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Results for “Synthetic Biology”

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bioRxiv2017

Histone demethylation and c-MYC activation enhance translational capacity in response to amino acid restriction

Nutrient limitation may elicit adaptive epigenetic changes but the nature and mechanisms of the cellular response to specific nutrient deficiencies are incompletely understood. We report that depriving human cells of amino acids (AAs) induces specific loss of H4K20me1 from gene bodies and elevated binding of c-MYC at promoters genome-wide. These effects are most pronounced at ribosomal protein and translation initiation genes, which are upregulated, leading to enhanced protein synthetic capacity. Combination of H4K20 methyltransferase depletion and c-MYC over-expression in rich media is required and sufficient to recapitulate the effects of AA restriction. Our data reveal an unexpected and epigenetically implemented increase in translational capacity when AAs are limiting, likely to safeguard the proteome by making effective use of limited resources.\n\nOne Sentence Summary: Combination of H4K20me1 demethylation and c-MYC activation enhance translational capacity in response to amino acid restriction.

molecular biology↗

bioRxiv2016

The Effects of Ribosome Autocatalysis and Negative Feedback in Resource Competition

BackgroundResource competition, and primarily competition for ribosomes, can lead to unexpected behavior of genetic circuits and has recently gained renewed attention with both experimental and theoretical studies. Current models studying the effects of resource competition assume a constant production of ribosomes and these models describe the experimental results well. However, ribosomes are also autocatalytic since they are partially made of protein and autocatalysis has been shown to have detrimental effects on a systems stability and robustness. Additionally, there are known feedback regulations on ribosome synthesis such as inhibition of rRNA synthesis via ppGpp.\n\nResultsHere, we develop two-state models of ribosome and protein synthesis incorporating autocatalysis and feedback to investigate conditions under which these regulatory actions have a significant effect in situations of increased ribosome demand. Our modeling results indicate that for sufficiently low demand, defined by the mRNA level of synthetic genes, autocatalysis has little or no effect. However, beyond a certain demand level, the system goes through a transcritical bifurcation at which the only non-negative steady state is at zero ribosome concentration. The presence of negative feedback, in turn, can shift this point to higher demand values, thus restoring the qualitative behavior observed in a model with a constant ribosome production at low demand. However, autocatalysis affects the dynamics of the system and can lead to an overshoot in the temporal response of the synthetic genes to changes in induction level.\n\nConclusionOur results show that ribosome autocatalysis has a significant effect on the system robustness to increases in ribosome demand, however the existing negative feedback on ribosome production compensates for the effects of the necessary autocatalytic loop and restores the behavior seen in the system with constant ribosome production. These findings explain why previous models with constant ribosome production reproduce the steady state behavior well, however incorporating autocatalysis and feedback is needed to capture the transient behavior.

Systems Biology↗

bioRxiv2018

Replication stress induced by CCNE1 overexpression creates a dependency on XRCC2 at the replication fork

Across multiple cancer types, genome instability has been linked to aberrant over-expression of CCNE1 due to premature cell cycle entry and replication stress. Using a gain-of-function screen, we found that XRCC2 cooperates with CCNE1 in the neoplastic transformation of TP53 mutant cells. A pan-cancer analysis of TCGA data revealed a striking correlation between CCNE1 and XRCC2 expression and knockdown of XRCC2 in Cyclin E1 overexpressing cell lines is synthetic lethal. Immunopurification of XRCC2 showed that it interacts with the Minichromosome Maintenance Complex Component 7 (MCM7) protein. This interaction appears to be critical for protecting replication forks as knockdown of XRCC2 leads to a strong increase in MCM7 ubiquitination with concomitant decrease in MCM7 protein levels, and reduced replication fork speed. Importantly, Overexpression of MCM7 rescues the effect of XRCC2 knockdown. Our data describe a new dependency of Cyclin E1 overexpressing tumors on factors that stabilize the replication fork.

molecular biology↗

bioRxiv2015

Evolutionary dynamics of roX lncRNA function and genomic occupancy

Many long noncoding RNAs (lncRNAs) can regulate chromatin states, but the evolutionary origin and dynamics driving lncRNA-genome interactions are unclear. We developed an integrative strategy that identifies lncRNA orthologs in different species despite limited sequence similarity that is applicable to fly and mammalian lncRNAs. Analysis of the roX lncRNAs, which are essential for dosage compensation of the single X-chromosome in Drosophila males, revealed 47 new roX orthologs in diverse Drosophilid species across ~40 million years of evolution. Genetic rescue by roX orthologs and engineered synthetic lncRNAs showed that evolutionary maintenance of focal structural repeats mediates roX function. Genomic occupancy maps of roX RNAs in four species revealed rapid turnover of individual binding sites but conservation within nearby chromosomal neighborhoods. Many new roX binding sites evolved from DNA encoding a pre-existing RNA splicing signal, effectively linking dosage compensation to transcribed genes. Thus, evolutionary analysis illuminates the principles for the birth and death of lncRNAs and their genomic targets.

