Project description:Methods to map small-molecule binding sites on cellular RNAs are important for understanding interactions with both endogenous and exogenous compounds. Pt(ii) reagents are well-known DNA and RNA crosslinking agents, but sequence-specific and genome-wide identification of Pt targets following in-cell treatment is challenging. Here we describe application of high-throughput 'Pt-Seq' to identify Pt-rRNA adducts following treatment of S. cerevisiae with cisplatin.
Project description:Deciphering immune recognition is critical for understanding a broad range of diseases and for the development of effective vaccines and immunotherapies. Efforts to do so are limited by a lack of technologies capable of simultaneously capturing the complexity of adaptive immunoreceptor repertoires and the landscape of potential antigens. To address this, we present receptor-antigen pairing by targeted retroviruses, which combines viral pseudotyping and molecular engineering approaches to enable one-pot library-on-library interaction screens by displaying antigens on the surface of lentiviruses and encoding their identity in the viral genome. Antigen-specific viral infection of cell lines expressing human T or B cell receptors allows readout of both antigen and receptor identities via single-cell sequencing. The resulting system is modular, scalable and compatible with any cell type. These techniques provide a suite of tools for targeted viral entry, molecular engineering and interaction screens with broad potential applications.
Project description:A major challenge in systems biology is to understand the gene regulatory networks that drive development, physiology and pathology. Interactions between transcription factors and regulatory genomic regions provide the first level of gene control. Gateway-compatible yeast one-hybrid (Y1H) assays present a convenient method to identify and characterize the repertoire of transcription factors that can bind a DNA sequence of interest. To delineate genome-scale regulatory networks, however, large sets of DNA fragments need to be processed at high throughput and high coverage. Here we present enhanced Y1H (eY1H) assays that use a robotic mating platform with a set of improved Y1H reagents and automated readout quantification. We demonstrate that eY1H assays provide excellent coverage and identify interacting transcription factors for multiple DNA fragments in a short time. eY1H assays will be an important tool for mapping gene regulatory networks in Caenorhabditis elegans and other model organisms as well as in humans.
Project description:BACKGROUND: The genus Flavivirus encompasses more than 50 distinct species of arthropod-borne viruses, including several major human pathogens, such as West Nile virus, yellow fever virus, Japanese encephalitis virus and the four serotypes of dengue viruses (DENV type 1-4). Each year, flaviviruses cause more than 100 million infections worldwide, some of which lead to life-threatening conditions such as encephalitis or haemorrhagic fever. Among the viral proteins, NS3 and NS5 proteins constitute the major enzymatic components of the viral replication complex and are essential to the flavivirus life cycle. RESULTS: We report here the results of a high-throughput yeast two-hybrid screen to identify the interactions between human host proteins and the flavivirus NS3 and NS5 proteins. Using our screen results and literature curation, we performed a global analysis of the NS3 and NS5 cellular targets based on functional annotation with the Gene Ontology features. We finally created the first flavivirus NS3 and NS5 proteins interaction network and analysed the topological features of this network. Our proteome mapping screen identified 108 human proteins interacting with NS3 or NS5 proteins or both. The global analysis of the cellular targets revealed the enrichment of host proteins involved in RNA binding, transcription regulation, vesicular transport or innate immune response regulation. CONCLUSIONS: We proposed that the selective disruption of these newly identified host/virus interactions could represent a novel and attractive therapeutic strategy in treating flavivirus infections. Our virus-host interaction map provides a basis to unravel fundamental processes about flavivirus subversion of the host replication machinery and/or immune defence strategy.
Project description:Quantifying the effects of cis-regulatory DNA on gene expression is a major challenge. Here, we present the multiplexed editing regulatory assay (MERA), a high-throughput CRISPR-Cas9-based approach that analyzes the functional impact of the regulatory genome in its native context. MERA tiles thousands of mutations across ∼40 kb of cis-regulatory genomic space and uses knock-in green fluorescent protein (GFP) reporters to read out gene activity. Using this approach, we obtain quantitative information on the contribution of cis-regulatory regions to gene expression. We identify proximal and distal regulatory elements necessary for expression of four embryonic stem cell-specific genes. We show a consistent contribution of neighboring gene promoters to gene expression and identify unmarked regulatory elements (UREs) that control gene expression but do not have typical enhancer epigenetic or chromatin features. We compare thousands of functional and nonfunctional genotypes at a genomic location and identify the base pair-resolution functional motifs of regulatory elements.
