Biology subjects

Boutros, M.

Publications and source records attributed to Boutros, M..

At least 37 records · Page 2Linked to original sources

bioRxiv2021

Glyoxal as alternative fixative for single cell RNA sequencing

Single cell RNA sequencing (scRNA-seq) has become an important method to identify cell types, delineate the trajectories of cell differentiation in whole organisms and understand the heterogeneity in cellular responses. Nevertheless, sample collection and processing remain a severe bottleneck for scRNA-seq experiments. Cell isolation protocols often lead to significant changes in the transcriptomes of cells, requiring novel methods to preserve cell states. Here, we developed and benchmarked protocols using glyoxal as a fixative for scRNA-seq application. Using Drop-seq methodology, we detected high numbers of transcripts and genes from glyoxal-fixed Drosophila cells after scRNA-seq. The effective glyoxal fixation of transcriptomes in Drosophila and human cells was further supported by a high correlation of gene expression data between glyoxal-fixed and unfixed samples. Accordingly, we also found highly expressed genes overlapping to a large extent between experimental conditions. These results indicated that our fixation protocol did not induce considerable changes in gene expression and conserved the transcriptome for subsequent single cell isolation procedures. In conclusion, we present glyoxal as a suitable fixative for Drosophila cells and potentially cells of other species that allows high-quality scRNA-seq applications.

genomics↗

bioRxiv2020

EVI/WLS function is regulated by ubiquitination and linked to ER-associated degradation by ERLIN2

Wnt signalling is important for development in all metazoan animals and associated with various human diseases. The Ubiquitin-Proteasome System (UPS) and regulatory ER-associated degradation (ERAD) has been implicated in the secretion of WNT proteins. Here, we investigated how the WNT secretory factor EVI/WLS is ubiquitinated, recognised by ERAD components, and subsequently removed from the secretory pathway. We performed a focused, immunoblot-based RNAi screen for factors that influence EVI/WLS protein stability. We identified the VCP-binding proteins FAF2 and UBXN4 as novel interaction partners of EVI/WLS and showed that ERLIN2 links EVI/WLS to the ubiquitination machinery. Interestingly, we found in addition that EVI/WLS is ubiquitinated and degraded in cells irrespective of their level of WNT production. K11, K48, and K63-linked ubiquitination is mediated by the E2 ubiquitin conjugating enzymes UBE2J2, UBE2N, and UBE2K, but independent of the E3 ligases HRD1/SYVN. Taken together, our study identified factors that link UPS to the WNT secretory pathway and provides mechanistic details on the fate of an endogenous substrate of regulatory ERAD in mammalian cells.

cell biology↗

bioRxiv2020

Establishment of a simplified preparation method for single-nucleus RNA-sequencing and its application to long-term frozen tumor tissues

Recent advances allowing the genomic analysis of individual cells from a bulk population have provided intriguing new insights into areas such as developmental processes and tumor heterogeneity. Most approaches to date, however, rely on the availability of fresh surgical specimens, thereby dramatically reducing the ability to profile particularly rare tissue types. Pediatric central nervous system tumors - the leading cause of childhood cancer deaths - represent one such example, where often only frozen rather than native material is available. Due to an increasing need for advanced techniques to understand the heterogeneity of these tumors, we optimized a method to isolate intact nuclei from long-term frozen pediatric glioma tissues. We performed a technical comparison between different single nucleus RNA-sequencing (snRNA-seq) systems using a patient-derived xenograft model as a test sample. Further, we applied the established nucleus isolation method to analyze frozen primary tissue from two pediatric central nervous system tumors - one pilocytic astrocytoma and one glioblastoma - allowing the identification of distinct tumor cell populations and infiltrating microglia. The results show that our fast, simple and low-cost nuclear isolation protocol provides intact nuclei, which can be used in both droplet-based 3 transcriptome amplification (10X Genomics) and plate-based whole transcriptome amplification (Fluidigm C1) single-cell sequencing platforms, thereby dramatically increasing the potential for application of such methods to rare entities.

cancer biology↗

bioRxiv2020

SARS-CoV-2 infection induces a pro-inflammatory cytokine response through cGAS-STING and NF-κB

