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Results for “Synthetic Biology”

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bioRxiv2017

Variable dose analysis: A novel RNAi-based method for detection of synthetic lethal interactions

Synthetic sick or synthetic lethal (SS/L) screens are a powerful way to identify candidate drug targets to specifically kill tumor cells but such screens generally suffer from low reproducibility. We found that many SS/L interactions involve essential genes and are therefore detectable within a limited range of knockdown efficiency. Such interactions are often missed by overly efficient RNAi reagents. We therefore developed an assay that measures viability over a range of knockdown efficiency within a cell population. This method, called variable dose analysis (VDA), is highly sensitive to viability phenotypes and reproducibly detects SS/L interactions. We applied the VDA method to search for SS/L interactions with TSC1 and TSC2, the two tumor suppressors underlying tuberous sclerosis complex (TSC) and generated a SS/L network for TSC. Using this network, we identified four FDA-approved drugs that selectively affect viability of TSC deficient cells, representing promising candidates for repurposing to treat TSC-related tumors.

cancer biology↗

bioRxiv2018

PARPi triggers STING-dependent immune response and enhances therapeutic efficacy of immune checkpoint blockade independent of BRCAness

Poly-(ADP-ribose) polymerase (PARP) inhibitors (PARPis) have shown remarkable therapeutic efficacy against BRCA1/2 mutant cancers through a synthetic lethal interaction. PARPis are believed to exert their therapeutic effects mainly through the blockade of single-strand DNA damage repair, which leads to the accumulation of toxic DNA double strand breaks, specifically in cancer cells with DNA repair deficiency (BCRAness), including those harboring BRCA1/2 mutations. Here, we show that PARPis modulate immune reposes, which contribute to their therapeutic effects independent of BRCA1/2 mutations. The mechanism underlying this PARPi-induced reprogramming of anti-tumor microenvironment involves a promoted accumulation of cytosolic DNA fragments due to unresolved DNA lesions. This in turn activates the DNA sensing cGAS-STING pathway and stimulates production of type I interferons. Ultimately, these events promote PARPi-induced antitumor immunity independent of BRCAness, which can be further enhanced by immune checkpoint blockade. Our results may provide a mechanistic rationale for using PARPis as immunomodulatory agents to harness therapeutic efficacy of immune checkpoint blockade.

cancer biology↗

bioRxiv2017

Clay-induced DNA double-strand breaks underlay genetic diversity, antibiotic resistance and molecular basis of asbestosis

Some natural clays and synthetic nanofibres present in the environment have a severe impact on human health. After several decades of research, the molecular mechanism of how asbestos induce cancers is not well understood. Different fibres, including asbestos, can penetrate the membrane and introduce DNA in both, bacterial and eukaryotic cells. Incubating Escherichia coli with sepiolite, a clayey material, and asbestos under friction forces, both fibres cause double-strand breaks in bacteria. Since antibiotics and clays are used together in animal husbandry, the mutagenic effect of these fibres might constitute a pathway to antibiotic resistance due to the friction provided by peristalsis of the gut from farm animals in addition to the previously proposed horizontal gene transfer. Moreover, we raise the possibility that the same mechanism could generate bacteria diversity in natural scenarios with a role in the evolution of species. Finally, we provide a new model on how asbestos may promote mutagenesis and cancer based on the observed mechanical genotoxicity.

evolutionary biology↗

bioRxiv2017

fastBMA: Scalable Network Inference and Transitive Reduction

BACKGROUNDInferring genetic networks from genome-wide expression data is extremely demanding computationally. We have developed fastBMA, a distributed, parallel and scalable implementation of Bayesian model averaging (BMA) for this purpose. fastBMA also includes a novel and computationally efficient method for eliminating redundant indirect edges in the network.\n\nFINDINGSWe evaluated the performance of fastBMA on synthetic data and experimental genome-wide yeast and human datasets. When using a single CPU core, fastBMA is up to 100 times faster than the next fastest method, LASSO, with increased accuracy. It is a memory efficient, parallel and distributed application that scales to human genome wide expression data. A 10,000-gene regulation network can be obtained in a matter of hours using a 32-core cloud cluster.\n\nCONCLUSIONSfastBMA is a significant improvement over its predecessor ScanBMA. It is orders of magnitude faster and more accurate than other fast network inference methods such as LASSO. The improved scalability allows it to calculate networks from genome scale data in a reasonable timeframe. The transitive reduction method can improve accuracy in denser networks. fastBMA is available as code (M.I.T. license) from GitHub (https://github.com/lhhunghimself/fastBMA), as part of the updated networkBMA Bioconductor package (https://www.bioconductor.org/packages/release/bioc/html/networkBMA.html) and as ready-to-deploy Docker images (https://hub.docker.com/r/biodepot/fastbma/).