Evolutionary Biology↗

bioRxiv2017

Endodermal differentiation is reconstructed by coordination of two parallel signaling systems derived from the stele in roots

The plant roots represent the exquisitely controlled cell fate map in which different cell types undergo a complete status transition from stem cell division and initial fate specification, to the terminal differentiation. The endodermis is initially specified in meristem but further differentiates to form Casparian strips (CSs), the apoplastic barrier in the mature zone for the selective transport between stele and outer tissues, and thus is regarded as plant inner skin. In the Arabidopsis thaliana root the transcription factors SHORTROOT (SHR) regulate asymmetric cell division in cortical initials to separate endodermal and cortex cell layer. In this paper, we utilized synthetic approach to examine the reconstruction of fully functional Casparian strips in plant roots. Our results revealed that SHR serves as a master regulator of a hierarchical signaling cascade that, combined with stele-derived small peptides, is sufficient to rebuild the functional CS in non-endodermal cells. This is a demonstration of the deployment of two parallel signaling systems, in which both apoplastic and symplastic communication were employed, for coordinately specifying the endodermal cell fate.

plant biology↗

bioRxiv2015

The effects of time-varying temperature on delays in genetic networks

Delays in gene networks result from the sequential nature of protein assembly. However, it is unclear how models of gene networks that use delays should be modified when considering time-dependent changes in temperature. This is important, as delay is often used in models of genetic oscillators that can be entrained by periodic fluctuations in temperature. Here, we analytically derive the time dependence of delay distributions in response to time-varying temperature changes. We find that the resulting time-varying delay is nonlinearly dependent on parameters of the time-varying temperature such as amplitude and frequency, therefore, applying an Arrhenius scaling may result in erroneous conclusions. We use these results to examine a model of a synthetic gene oscillator with temperature compensation. We show that temperature entrainment follows from the same mechanism that results in temperature compensation. Under a common Arrhenius scaling alone, the frequency of the oscillator is sensitive to changes in the mean temperature but robust to changes in the frequency of a periodically time-varying temperature. When a mechanism for temperature compensation is included in the model, however, we show that the oscillator is entrained by periodically varying temperature even when maintaining insensitivity to the mean temperature.

Systems Biology↗

bioRxiv2016

Exploitation by cheaters facilitates the preservation of essential public goods in microbial communities

How are public goods1-4 maintained in bacterial cooperative populations? The presence of these compounds is usually threatened by the rise of cheaters that do not contribute but just exploit the common resource5,6. Minimizing cheater invasions appears then as a necessary maintenance mechanism7,8. However, that invasions can instead add to the persistence of cooperation is a prospect that has yet remained largely unexplored6. Here, we show that the detrimental consequences of cheaters can actually preserve public goods, at the cost of recurrent collapses and revivals of the population. The result is made possible by the interplay between spatial constraints and the essentiality of the shared resource. We validate this counter-intuitive effect by carefully combining theory and experiment, with the engineering of an explicit synthetic community in which the public compound allows survival to a bactericidal stress. Notably, the characterization of the experimental system identifies additional factors that can matter, like the impact of the lag phase on the tolerance to stress, or the appearance of spontaneous mutants. Our work emphasizes the unanticipated consequences of the eco-evolutionary feedbacks that emerge in microbial communities relying on essential public goods to function, feedbacks that reveal fundamental for the adaptive change of ecosystems at all scales.

Systems Biology↗

bioRxiv2015

SiteOut: an online tool to design binding site-free DNA sequences

DNA-binding proteins control many fundamental biological processes such as transcription, recombination and replication. A major goal is to decipher the role that DNA sequence plays in orchestrating the binding and activity of such regulatory proteins. To address this goal, it is useful to rationally design DNA sequences with desired numbers, affinities and arrangements of protein binding sites. However, removing binding sites from DNA is computationally non-trivial since one risks creating new sites in the process of deleting or moving others. Here we present an online binding site removal tool, SiteOut, that enables users to design arbitrary DNA sequences that entirely lack binding sites for factors of interest. SiteOut can also be used to delete sites from a specific sequence, or to introduce site-free spacers between functional sequences without creating new sites at the junctions. In combination with commercial DNA synthesis services, SiteOut provides a powerful and flexible platform for synthetic projects that interrogate regulatory DNA. Here we describe the algorithm and illustrate the ways in which SiteOut can be used; it is publicly available at https://depace.med.harvard.edu/siteout/.

