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1.  Amplicon Sequencing of Colorectal Cancer: Variant Calling in Frozen and Formalin-Fixed Samples 
PLoS ONE  2015;10(5):e0127146.
Next generation sequencing (NGS) is an emerging technology becoming relevant for genotyping of clinical samples. Here, we assessed the stability of amplicon sequencing from formalin-fixed paraffin-embedded (FFPE) and paired frozen samples from colorectal cancer metastases with different analysis pipelines. 212 amplicon regions in 48 cancer related genes were sequenced with Illumina MiSeq using DNA isolated from resection specimens from 17 patients with colorectal cancer liver metastases. From ten of these patients, paired fresh frozen and routinely processed FFPE tissue was available for comparative study. Sample quality of FFPE tissues was determined by the amount of amplifiable DNA using qPCR, sequencing libraries were evaluated using Bioanalyzer. Three bioinformatic pipelines were compared for analysis of amplicon sequencing data. Selected hot spot mutations were reviewed using Sanger sequencing. In the sequenced samples from 16 patients, 29 non-synonymous coding mutations were identified in eleven genes. Most frequent were mutations in TP53 (10), APC (7), PIK3CA (3) and KRAS (2). A high concordance of FFPE and paired frozen tissue samples was observed in ten matched samples, revealing 21 identical mutation calls and only two mutations differing. Comparison of these results with two other commonly used variant calling tools, however, showed high discrepancies. Hence, amplicon sequencing can potentially be used to identify hot spot mutations in colorectal cancer metastases in frozen and FFPE tissue. However, remarkable differences exist among results of different variant calling tools, which are not only related to DNA sample quality. Our study highlights the need for standardization and benchmarking of variant calling pipelines, which will be required for translational and clinical applications.
doi:10.1371/journal.pone.0127146
PMCID: PMC4444292  PMID: 26010451
2.  Functional fingerprinting of human mesenchymal stem cells using high-throughput RNAi screening 
Genome Medicine  2015;7(1):46.
Mesenchymal stem cells (MSCs) are promising candidates for cellular therapies ranging from tissue repair in regenerative medicine to immunomodulation in graft versus host disease after allogeneic transplantation or in autoimmune diseases. Nonetheless, progress has been hampered by their enormous phenotypic as well as functional heterogeneity and the lack of uniform standards and guidelines for quality control. In this study, we describe a method to perform cellular phenotyping by high-throughput RNA interference in primary human bone marrow MSCs. We have shown that despite heterogeneity of MSC populations, robust functional assays can be established that are suitable for high-throughput and high-content screening. We profiled primary human MSCs against human fibroblasts. Network analysis showed a kinome fingerprint that differs from human primary fibroblasts as well as fibroblast cell lines. In conclusion, this study shows that high-throughput screening in primary human MSCs can be reliably used for kinome fingerprinting.
Electronic supplementary material
The online version of this article (doi:10.1186/s13073-015-0170-2) contains supplementary material, which is available to authorized users.
doi:10.1186/s13073-015-0170-2
PMCID: PMC4481116  PMID: 26120366
3.  A high-throughput RNAi screen for detection of immune-checkpoint molecules that mediate tumor resistance to cytotoxic T lymphocytes 
EMBO Molecular Medicine  2015;7(4):450-463.
The success of T cell-based cancer immunotherapy is limited by tumor's resistance against killing by cytotoxic T lymphocytes (CTLs). Tumor-immune resistance is mediated by cell surface ligands that engage immune-inhibitory receptors on T cells. These ligands represent potent targets for therapeutic inhibition. So far, only few immune-suppressive ligands have been identified. We here describe a rapid high-throughput siRNA-based screening approach that allows a comprehensive identification of ligands on human cancer cells that inhibit CTL-mediated tumor cell killing. We exemplarily demonstrate that CCR9, which is expressed in many cancers, exerts strong immune-regulatory effects on T cell responses in multiple tumors. Unlike PDL1, which inhibits TCR signaling, CCR9 regulates STAT signaling in T cells, resulting in reduced T-helper-1 cytokine secretion and reduced cytotoxic capacity. Moreover, inhibition of CCR9 expression on tumor cells facilitated immunotherapy of human tumors by tumor-specific T cells in vivo. Taken together, this method allows a rapid and comprehensive determination of immune-modulatory genes in human tumors which, as an entity, represent the ‘immune modulatome’ of cancer.
doi:10.15252/emmm.201404414
PMCID: PMC4403046  PMID: 25691366
cancer immunotherapy; immune suppression; RNAi screen
4.  A map of directional genetic interactions in a metazoan cell 
eLife  null;4:e05464.