systems biology↗

bioRxiv2018

An essential role for dNTP homeostasis following CDK-induced replication stress

Replication stress is a common feature of cancer cells, and thus a potentially important therapeutic target. Here we show that CDK-induced replication stress is synthetic lethal with mutations disrupting dNTP homeostasis in fission yeast. Wee1 inactivation leads to increased dNTP demand and replication stress through CDK-induced firing of dormant replication origins. Subsequent dNTP depletion leads to inefficient DNA replication, Mus81-dependent DNA damage, and to genome instability. Cells respond to this replication stress by increasing dNTP supply through Set2-dependent MBF-induced expression of Cdc22, the catalytic subunit of ribonucleotide reductase (RNR). Disrupting dNTP synthesis following Wee1 inactivation, through abrogating Set2-dependent H3K36 tri-methylation or DNA integrity checkpoint inactivation results in critically low dNTP levels, replication collapse and cell death, which can be rescued by increasing dNTP levels. These findings support a dNTP supply and demand model in which maintaining dNTP homeostasis is essential to prevent replication catastrophe in response to CDK-induced replication stress.

molecular biology↗

bioRxiv2016

Extracting a low-dimensional description of multiple gene expression datasets reveals a potential driver for tumor-associated stroma in ovarian cancer

BackgroundDiscovering patient subtypes and molecular drivers of a subtype are difficult and driving problems underlying most modern disease expression studies collected across patient populations. Expression patterns conserved across multiple expression datasets from independent disease studies are likely to represent important molecular events underlying the disease.\n\nMethodsWe present the INSPIRE (INferring Shared modules from multiPle gene expREssion datasets) method to infer highly coherent and robust modules of co-expressed genes and the dependencies among the modules from multiple expression datasets. Focusing on inferring modules and their dependencies conserved across multiple expression datasets is important for several reasons. First, using multiple datasets will increase the power to detect robust and relevant patterns (modules and dependencies among modules). Second, INSPIRE enables the use of multiple datasets that contain different sets of genes due to, e.g., the difference in microarray platforms. Many methods designed for expression data analysis cannot integrate multiple datasets with variable discrepancy to infer a single combined model, whereas INSPIRE can naturally model the dependencies among the modules even when a large proportion of genes are not observed on a certain platform.\n\nResultsWe evaluated INSPIRE on synthetically generated datasets with known underlying network structure among modules, and gene expression datasets from multiple ovarian cancer studies. We show that the model learned by INSPIRE can explain unseen data better and can reveal prior knowledge on gene functions more accurately than alternative methods. We demonstrate that applying INSPIRE to nine ovarian cancer datasets leads to the identification of a new marker and potential molecular driver of tumor-associated stroma - HOPX. We also demonstrate that the HOPXmodule strongly overlaps with the genes defining the mesenchymal patient subtype identified in The Cancer Genome Atlas (TCGA) ovarian cancer data. We provide evidence for a previously unknown molecular basis of tumor resectability efficacy involving tumor-associated mesenchymal stem cells represented by HOPX.\n\nConclusionsINSPIRE extracts a low-dimensional description from multiple gene expression data, which consists of modules and their dependencies. The discovery of a new tumor-associated stroma marker, HOPX, and its module suggests a previously unknown mechanism underlying tumor-associated stroma.

