends on a critical concentration of G-actin, implying a polymerization mechanism similar to mammalian actin.\n\nOne Sentence SummaryToxoplasma establishes a stable F-actin network that is essential for replication and material transport between individual parasites.

Cell Biology↗

bioRxiv2016

The role of vertical symbiont transmission in altering cooperation and fitness of coral-Symbiodinium symbioses

The coral symbiosis is the linchpin of the reef ecosystem, yet the mechanisms that promote and maintain cooperation between hosts and symbionts have not been fully resolved. We used a phylogenetically controlled design to investigate the role of vertical symbiont transmission, an evolutionary mechanism in which symbionts are inherited directly from parents, predicted to enhance cooperation and holobiont fitness. Six species of coral, three vertical transmitters and their closest horizontally transmitting relatives, which exhibit environmental acquisition of symbionts, were fragmented and subjected to a two-week thermal stress experiment. Symbiont cell density, photosynthetic function and translocation of photosynthetically fixed carbon between symbionts and hosts were quantified to assess changes in physiological performance and cooperation. All species exhibited similar decreases in symbiont cell density and net photosynthesis in response to elevated temperature, consistent with the onset of bleaching. Yet baseline cooperation, i.e. translocation of photosynthate, in ambient conditions and the reduction in cooperation in response to elevated temperature differed among species. Although Porites lobata and Galaxea acrhelia did exhibit the highest levels of baseline cooperation, we did not observe universally higher levels of cooperation in vertically transmitting species. Post hoc sequencing of the Symbiodinium ITS-2 locus was used to investigate the potential role of differences in symbiont community composition. Interestingly, reductions in cooperation at the onset of bleaching tended to be associated with increased symbiont community diversity among coral species. The theoretical benefits of evolving vertical transmission are based on the underlying assumption that the host-symbiont relationship becomes genetically uniform, thereby reducing competition among symbionts. Taken together, our results suggest that it may not be vertical transmission per se that influences host-symbiont cooperation, but genetic uniformity of the symbiont community, although additional work is needed to test this hypothesis.

Evolutionary Biology↗

bioRxiv2016

Comprehensive population-based genome sequencing provides insight into hematopoietic regulatory mechanisms

Genetic variants affecting hematopoiesis can influence commonly measured blood cell traits. To identify factors that affect hematopoiesis, we performed association studies for blood cell traits in the population-based Estonian Biobank using high coverage whole genome sequencing (WGS) in 2,284 samples and SNP genotyping in an additional ~17,000 samples. Our analyses identified 17 associations across 14 blood cell traits. Integration of WGS-based fine-mapping and complementary epigenomic data sets provided evidence for causal mechanisms at several loci, including at a novel basophil count-associated locus near the master hematopoietic transcription factor CEBPA. The fine-mapped variant at this basophil count association near CEBPA overlapped an enhancer active in common myeloid progenitors and influenced its activity. In situ perturbation of this enhancer by CRISPR/Cas9 mutagenesis in hematopoietic stem and progenitor cells demonstrated that it is necessary for and specifically regulates CEBPA expression during basophil differentiation. We additionally identified basophil count-associated variation at another more pleiotropic myeloid enhancer near GATA2, highlighting regulatory mechanisms for ordered expression of master hematopoietic regulators during lineage specification. Our study illustrates how population-based genetic studies can provide key insights into poorly understood cell differentiation processes of considerable physiologic relevance.

Genetics↗

bioRxiv2016

Excitability in the p53 network mediates robust signaling with tunable activation thresholds in single cells

Cellular signaling systems precisely transmit information in the presence of molecular noise while retaining flexibility to accommodate the needs of individual cells. To understand design principles underlying such versatile signaling, we analyzed the response of the tumor suppressor p53 to varying levels of DNA damage in hundreds of individual cells and observed a switch between distinct signaling modes characterized by isolated pulses and sustained oscillations of p53 accumulation. Guided by dynamic systems theory we show that this requires an excitable network structure comprising positive feedback and provide experimental evidence for its molecular identity. The resulting data-driven model reproduced all features of measured signaling responses and is sufficient to explain their heterogeneity in individual cells. We present evidence that heterogeneity in the levels of the feedback regulator Wip1 sets cell-specific thresholds for p53 activation, providing means to modulate its response through interacting signaling pathways. Our results demonstrate how excitable signaling networks can provide high specificity, sensitivity and robustness while retaining unique possibilities to adjust their function to the physiology of individual cells.

Cell Biology↗

bioRxiv2016

Inferring Fitness Landscapes and Selection on Phenotypic States from Single-Cell Genealogical Data

Recent advances in single-cell time-lapse microscopy have revealed non-genetic heterogeneity and temporal fluctuations of cellular phenotypes. While different phenotypic traits such as abundance of growth-related proteins in single cells may have differential effects on the reproductive success of cells, rigorous experimental quantification of this process has remained elusive due to the complexity of single cell physiology within the context of a proliferating population. We introduce and apply a practical empirical method to quantify the fitness landscapes of arbitrary phenotypic traits, using genealogical data in the form of population lineage trees which can include phenotypic data of various kinds. Our inference methodology for fitness landscapes determines how reproductivity is correlated to cellular phenotypes, and provides a natural generalization of bulk growth rate measures for single-cell histories. Using this technique, we quantify the strength of selection acting on different cellular phenotypic traits within populations, which allows us to determine whether a change in population growth is caused by individual cells response, selection within a population, or by a mixture of these two processes. By applying these methods to single-cell time-lapse data of growing bacterial populations that express a resistance-conferring protein under antibiotic stress, we show how the distributions, fitness landscapes, and selection strength of single-cell phenotypes are affected by the drug. Our work provides a unified and practical framework for quantitative measurements of fitness landscapes and selection strength for any statistical quantities definable on lineages, and thus elucidates the adaptive significance of phenotypic states in time series data. The method is applicable in diverse fields, from single cell biology to stem cell differentiation and viral evolution.

Systems Biology↗

bioRxiv2016

A Single Nucleotide Resolution Model for Large-Scale Simulations of Double Stranded DNA

The computational modelling of DNA is becoming crucial in light of new advances in DNA nanotechnology, single-molecule experiments and in vivo DNA tampering. Here we present a mesoscopic model for double stranded DNA (dsDNA) at the single nucleotide level which retains the characteristic helical structure, while being able to simulate large molecules -- up to a million base pairs -- for time-scales which are relevant to physiological processes. This is made possible by an efficient and highly-parallelised implementation of the model which we discuss here. We compare the behaviour of our model with single molecule experiments where dsDNA is manipulated by external forces or torques. We also present some results on the kinetics of denaturation of linear DNA.

Biophysics↗