Genetics↗

bioRxiv2017

A Bulky Glycocalyx Fosters Metastasis Formation by Promoting G1 Cell Cycle Progression

Metastasis depends upon cancer cell growth and survival within the metastatic niche. Tumors which remodel their glycocalyces, by overexpressing bulky glycoproteins like mucins, exhibit a higher predisposition to metastasize, but the role of mucins in oncogenesis remains poorly understood. Here we report that a bulky glycocalyx promotes the expansion of disseminated tumor cells in vivo by fostering integrin adhesion assembly to permit G1 cell cycle progression. We engineered tumor cells to display glycocalyces of various thicknesses by coating them with synthetic mucin-mimetic glycopolymers. Cells adorned with longer glycopolymers showed increased metastatic potential, enhanced cell cycle progression, and greater levels of integrin-FAK mechanosignaling and Akt signaling in a syngeneic mouse model of metastasis. These effects were mirrored by expression of the ectodomain of cancer-associated mucin MUC1. These findings functionally link mucinous proteins with tumor aggression, and offer a new view of the cancer glycocalyx as a major driver of disease progression.

cancer biology↗

bioRxiv2017

The use of salivary α-amylase as an evolutionary solution to host selection in parasitoids

Foraging parasitoids use chemical signals in host recognition and selection processes. Thereby, chemicals from the herbivore hosts play a crucial role. When different herbivores are present in the same plant or field, the perception of specific volatiles and contact compounds emitted from the host itself enable the parasitoids both to differentiate between hosts and non-hosts and to estimate the health status of its host. During the host feeding process, contact between the parasitoid and its host is very crucial, and oral secretions from the host play a key role during the first contact for such evaluation by the parasitoid. Using an integration of behavioral observations, biochemical and sensory physiological approaches we demonstrate that female parasitoids of Cotesia flavipes recognize their host and oviposit in reaction to an -amylase, which is present in the oral secretions of the larvae of their host, Chilo partellus. This activity was also mediated by a purified -amylase synthetized from Drosophila melanogaster. Using this synthetized enzyme, we further demonstrate that the conformation of the enzyme rather than its catalytic site is responsible for this activity. This enzyme is activating gustatory neurons of the terminal antennal sensilla chaetica of C. flavipes females. -amylases are therefore good candidates for an evolutionary solution to host selection in parasitoids, thus opening new avenues for investigations in hosts-parasitoids interactions.

evolutionary biology↗

bioRxiv2016

Active modules for multilayer weighted gene co-expression networks: a continuous optimization approach

MotivationSearching for active connected subgraphs in biological networks has shown important to identifying functional modules. Most existing active modules identification methods need both network structural information and gene activity measures, typically requiring prior knowledge database and high-throughput data. As a pure data-driven gene network, weighted gene co-expression network (WGCN) could be constructed only from expression profile. Searching for modules on WGCN thus has potential values. While traditional clustering based modules detection on WGCN method covers all genes, unavoidable introducing many uninformative ones when annotating modules. We need to find more accurate part of them.\n\nResultsWe propose a fine-grained method to identify active modules on the multi-layer weighted (co-expression gene) network, based on a continuous optimization approach (AMOUNTAIN). The multilayer network are also considered under the unified framework, as a natural extension to single layer network case. The effectiveness is validated on both synthetic data and real-world data. And the software is provided as a user-friendly R package.\n\nAvailabilityAvailable at https://github.com/fairmiracle/AMOUNTAIN\n\nContacts.he@cs.bham.ac.uk\n\nSupplementary informationSupplementary data are available at Bioin-formatics online.