Project description:Lis1, Nudel/NudE, and dynactin are regulators of cytoplasmic dynein, a minus end-directed, microtubule (MT)-based motor required for proper spindle assembly and orientation. In vitro studies have shown that dynactin promotes processive movement of dynein on MTs, whereas Lis1 causes dynein to enter a persistent force-generating state (referred to here as dynein stall). Yet how the activities of Lis1, Nudel/NudE, and dynactin are coordinated to regulate dynein remains poorly understood in vivo. Working in Xenopus egg extracts, we show that Nudel/NudE facilitates the binding of Lis1 to dynein, which enhances the recruitment of dynactin to dynein. We further report a novel Lis1-dependent dynein-dynactin interaction that is essential for the organization of mitotic spindle poles. Finally, using assays for MT gliding and spindle assembly, we demonstrate an antagonistic relationship between Lis1 and dynactin that allows dynactin to relieve Lis1-induced dynein stall on MTs. Our findings suggest the interesting possibility that Lis1 and dynactin could alternately engage with dynein to allow the motor to promote spindle assembly.
Project description:We have designed the most efficient strategy to knock out genes in fission yeast Schizosaccharomyces pombe on a large scale. Our technique is based on knockout constructs that contain regions homologous to the target gene cloned into vectors carrying dominant drug-resistance markers. Most of the steps are carried out in a 96-well format, allowing simultaneous deletion of 96 genes in one batch. Based on our knockout technique, we designed a strategy for cloning knockout constructs for all predicted fission yeast genes, which is available in a form of a searchable database http://mendel.imp.ac.at/Pombe_deletion/. We validated this technique in a screen where we identified novel genes required for chromosome segregation during meiosis. Here, we present our protocol with detailed instructions. Using this protocol, one person can knock out 96 S. pombe genes in 8 days.
Project description:The race to discover and isolate giant viruses began 15 years ago. Metagenomics is counterbalancing coculture, with the detection of giant virus genomes becoming faster as sequencing technologies develop. Since the discovery of giant viruses, many efforts have been made to improve methods for coculturing amebas and giant viruses, which remains the key engine of isolation of these microorganisms. However, these techniques still lack the proper tools for high-speed detection. In this paper, we present advances in the isolation of giant viruses. A new strategy was developed using a high-throughput microscope for real-time monitoring of cocultures using optimized algorithms targeting infected amebas. After validating the strategy, we adapted a new tabletop scanning electron microscope for high-speed identification of giant viruses directly from culture. The speed and isolation rate of this strategy has raised the coculture to almost the same level as sequencing techniques in terms of detection speed and sensitivity.
Project description:Macrophages are plastic cells of the innate immune system that perform a wide range of immune- and homeostasis-related functions. Due to their plasticity, macrophages can polarize into a spectrum of activated phenotypes. Rapid identification of macrophage polarization states provides valuable information for drug discovery, toxicological screening, and immunotherapy evaluation. The complexity associated with macrophage activation limits the ability of current biomarker-based methods to rapidly identify unique activation states. In this study, we demonstrate the ability of a 2-element sensor array that provides an information-rich 5-channel output to successfully determine macrophage polarization phenotypes in a matter of minutes. The simple and robust sensor generates a high dimensional data array which enables accurate macrophage evaluations in standard cell lines and primary cells after cytokine treatment, as well as following exposure to a model disease environment.
Project description:BackgroundHigh-quality quantitative data is a major limitation in systems biology. The experimental data used in systems biology can be assigned to one of the following categories: assays yielding average data of a cell population, high-content single cell measurements and high-throughput techniques generating single cell data for large cell populations. For modeling purposes, a combination of data from different categories is highly desirable in order to increase the number of observable species and processes and thereby maximize the identifiability of parameters.ResultsIn this article we present a method that combines the power of high-content single cell measurements with the efficiency of high-throughput techniques. A calibration on the basis of identical cell populations measured by both approaches connects the two techniques. We develop a mathematical model to relate quantities exclusively observable by high-content single cell techniques to those measurable with high-content as well as high-throughput methods. The latter are defined as free variables, while the variables measurable with only one technique are described in dependence of those. It is the combination of data calibration and model into a single method that makes it possible to determine quantities only accessible by single cell assays but using high-throughput techniques. As an example, we apply our approach to the nucleocytoplasmic transport of STAT5B in eukaryotic cells.ConclusionsThe presented procedure can be generally applied to systems that allow for dividing observables into sets of free quantities, which are easily measurable, and variables dependent on those. Hence, it extends the information content of high-throughput methods by incorporating data from high-content measurements.