Systems Biology↗

bioRxiv2018

LADL: Light-activated dynamic looping for endogenous gene expression control

Mammalian genomes are folded into tens of thousands of long-range looping interactions1,2. The cause and effect relationship between looping and genome function is poorly understood, and the extent to which chromatin loops are dynamic on short time scales remains a fundamental unanswered question. Currently available strategies for loop engineering involve synthetic transcription factors tethered to dCas93,4 or zinc fingers5,6, which are constitutively expressed5,6 or induced on long time scales by the presence of a small molecule3. Here we report a new class of 3-D optoepigenetic tools for the directed rearrangement of 3-D chromatin looping on short time scales using blue light. We create synthetic architectural proteins by fusing the CIBN protein subunit from Arabidopsis thaliana with enzymatically dead Cas9 (dCas9). We target our light-activated dynamic looping system (LADL) to two genomic anchors with CRISPR guide RNAs and engineer their spatial co-localization via light-induced heterodimerization of the cryptochrome 2 (CRY2) protein with dCas9-CIBN. We apply LADL to redirect a stretch enhancer (SE) away from its endogenous Klf4 target gene and to the Zfp462 promoter. Looping changes occur as early as four hours after light induction. Using single molecule RNA FISH, we observe a LADL-induced increase in the total nascent Zfp462 transcripts and the number of Zfp462 alleles expressing simultaneously per cell. Moreover, LADL also increased synchronous Sox2 expression after reinforcement of a known Sox2-SE looping interaction. LADL facilitates loop synchronization across a large population of cells without exogenous chemical cofactors and can enable future efforts to engineer reversible and oscillatory looping on short time scales.

molecular biology↗

bioRxiv2018

Genome-wide prediction of synthetic rescue mediators of resistance to targeted and immunotherapy

Most patients with advanced cancer eventually acquire resistance to targeted therapies, spurring extensive efforts to identify molecular events mediating therapy resistance. Many of these events involve synthetic rescue (SR) interactions, where the reduction in cancer cell viability caused by targeted gene inactivation is rescued by an adaptive alteration of another gene (the rescuer). Here we perform a genome-wide prediction of SR rescuer genes by analyzing tumor transcriptomics and survival data of 10,000 TCGA cancer patients. Predicted SR interactions are validated in new experimental screens. We show that SR interactions can successfully predict cancer patients response and emerging resistance. Inhibiting predicted rescuer genes sensitizes resistant cancer cells to therapies synergistically, providing initial leads for developing combinatorial approaches to overcome resistance proactively. Finally, we show that the SR analysis of melanoma patients successfully identifies known mediators of resistance to immunotherapy and predicts novel rescuers.

cancer biology↗

bioRxiv2018

Environmental factors shape methionine metabolism in p16/MTAP deleted cells

The co-deletion of a common tumor suppressor locus and neighboring metabolic gene is an attractive possible synthetic dependency of tumor suppression on metabolism. However, the general impact that these co-deletions have on metabolism, which also dependent on nutrient availability and the tissue of origin, is unknown. As a model to investigate this question, we considered a set of tissue-matched cancer cells with homozygous co-deletions in CDKN2a and MTAP, genes respectively encoding the most commonly deleted tumor suppressor p16 and an enzyme involved in methionine metabolism. A comparative metabolomics analysis revealed that while there exists a definite pan-cancer metabolic signature of MTAP-deletion, this signature was not preserved when cells were subjected to changes in the availability of methionine, serine, or cysteine, nutrients related to methionine metabolism. Notably, the heterogeneity exhibited by these cells in their responsiveness to nutrient availability dominated both MTAP status and tissue-of-origin. Furthermore, re-expression of MTAP exerted a modest effect on metabolism. Together these findings demonstrate that environmental factors, particularly nutrition and tissue identity, may overwhelm the genetic effects of collateral deletions of metabolic genes.

molecular biology↗

bioRxiv2018

3’ Branch Ligation: A Novel Method to Ligate Non-Complementary DNA to Recessed or Internal 3’OH Ends in DNA or RNA