SARS-CoV-2 is a novel virus that has rapidly spread, causing a global pandemic. In the majority of infected patients, SARS-CoV-2 leads to mild disease; however, in a significant proportion of infections, individuals develop severe symptoms that can lead to permanent lung damage or death. These severe cases are often associated with high levels of pro-inflammatory cytokines and low antiviral responses which can lead to systemic complications. We have evaluated transcriptional and cytokine secretion profiles from infected cell cultures and detected a distinct upregulation of inflammatory cytokines that parallels samples taken from infected patients. Building on these observations, we found a specific activation of NF-{kappa}B and a block of IRF3 nuclear translocation in SARS-CoV-2 infected cells. This NF-{kappa}B response is mediated by cGAS-STING activation and could be attenuated through STING targeting drugs. Our results show that SARS-CoV-2 curates a cGAS-STING mediated NF-{kappa}B driven inflammatory immune response in epithelial cells that likely contributes to inflammatory responses seen in patients and might be a target to suppress severe disease symptoms.

microbiology↗

bioRxiv2020

Allele-specific endogenous tagging and quantitative analysis of β-catenin in colon cancer cells

Wnt signaling plays important roles in development, homeostasis, and tumorigenesis. Mutations in {beta}-catenin that activate Wnt signaling have been found in colorectal and hepatocellular carcinomas. However, the dynamics of wild-type and mutant forms of {beta}-catenin are not fully understood. Here, we genome-engineered fluorescently tagged alleles of the endogenous {beta}-catenin in a colorectal cancer cell line. Wild-type and oncogenic mutant alleles were tagged with different fluorescent proteins, enabling the analysis of both variants in the same cell. We analyzed the properties of both {beta}-catenin alleles using immunoprecipitation, immunofluorescence and fluorescence correlation spectroscopy approaches, revealing distinctly different biophysical properties. In addition, activation of Wnt signaling by treatment with a GSK3{beta} inhibitor or a truncating APC mutation modulated the wild-type allele to mimic the properties of the mutant {beta}-catenin allele. The one-step tagging strategy demonstrates how genome engineering can be employed for the parallel functional analysis of different genetic variants.

genetics↗

bioRxiv2020

Pooled CRISPR screening at high sensitivity with an empirically designed sgRNA library

Given their broad utility in functionally annotating genomes, the experimental design of genome-scale CRISPR screens can vary greatly and criteria for optimal experimental implementation and library composition are still emerging. In this study, we report advantages of conducting viability screens in selected Cas9 single cell clones in contrast to Cas9 bulk populations. We further systematically analyzed published CRISPR screens in human cells to identify single-guide (sg)RNAs with consistent high on-target and low off-target activity. Selected guides were collected in a new genome-scale sgRNA library, which efficiently identifies core and context-dependent essential genes. In summary, we show how empirically designed libraries in combination with an optimised experimental design increase the dynamic range in gene essentiality screens at reduced library coverage.

genomics↗

bioRxiv2020

Multiplexed conditional genome editing with Cas12a in Drosophila

CRISPR-Cas genome engineering has revolutionised biomedical research by enabling targeted genome modification with unprecedented ease. In the popular model organism Drosophila melanogaster gene editing has so far relied exclusively on the prototypical CRISPR nuclease Cas9. The availability of additional CRISPR systems could expand the genomic target space, offer additional modes of regulation and enable the independent manipulation of genes in different cell populations of the same animal. Here we describe a platform for efficient Cas12a gene editing in Drosophila. We show that Cas12a from Lachnospiraceae bacterium, but not Acidaminococcus spec., can mediate robust gene editing in vivo. In combination with most crRNAs, LbCas12a activity is strongly suppressed at lower temperatures, enabling control of gene editing by simply modulating temperature. LbCas12a can directly utilize compact crRNAs arrays that are substantially easier to construct than Cas9 sgRNA arrays, facilitating multiplex genome engineering of several target sites in parallel. Targeting genes with arrays of three crRNAs results in the induction of loss-of function phenotypes with comparable efficiencies than a state-of-the-art Cas9 system. Lastly, we show that cell type-specific expression of LbCas12a is sufficient to mediate tightly controlled gene editing in a variety of tissues, allowing detailed analysis of gene function in this multicellular organism. Cas12a gene editing substantially expands the genome engineering toolbox in this organism and will be a powerful method for the functional annotation of the Drosophila genome. This work also lays out principles for the development of multiplexed transgenic Cas12a genome engineering systems in other genetically tractable organisms.