Gene–gene interactions shape complex phenotypes and modify the effects of mutations during development and disease. The effects of statistical gene–gene interactions on phenotypes have been used to assign genes to functional modules. However, directional, epistatic interactions, which reflect regulatory relationships between genes, have been challenging to map at large-scale. Here, we used combinatorial RNA interference and automated single-cell phenotyping to generate a large genetic interaction map for 21 phenotypic features of Drosophila cells. We devised a method that combines genetic interactions on multiple phenotypes to reveal directional relationships. This network reconstructed the sequence of protein activities in mitosis. Moreover, it revealed that the Ras pathway interacts with the SWI/SNF chromatin-remodelling complex, an interaction that we show is conserved in human cancer cells. Our study presents a powerful approach for reconstructing directional regulatory networks and provides a resource for the interpretation of functional consequences of genetic alterations.
DOI: http://dx.doi.org/10.7554/eLife.05464.001
eLife digest
Genes encode instructions that control our physical characteristics, known as traits. Although some traits are controlled by the activity of a single gene, most traits are influenced by the activities of multiple genes.
The genes that influence a particular trait may work independently of each other. However, it is also possible for the genes to interact so that one gene may mask or amplify the effect of another gene. Although gene interactions were first described almost 100 years ago, it has been difficult to identify them and work out the direction of these interactions (i.e., does gene A affect gene B, or vice versa?).
Fischer, Sandmann et al. have now studied the interactions between the genes involved in 21 different traits of fruit fly cells. A technique called RNA interference was used to lower the expression of the genes in different combinations, which made it possible to analyze any changes in the traits that occurred when particular genes were not working properly. Fischer, Sandmann et al. took hundreds of thousands images of the cells and analyzed the changes in cell shape, cell size, cell division and other traits. Next, they developed a method to infer the directions of the interactions between individual pairs of genes from the data and then made a map of the genetic interactions for the traits.
This map was able to reconstruct the known order of activity of genes during cell division and other cell processes. Furthermore, it revealed previously unknown interactions between genes. For example, genes involved in the Ras signaling pathway—which promotes cell growth and is frequently mutated in human tumors—interacted with genes that encode a group of proteins called the SWI/SNF complex. This complex alters how DNA is packaged in cells to control the expression of genes, and these gene interactions may play an important role in the control of cell growth by Ras signaling.
The approach developed by Fischer, Sandmann et al. can shed light on the interactions between genes that produce complex traits of cells. In future, this approach might be helpful to find out which genetic differences between individuals alter the effectiveness of drug treatments, and the impact of using combinations of drugs to treat diseases.
DOI: http://dx.doi.org/10.7554/eLife.05464.002
doi:10.7554/eLife.05464
PMCID: PMC4384530  PMID: 25748138
genetic interactions; epistasis; image-based phenotyping; D. melanogaster
5.  A synthetic lethal screen identifies FAT1 as an antagonist of caspase-8 in extrinsic apoptosis 
The EMBO Journal  2014;33(3):181-197.
The extrinsic apoptosis pathway is initiated by binding of death ligands to death receptors resulting in the formation of the death-inducing signaling complex (DISC). Activation of procaspase-8 within the DISC and its release from the signaling complex is required for processing executor caspases and commiting cell death. Here, we report that the atypical cadherin FAT1 interacts with caspase-8 preventing the association of caspase-8 with the DISC. We identified FAT1 in a genome-wide siRNA screen for synthetic lethal interactions with death receptor-mediated apoptosis. Knockdown of FAT1 sensitized established and patient-derived glioblastoma cell lines for apoptosis transduced by cell death ligands. Depletion of FAT1 resulted in enhanced procaspase-8 recruitment to the DISC and increased formation of caspase-8 containing secondary signaling complexes. In addition, FAT1 knockout cell lines generated by CRISPR/Cas9-mediated genome engineering were more susceptible for death receptor-mediated apoptosis. Our findings provide evidence for a mechanism to control caspase-8-dependent cell death by the atypical cadherin FAT1. These results contribute towards the understanding of effector caspase regulation in physiological conditions.
doi:10.1002/embj.201385686
PMCID: PMC3983683  PMID: 24442637
apoptosis; FAT1; glioblastoma; TRAIL
6.  Functional Analysis of the Drosophila Embryonic Germ Cell Transcriptome by RNA Interference 
PLoS ONE  2014;9(6):e98579.