Nucleic acid ligases are crucial enzymes that repair breaks in DNA or RNA during synthesis, repair and recombination. Various molecular tools have been developed using the diverse activities of DNA/RNA ligases. Herein, we demonstrate a non-conventional ability of T4 DNA ligase to join 5 phosphorylated blunt-end double-stranded DNA to DNA breaks at 3 recessive ends, gaps, or nicks to form a 3 branch structure. Therefore, this base pairing-independent ligation is termed 3 branch ligation (3BL). In an extensive study of optimal ligation conditions, similar to blunt-end ligation, the presence of 10% PEG-8000 in the ligation buffer significantly increased ligation efficiency. A low level of nucleotide preference was observed at the junction sites using different synthetic DNAs. Furthermore, we discovered that T4 DNA ligase efficiently ligated DNA to the 3 recessed end of RNA, not to that of DNA, in a DNA/RNA hybrid, whereas RNA ligases are less efficient in this reaction. These novel properties of T4 DNA ligase can be utilized as a broad molecular technique in many important applications. We performed a proof-of-concept study of a new directional tagmentation protocol for next generation sequencing (NGS) library construction that eliminates inverted adapters and allows sample barcode insertion adjacent to genomic DNA. 3BL after single transposon tagmentation can theoretically achieve 100% usable template, and our empirical data demonstrate that the new approach produced higher yield compared with traditional double transposon or Y transposon tagmentation. We further explore the potential use of 3BL for preparing targeted RNA NGS libraries with mitigated structure-based bias and adapter dimer problems.

molecular biology↗

bioRxiv2018

The Trithorax group protein dMLL3/4 instructs the assembly of the zygotic genome at fertilization

The transition from fertilized oocyte to totipotent embryo relies on maternally-provided factors that are synthetized and accumulated in developing oocytes. Yet, it is still unclear how oocytes regulate the expression of these embryo fate-promoting genes within the general transcriptional program of oogenesis. Here we report that the Drosophila Trithorax group protein MLL3/4 (dMLL3/4, also known as Trr) is essential for the transition to embryo fate at fertilization. In the absence of dMLL3/4, oocytes develop normally but fail to initiate the embryo mitotic divisions after fertilization. This incapability results from defects in both paternal genome reprogramming and maternal meiotic completion. We show that, during oogenesis, dMLL3/4 promotes the expression of a functionally coherent gene subset that is later required for the correct assembly of the zygotic genome. Accordingly, we identify the evolutionarily-conserved IDGF4 glycoprotein (known as oviductin in mammals) as a new oocyte-to-embryo transition gene under dMLL3/4 transcriptional control. Based on these observations, we propose that dMLL3/4 plays an instructive role in the oocyte-to-embryo transition that is functionally uncoupled from the requirements of normal oocyte differentiation.

developmental biology↗

bioRxiv2026

INFORME: coupling information-theoretic experimental design with nonlinear mixed-effects modeling for efficient observation scheduling

Mathematical models of treatment response can inform individualized therapy, but their calibration often requires longitudinal measurements that are costly, burdensome, and collected on fixed schedules. Such schedules may be inefficient, over-sampling patients whose response is already well characterized while delaying informative measurements for those whose model parameters remain uncertain. We present INFORME (INFORmation-theoretic design with Mixed Effects), a framework that combines Bayesian information-theoretic experimental design with nonlinear mixed-effects modeling to adaptively select each patients next measurement time. Population and response-subgroup parameter distributions learned from an existing cohort provide informative priors, allowing candidate measurement times to be ranked by their expected reduction in patient-specific parameter uncertainty. As observations accumulate, priors can be updated to reflect the response subgroup most consistent with the patients data. We evaluate INFORME in two radiotherapy datasets: 150 synthetic tumor volume trajectories from a hybrid cellular automaton model of prostate cancer spheroids (HD1) and longitudinal tumor volumes from 39 patients with head-and-neck cancer (HD2). In HD1, population priors allowed omission of both pretreatment scans, while adaptive scheduling reduced the protocol from nine scans to three or four, with the response group identified from a single post-treatment scan on day 27. In HD2, the adaptive schedule used three scans instead of six and improved prediction by delaying the first on-treatment scan from week 1 to week 2, avoiding transient dynamics that produced false-positive and false-negative response projections. Across both datasets, the adaptive schedules used a mean of 2.7 scans in stead of seven and advanced completion of the patient-specific prediction by a mean of 15.5 days (95% CI, 6.7-24.3) relative to the equidistant protocol, while treatment duration remained unchanged. INFORME therefore reduces measurement burden and accelerates patient-specific prediction by concentrating observations at times that are most informative for model calibration.