genetics↗

bioRxiv2020

Systematic functional analysis of Rab GTPasesreveals limits of neuronal robustness in Drosophila

Rab GTPases are molecular switches that regulate membrane trafficking in all cells. Neurons have particular demands on membrane trafficking and express numerous Rab GTPases of unknown function. Here we report the generation and characterization of molecularly defined null mutants for all 26 rab genes in Drosophila. In addition, we created a transgenic fly collection for the acute, synchronous release system RUSH for all 26 Rabs. In flies, all rab genes are expressed in the nervous system where at least half exhibit particularly high levels compared to other tissues. Surprisingly, loss of any of these 13 nervous-system enriched Rabs yields viable and fertile flies without obvious morphological defects. However, 9 of these 13 affect either developmental timing when challenged with different temperatures, or neuronal function when challenged with continuous stimulation. These defects are non-lethal under laboratory conditions, but represent sensitized genetic backgrounds that reveal limits of developmental and functional robustness to environmental challenges. Interestingly, the neuronal rab26 was previously proposed to function in synaptic maintenance by linking autophagy and synaptic vesicle recycling and we identified rab26 as one of six rab mutants with reduced synaptic function under continuous stimulation conditions. However, we found no changes to autophagy or synaptic vesicle markers in the rab26 mutant, but instead a cell-specific role in membrane receptor turnover associated with cholinergic synapses in the fly visual system. Our systematic functional analyses suggest that several Rabs ensure robust development and function under varying environmental conditions. The mutant and transgenic fly collections generated in this study provide a basis for further studies of Rabs during development and homeostasis in vivo.

neuroscience↗

bioRxiv2019

JNK-dependent intestinal barrier failure disrupts host-microbe homeostasis during tumorigenesis

In all animals, the intestinal epithelium forms a tight barrier to the environment. The epithelium regulates the absorption of nutrients, mounts immune responses and prevents systemic infections. Here, we investigate the consequences of tumorigenesis on the microbiome using a Drosophila intestinal tumor model. We show that upon loss-of BMP signaling, tumors lead to aberrant activation of JNK signaling, followed by intestinal barrier dysfunction and commensal imbalance. In turn, the dysbiotic microbiome triggers a regenerative response and stimulates tumor growth. We find that inhibiting JNK signaling or depletion of the microbiome restores barrier function of the intestinal epithelium, leading to a reestablishment of host-microbe homeostasis, and organismic lifespan extension. Our experiments identify a JNK-dependent feedback amplification loop between intestinal tumors and the microbiome. They also highlight the importance of controlling the activity level of JNK signaling to maintain epithelial barrier function and host-microbe homeostasis.

developmental biology↗

bioRxiv2019

Modelling asymmetric count ratios in CRISPR screens to decrease experiment size and improve phenotype detection

Pooled CRISPR screens are a powerful tool to probe genotype-phenotype relationships at genome-wide scale. However, criteria for optimal design are missing, and it remains unclear how experimental parameters affect results. Here, we report that random decreases in gRNA abundance are more likely than increases due to bottle-neck effects during the cell proliferation phase. Failure to consider this asymmetry leads to loss of detection power. We provide a new statistical test that addresses this problem and improves hit detection at reduced experiment size. The method is implemented in the open source package gscreend (submission to Bioconductor pending).

bioinformatics↗

bioRxiv2019

Multiparametric phenotyping of compound effects on patient derived organoids

Patient derived organoids resemble the biology of tissues and tumors, enabling ex vivo modeling of human diseases from primary patient samples. Organoids can be used as models for drug discovery and are being explored to guide clinical decision making. Patient derived organoids can have heterogeneous morphologies with unclear biological causes and relationship to treatment response. Here, we used high-throughput, image-based profiling to quantify phenotypes of over 5 million individual colorectal cancer organoids after treatment with more than 500 small molecules. Integration of data using a joint multi-omics modelling framework identified organoid size and cystic vs. solid organoid architecture as axes of morphological variation across organoids. Mechanistically, we found that organoid size was linked to IGF1 receptor signaling, while a cystic organoid architecture was associated with an LGR5+ stemness program. Treatment-induced organoid morphology reflected organoid viability, drug mechanism of action, and was biologically interpretable using joint modelling. Inhibition of MEK led to cystic reorganization of organoids and increased expression of LGR5, while inhibition of mTOR induced IGF1 receptor signaling. In conclusion, we identified shared axes of variation for colorectal cancer organoid morphology, their underlying biological mechanisms, and pharmacological interventions with the ability to move organoids along them. Image-based profiling of patient derived organoids coupled with multi-omics integration facilitates drug discovery by linking drug responses with underlying biological mechanisms.