Bioinformatics↗

bioRxiv2026

A reproducibility-audit framework for generalizable versus dataset-specific molecular transition boundaries in Alzheimer's disease

Molecular staging of Alzheimer's disease (AD) increasingly defines transition boundaries along single-cell pseudo-progression trajectories, yet whether such boundaries reproduce across brain regions, cohorts and molecular modalities is rarely tested. We present a permutation-controlled audit that combines nine boundary-detection algorithms with a fixed marker panel and four orthogonal reproducibility axes-algorithmic consensus, region, cohort and modality. On synthetic data with planted ground-truth boundaries the audit reaches 100% sensitivity and 94% specificity, rejecting four distinct artefact classes each by a different axis. Applied to the Seattle Alzheimer's Disease Brain Cell Atlas middle temporal gyrus, it localizes a transition that is robust across algorithms and recovered in most cell types but does not generalize: its leading marker is attenuated or absent in prefrontal cortex, entorhinal cortex and cerebrospinal fluid, and an apparent cross-region conservation of glial metabolic genes proves to be a global-expression offset rather than a shared program. The same audit nonetheless certifies an externally validated marker (astrocytic PTGDS) as reproducible across regions and modalities, showing that it separates generalizable anchors from dataset-specific ones rather than rejecting all signals. We provide this four-axis audit as a transferable, code-available standard to apply before a trajectory boundary is read as a biological stage, in AD and other progressive proteinopathies.

neuroscience↗

bioRxiv2016

Removing bias against short sequences enables northern blotting to better complement RNA-seq for the study of small RNAs

Changes in small non-coding RNAs such as micro RNAs (miRNAs) can serve as indicators of disease and can be measured using next-generation sequencing of RNA (RNA-seq). Here, we highlight the need for approaches that complement RNA-seq, discover that northern blotting of small RNAs is biased against short sequences, and develop a protocol that removes this bias. We found that multiple small RNA-seq datasets from the worm C. elegans had shorter forms of miRNAs that appear to be degradation products that arose during the preparatory steps required for RNA-seq. When using northern blotting during these studies, we discovered that miRNA-length probes can have a ~360-fold bias against detecting even synthetic sequences that are 8 nt shorter. By using shorter probes and by performing hybridization and washes at low temperatures, we greatly reduced this bias to enable equivalent detection of 24 nt to 14 nt RNAs. Our protocol can better discriminate RNAs that differ by a single nucleotide and can detect specific miRNAs present in total RNA from C. elegans. This improved northern blotting is particularly useful to obtain a measure of small RNA integrity, analyze products of RNA processing or turnover, and analyze functional RNAs that are shorter than typical miRNAs.

Molecular Biology↗

bioRxiv2017

Evaluating Strategies For Reversing CRISPR-Cas9 Gene Drives

A gene drive biases inheritance of a gene so that it increases in frequency within a population even when the gene confers no fitness benefit. There has been renewed interest in environmental releases of engineered gene drives due to recent proof of principle experiments with the CRISPR-Cas9 system as a drive mechanism. Release of modified organisms, however, is controversial, especially when the drive mechanism could theoretically alter all individuals of a species. Thus, it is desirable to have countermeasures to reverse a drive if a problem arises. Several genetic mechanisms for limiting or eliminating gene drives have been proposed and/or developed, including synthetic resistance, reversal drives, and immunizing reversal drives. While predictions about efficacy of these mechanisms have been optimistic, we lack detailed analyses of their expected dynamics. We develop a discrete time model for population genetics of a drive and proposed genetic countermeasures. Efficacy of drive reversal varies between countermeasures. For some parameter values, the model predicts unexpected behavior including polymorphic equilibria and oscillatory dynamics. The timing and number of released individuals containing a genetic countermeasure can substantially impact outcomes. The choice among countermeasures by researchers and regulators will depend on specific goals and population parameters of target populations.

evolutionary biology↗

bioRxiv2017

Fibroblast Growth Factor 2 lethally sensitizes cancer cells to stress-targeted therapeutic inhibitors

In malignant transformation, cellular stress response pathways are dynamically mobilized to counterbalance oncogenic activity, keeping cancer cells viable. Therapeutic disruption of this riskily balanced homeostasis might change the outcome of many human cancers, particularly those for which no effective therapy is available. Here, we report the use of Fibroblast Growth Factor 2 (FGF2) to demonstrate that further mitogenic activation disrupts cellular homeostasis and strongly sensitizes cancer cells to stress-targeted therapeutic inhibitors. We show that FGF2 enhanced replication and proteotoxic stresses in a K-Ras-driven murine cancer cell model, and combinations of FGF2 and proteasome or DNA damage response-checkpoint inhibitors triggered cell death. CRISPR/Cas9-mediated K-Ras depletion suppressed the malignant phenotype and prevented these synergic toxicities in these murine cells. Moreover, in a panel of human Ewings sarcoma family tumor cells, sub-lethal concentrations of bortezomib (proteasome-inhibitor) or VE-821 (ATR-inhibitor) induced cell death when combined with FGF2. Sustained MAPK-ERK1/2 overactivation induced by FGF2 underlies these synthetic lethalities, once late pharmacological inhibition of this pathway restored cell homeostasis and prevented these described synergies. Our results highlight how mitotic signaling pathways frequently overridden in malignant transformation might be exploited to disrupt the risky robustness of cancer cells, ultimately sensitizing them to stress-targeted therapies. This approach provides a new therapeutic rationale for human cancers, with important implications for tumors still lacking effective treatment, and for those that frequently relapse after available therapies.