systems biology↗

bioRxiv2017

A Map Of Genetic Interactions In Cancer Cells

Cancer genomes often harbor hundreds of molecular aberrations. Such genetic variants can be drivers or passengers of tumorigenesis and, as a side effect, create new vulnerabilities for potential therapeutic exploitation. To systematically identify genotype-dependent vulnerabilities and synthetic lethal interactions, forward genetic screens in different genetic backgrounds have been conducted. We devised MINGLE, a computational framework that integrates CRISPR/Cas9 screens originating from many different libraries and laboratories to build genetic interaction maps. It builds on analytical approaches that were established for genetic network discovery in model organisms. We applied this method to integrate and analyze data from 85 CRISPR/Cas9 screens in human cancer cell lines combining functional data with information on genetic variants to explore the relationships of more than 2.1 million gene-background relationships. In addition to known dependencies, our analysis identified new genotype-specific vulnerabilities of cancer cells. Experimental validation of predicted vulnerabilities associated with aberrant Wnt/{beta}-catenin signaling identified GANAB and PRKCSH as new positive regulators of Wnt/{beta}-catenin signaling. By clustering genes with similar genetic interaction profiles, we drew the largest genetic network in cancer cells to date. Our scalable approach highlights how diverse genetic screens can be integrated to systematically build informative maps of genetic interactions in cancer, which can grow dynamically as more data is included.

systems biology↗

bioRxiv2018

Synthetic STARR-seq reveals how DNA shape and sequence modulate transcriptional output and noise.

The binding of transcription factors to short recognition sequences plays a pivotal role in controlling the expression of genes. The sequence and shape characteristics of binding sites influence DNA binding specificity and have also been implicated in modulating the activity of transcription factors downstream of binding. To quantitatively assess the transcriptional activity of dozens of thousands of designed synthetic sites in parallel, we developed a synthetic version of STARR-seq (synSTARR-seq). We used the approach to systematically analyze how variations in the recognition sequence of the glucocorticoid receptor (GR) affect transcriptional regulation. Our approach resulted in the identification of a novel highly active functional GR binding sequence and revealed that sequence variation both within and flanking GRs core binding site can modulate GR activity without apparent changes in DNA binding affinity. Notably, we found that the sequence composition of variants with similar activity profiles was highly diverse. In contrast, groups of variants with similar activity profiles showed distinct DNA shape characteristics indicating that DNA shape may be a better predictor of activity than DNA sequence. Finally, using single cell experiments with individual enhancer variants, we obtained clues indicating that the architecture of the response element can independently tune expression mean and cell-to cell variability in gene expression (noise). Together, our studies establish synSTARR as a powerful method to systematically study how DNA sequence and shape modulate transcriptional output and noise.

systems biology↗

bioRxiv2018

Temporal Control of Transcription by Zelda in living Drosophila embryos

Abstract/introPioneer factors have the exquisite ability to engage their target sites at nucleosomal DNA, which leads to a local remodeling of chromatin and the establishment of a transcriptional competence. However, the direct impact of enhancer priming by pioneer factors on the temporal control of gene expression and on mitotic memory remains elusive. In Drosophila embryos, the maternally deposited activator Zelda (Zld) exhibits key pioneer factor properties and indeed regulates the awakening of the zygotic genome. The analysis of thousands of endogenous Zld bound regions in various genetic contexts, as well as the study of isolated synthetic enhancers with static approaches, led to the proposal that Zld could act as a quantitative developmental timer. Here we employ quantitative live imaging methods and mathematical modeling to directly test the effect of Zld on temporal coordination in gene activation and on mitotic memory. Using an automatic tracking software, we quantified the timing of activation in hundreds of nuclei and their progeny in Drosophila embryos. We demonstrate that increasing the number of Zld binding sites accelerates the kinetics of transcriptional activation regardless of their past transcriptional state. In spite of its known pioneering activities, we show that Zld is not a mitotic bookmarker and is neither necessary nor sufficient to foster mitotic memory. Fluorescent recovery after photo-bleaching and fluorescent correlation spectroscopy experiments reveal that, Zld is highly dynamic and exhibits transient binding to chromatin. We propose that Zld low binding rates could be compensated for by local accumulation of Zld in nuclear microenvironments in vivo, thus allowing rapid and coordinated gene activation.