genomics↗

bioRxiv2019

A large-scale resource for tissue-specific CRISPR mutagenesis in Drosophila

Genetic screens are powerful tools for the functional annotation of genomes. In the context of multicellular organisms, interrogation of gene function is greatly facilitated by methods that allow spatial and temporal control of gene abrogation. Here, we describe a large-scale transgenic short guide (sg) RNA library for efficient CRISPR-based disruption of specific target genes in a constitutive or conditional manner. The library consists currently of more than 2600 plasmids and 1600 fly lines with a focus on targeting kinases, phosphatases and transcription factors, each expressing two sgRNAs under control of the Gal4/UAS system. We show that conditional CRISPR mutagenesis is robust across many target genes and can be efficiently employed in various somatic tissues, as well as the germline. In order to prevent artefacts commonly associated with excessive amounts of Cas9 protein, we have developed a series of novel UAS-Cas9 transgenes, which allow fine tuning of Cas9 expression to achieve high gene editing activity without detectable toxicity. Functional assays, as well as direct sequencing of genomic sgRNA target sites, indicates that the vast majority of transgenic sgRNA lines mediate efficient gene disruption. Furthermore, we conducted the so far largest fully transgenic CRISPR screen in any metazoan organism, which further supported the high efficiency and accuracy of our library and revealed many so far uncharacterized genes essential for development.

genetics↗

bioRxiv2019

Lineage specific core-regulatory circuits determine gene essentiality in cancer cells

Cancer cells rely on dysregulated gene expression programs to maintain their malignant phenotype. A cells transcriptional state is controlled by a small set of interconnected transcription factors that form its core-regulatory circuit (CRC). Previous work in pediatric cancers has shown, that disruption of the CRC by genetic alterations causes tumor cells to become highly dependent on its components creating new opportunities for therapeutic intervention. However, the role of CRCs and the mechanisms by which they are controlled remain largely unknown for most tumor types. Here, we developed a method that infers lineage dependency scores to systematically predict functional CRCs and associated biological processes from context-dependent essentiality data sets. Analysis of genome-scale CRISPR-Cas9 screens in 558 cancer cell lines showed that most tumor types specifically depend on a small number of transcription factors for proliferation. We found that these transcription factors compose the CRCs in these tumor types. Moreover, they are frequently altered in patient tumor samples indicating their oncogenic potential. Finally, we show that biological processes associated with each CRC are revealed by analyzing codependency between lineage-specific essential genes. Our results demonstrate that genetic addiction to lineage-specific core transcriptional mechanisms occurs across a broad range of tumor types. We exploit this phenomenon to systematically infer CRCs from lineage specific gene essentiality. Furthermore, our findings shed light on the selective genetic vulnerabilities that arise as the consequence of transcriptional dysregulation in different tumor types and show how the plasticity of regulatory circuits might influence drug resistance and metastatic potential.

systems biology↗

bioRxiv2018

Gene expression atlas of a developing tissue by single cell expression correlation analysis

The Drosophila wing disc has been a fundamental model system for the discovery of key signaling pathways and for our understanding of developmental processes. However, a complete map of gene expression in this tissue is lacking. To obtain a complete gene expression atlas in the wing disc, we employed single-cell sequencing (scRNA-seq) and developed a new method for analyzing scRNA-seq data based on gene expression correlations rather than cell mappings. This enables us to discover 824 genes with spatially restricted expression patterns, and to compute expression maps for all genes in the wing disc. This approach identifies both known and new clusters of genes with similar expression patterns and functional relevance. As proof of concept, we characterize the previously unstudied gene CG5151 and show it regulates Wnt signaling. This novel method will enable the leveraging of scRNA-seq data for generating expression atlases of undifferentiated tissues during development.

developmental biology↗

bioRxiv2018

Evidence of functional long-range Wnt/Wg in the developing Drosophila wing epithelium