cancer biology↗

bioRxiv2017

Multi-scale Bayesian modeling of cryo-electron microscopy density maps

SummaryCryo-electron microscopy (cryo-EM) has become a mainstream technique for determining the structures of complex biological systems. However, accurate integrative structural modeling has been hampered by the challenges in objectively weighing cryo-EM data against other sources of information due to the presence of random and systematic errors, as well as correlations, in the data. To address these challenges, we introduce a Bayesian scoring function that efficiently and accurately ranks alternative structural models of a macromolecular system based on their consistency with a cryo-EM density map and other experimental and prior information. The accuracy of this approach is benchmarked using complexes of known structure and illustrated in three applications: the structural determination of the GroEL/GroES, RNA polymerase II, and exosome complexes. The approach is implemented in the open-source Integrative Modeling Platform (http://integrativemodeling.org), thus enabling integrative structure determination by combining cryo-EM data with other sources of information.\n\nHighlightsO_LIWe present a modeling approach to integrate cryo-EM data with other sources of information\nC_LIO_LIWe benchmark our approach using synthetic data on 21 complexes of known structure\nC_LIO_LIWe apply our approach to the GroEL/GroES, RNA polymerase II, and exosome complexes\nC_LI

bioinformatics↗

bioRxiv2018

Synthetic-evolution reveals that phosphoregulation of the mitotic kinesin-5 Cin8 is constrained

Cdk1 has been found to phosphorylate the majority of its substrates in disordered regions. These phosphorylation sites do not appear to require precise positioning for their function. The mitotic kinesin-5 Cin8 was shown to be phosphoregulated at three Cdk1 sites in disordered loops within its catalytic motor domain. Here, we examined the flexibility of Cin8 phosphoregulation by analyzing the phenotypes of synthetic Cdk1-sites that were systematically generated by single amino-acid substitutions, starting from a phosphodeficient variant of Cin8. Out of 29 synthetic Cdk1 sites that we created, eight were non-functional; 19 were neutral, similar to the phosphodeficient variant; and two gave rise to phosphorylation-dependent spindle phenotypes. Of these two, one site resulted in novel phosphoregulation, and only one site, immediately adjacent to a native Cdk1 site, produced phosphoregulation similar to wild-type. This study shows that, while the gain of a single phosphorylation site can confer regulation and modulate the dynamics of the spindle, to achieve optimal regulation of a mitotic kinesin-5 motor protein, phosphoregulation has to be site-specific and precise.

cell biology↗

bioRxiv2017

The ubiquity of directional and reciprocating motion in enzymes out of equilibrium

Molecular motors are thought to generate force and directional motion via nonequilibrium switching between energy surfaces. Because all enzymes can undergo such switching, we hypothesized that the ability to generate rotary motion and torque is not unique to highly adapted biological motor proteins, but is instead a common feature of enzymes. We used molecular dynamics simulations to compute energy surfaces for hundreds of torsions in three enzymes, adenosine kinase, protein kinase A, and HIV-1 protease, and used these energy surfaces within a kinetic model that accounts for intersurface switching and intrasurface probability flows. When substrate is out of equilibrium with product, we find computed torsion rotation rates up ~140 cycle s-1, with stall torques up to ~2 kcal mol-1 cycle-1, and power outputs up to ~50 kcal mol-1 s-1. We argue that these enzymes are instances of a general phenomenon of directional probability flows on asymmetric energy surfaces for systems out of equilibrium. Thus, we conjecture that cyclic probability fluxes, corresponding to rotations of torsions and higher-order collective variables, exist in any chiral molecule driven between states in a non-equilibrium manner; we call this the Asymmetry-Directionality conjecture. This is expected to apply as well to synthetic chiral molecules switched in a nonequilibrium manner between energy surfaces by light, redox chemistry, or catalysis.

biophysics↗