developmental biology↗

bioRxiv2017

Genome-wide and high-density CRISPR-Cas9 screens identify point mutations in PARP1 causing PARP inhibitor resistance

PARP inhibitors (PARPi) target homologous recombination defective tumour cells via synthetic lethality. Genome-wide and high-density CRISPR-Cas9 \"tag, mutate and enrich\" mutagenesis screens identified single amino acid mutations in PARP1 that cause profound PARPi-resistance. These included PARP1 mutations outside of the DNA interacting regions of the protein, such as mutations in solvent exposed regions of the catalytic domain and clusters of mutations around points of contact between ZnF, WGR and HD domains. These mutations altered PARP1 trapping, as did a mutation found in a clinical case of PARPi resistance. These genetic studies reinforce the importance of trapped PARP1 as a key cytotoxic DNA lesion and suggest that interactions between non-DNA binding domains of PARP1 influence cytotoxicity. Finally, different mechanisms of PARPi resistance (BRCA1 reversion, PARP1, 53BP1, REV7 mutation) had differing effects on chemotherapy sensitivity, suggesting that the underlying mechanism of PARPi resistance likely influences the success of subsequent therapies.

cancer biology↗

bioRxiv2017

Deciphering microbial interactions in synthetic human gut microbiome communities

The human gut microbiota comprises a dynamic ecological system that contributes significantly to human health and disease. The ecological forces that govern community assembly and stability in the gut microbiota remain unresolved. We developed a generalizable model-guided framework to predict higher-order consortia from time-resolved measurements of lower-order assemblages. This method was employed to decipher microbial interactions in a diverse 12-member human gut microbiome synthetic community. We show that microbial growth parameters and pairwise interactions are the major drivers of multi-species community dynamics, as opposed to context-dependent (conditional) interactions. The inferred microbial interaction network as well as a top-down approach to community assembly pinpointed both ecological driver and responsive species that were significantly modulated by microbial inter-relationships. Our model demonstrated that negative pairwise interactions could generate history-dependent responses of initial species proportions on physiological timescales that frequently does not originate from bistability. The model elucidated a topology for robust coexistence in pairwise assemblages consisting of a negative feedback loop that balances disparities in monospecies fitness levels. Bayesian statistical methods were used to evaluate the constraint of model parameters by the experimental data. Measurements of extracellular metabolites illuminated the metabolic capabilities of monospecies and potential molecular basis for competitive and cooperative interactions in the community. However, these data failed to predict influential organisms shaping community assembly. In sum, these methods defined the ecological roles of key species shaping community assembly and illuminated network design principles of microbial communities.

systems biology↗

bioRxiv2018

MicroRNAs as potential therapeutics to enhance chemosensitivity in advancedprostate cancer

Docetaxel and cabazitaxel are taxane chemotherapy treatments for metastatic castration-resistant prostate cancer (CRPC). However, therapeutic resistance remains a major issue. MicroRNAs are short non-coding RNAs that can silence multiple genes, regulating several signalling pathways simultaneously. Therefore, synthetic microRNAs may have therapeutic potential in CRPC by regulating genes involved in taxane response and minimise compensatory mechanisms that cause taxane resistance. To identify microRNAs that can improve the efficacy of taxanes in CRPC, we performed a genome-wide screen of 1280 microRNAs in the CRPC cell lines PC3 and DU145 in combination with docetaxel or cabazitaxel treatment. Mimics of miR-217 and miR-181b-5p enhanced apoptosis significantly in PC3 cells in the presence of these taxanes. These mimics downregulated at least a thousand different transcripts, which were enriched for genes with cell proliferation and focal adhesion functions. Individual knockdown of a selection of 46 genes representing these transcripts resulted in toxic or taxane sensitisation effects, indicating that these genes may be mediating the effects of the microRNA mimics. A range of these genes are expressed in CRPC metastases, suggesting that these microRNA mimics may be functional in CRPC. With further development, these microRNA mimics may have therapeutic potential to improve taxane response in CRPC patients.

cancer biology↗