Wnts are secreted proteins that regulate cell fate specification during development of all metazoans. Wnt proteins were proposed to spread over several cell diameters to activate signalling directly at a distance. In the Drosophila wing epithelium, an extracellular gradient of Wingless (Wg, the homolog of Wnt1) was observed extending over several cells away from producing cells. However, it was also recently shown that a membrane-tethered Neurotactin-Wg fusion protein (NRT-Wg) can rescue the loss-of endogenous Wg, leading to proper patterning of the wing. Therefore, whether Wg spreading is required for correct tissue patterning during development remains controversial and the functional range of wild-type Wg is unclear. Here, by capturing secreted Wg on distally located cells we show that the Wg gradient acts directly up to eleven cell distances. Cells located outside the reach of extracellular Wg depend on the Frizzled2 receptor to maintain target gene expression. We find that NRT-Wg is not restricted to the producing cells and propose that it can rescue signalling defects by perdurance in the receiving cells. These results provide insight into the mechanisms by which Wnt proteins mediate patterning of a rapidly growing tissue.

developmental biology↗

bioRxiv2018

Time-resolved mapping of genetic interactions to model rewiring of signaling pathways

Context-dependent changes in genetic vulnerabilities are important to understand the wiring of cellular pathways and variations in different environmental conditions. However, methodological frameworks to investigate the plasticity of genetic networks over time or in response to external stresses are lacking. To analyze the plasticity of genetic interactions, we performed an arrayed combinatorial RNAi screen in Drosophila cells at multiple time points and after pharmacological inhibition of Ras signaling activity. Using an image-based morphology assay to capture a broad range of phenotypes, we assessed the effect of 12768 pairwise RNAi perturbations in six different conditions. We found that genetic interactions form in different trajectories and developed an algorithm, termed MODIFI, to analyze how genetic interactions rewire over time. Using this framework, we identified more statistically significant interactions compared to endpoints assays and further observed several examples of context-dependent crosstalk between signaling pathways such as an interaction between Ras and Rel which is dependent on MEK activity.

genomics↗

bioRxiv2018

WEADE: a workflow for enrichment analysis and data exploration

Data analysis based on enrichment of Gene Ontology terms has become an important step in exploring large gene or protein expression datasets and several stand-alone or web tools exist for that purpose. However, a comprehensive and consistent analysis downstream of the enrichment calculation is missing so far. With WEADE we present a free web application that offers an integrated workflow for the exploration of genomic data combining enrichment analysis with a versatile set of tools to directly compare and intersect experiments or candidate gene lists of any size or origin including cross-species data. Lastly, WEADE supports the graphical representation of output data in the form of functional interaction networks based on prior knowledge, allowing users to go from plain expression data to functionally relevant candidate sub-lists in an interactive and consistent manner.

bioinformatics↗

bioRxiv2017

Decoding the regulatory logic of the Drosophila male stem cell system

In the past decade, the importance of the niche to provide regulatory inputs to balance stem cell self-renewal and differentiation has become clear. However, the regulatory interplay between stem cells and their niche at the whole genome level is still poorly understood. To elucidate the mechanisms controlling stem cells and their progenies as they progress through their developmental program at the transcriptional level, we recorded the regulatory program of two independent cell lineages in the Drosophila testis stem cell model. To this end, we identified genes active in the soma or germline as well as genome-wide binding profiles of two essential transcription factors, Zfh-1 and Abd-A, expressed in somatic support cells and crucial for fate acquisition of both cell lineages. Our data identified key roles for TOR signalling, signal processing V-ATPase proton pumps and the nuclear transport engaged nucleoporins and we demonstrate their importance in controlling germline maintenance, proliferation and differentiation from the support side. To make our dataset publicly available and support quick and intuitive data mining, we generated an interactive online analysis tool. Applying our tool for comparative analysis, we uncovered conserved core gene sets of adult stem cells across species boundaries. We have tested the functional relevance of these genes in the Drosophila testis and intestine and find a striking overrepresentation of stem cell defects when the corresponding genes were depleted. In summary, our dataset and interactive platform represents a versatile tool for identifying novel gene networks active in diverse stem cell types and provides a valuable resource for elucidating the multifaceted regulatory inputs required to guide proper stem cell behaviour.

developmental biology↗