In Drosophila melanogaster, primordial germ cells are specified at the posterior pole of the very early embryo. This process is regulated by the posterior localized germ plasm that contains a large number of RNAs of maternal origin. Transcription in the primordial germ cells is actively down-regulated until germ cell fate is established. Bulk expression of the zygotic genes commences concomitantly with the degradation of the maternal transcripts. Thus, during embryogenesis, maternally provided and zygotically transcribed mRNAs determine germ cell development collectively. In an effort to identify novel genes involved in the regulation of germ cell behavior, we carried out a large-scale RNAi screen targeting both maternal and zygotic components of the embryonic germ line transcriptome. We identified 48 genes necessary for distinct stages in germ cell development. We found pebble and fascetto to be essential for germ cell migration and germ cell division, respectively. Our data uncover a previously unanticipated role of mei-P26 in maintenance of embryonic germ cell fate. We also performed systematic co-RNAi experiments, through which we found a low rate of functional redundancy among homologous gene pairs. As our data indicate a high degree of evolutionary conservation in genetic regulation of germ cell development, they are likely to provide valuable insights into the biology of the germ line in general.
doi:10.1371/journal.pone.0098579
PMCID: PMC4045815  PMID: 24896584
7.  Molecular dissection of Wnt3a-Frizzled8 interaction reveals essential and modulatory determinants of Wnt signaling activity 
BMC Biology  2014;12:44.
Background
Wnt proteins are a family of secreted signaling molecules that regulate key developmental processes in metazoans. The molecular basis of Wnt binding to Frizzled and LRP5/6 co-receptors has long been unknown due to the lack of structural data on Wnt ligands. Only recently, the crystal structure of the Wnt8-Frizzled8-cysteine-rich-domain (CRD) complex was solved, but the significance of interaction sites that influence Wnt signaling has not been assessed.
Results
Here, we present an extensive structure-function analysis of mouse Wnt3a in vitro and in vivo. We provide evidence for the essential role of serine 209, glycine 210 (site 1) and tryptophan 333 (site 2) in Fz binding. Importantly, we discovered that valine 337 in the site 2 binding loop is critical for signaling without contributing to binding. Mutations in the presumptive second CRD binding site (site 3) partly abolished Wnt binding. Intriguingly, most site 3 mutations increased Wnt signaling, probably by inhibiting Wnt-CRD oligomerization. In accordance, increasing amounts of soluble Frizzled8-CRD protein modulated Wnt3a signaling in a biphasic manner.
Conclusions
We propose a concentration-dependent switch in Wnt-CRD complex formation from an inactive aggregation state to an activated high mobility state as a possible modulatory mechanism in Wnt signaling gradients.
doi:10.1186/1741-7007-12-44
PMCID: PMC4068752  PMID: 24885675
Wnt signaling; Wnt; Frizzled; Wnt3a mutation analysis
8.  Loss of epidermal Evi/Wls results in a phenotype resembling psoriasiform dermatitis 
The Journal of Experimental Medicine  2013;210(9):1761-1777.
The Wnt cargo receptor Evi maintains normal skin homeostasis and barrier function via Wnt secretion in the epidermis.
Cells of the epidermis renew constantly from germinal layer stem cells. Although epithelial cell differentiation has been studied in great detail and the role of Wnt signaling in this process is well described, the contribution of epidermal Wnt secretion in epithelial cell homeostasis remains poorly understood. To analyze the role of Wnt proteins in this process, we created a conditional knockout allele of the Wnt cargo receptor Evi/Gpr177/Wntless and studied mice that lacked Evi expression in the epidermis. We found that K14-Cre, Evi-LOF mice lost their hair during the first hair cycle, showing a reddish skin with impaired skin barrier function. Expression profiling of mutant and wild-type skin revealed up-regulation of inflammation-associated genes. Furthermore, we found that Evi expression in psoriatic skin biopsies is down-regulated, suggesting that Evi-deficient mice developed skin lesions that resemble human psoriasis. Immune cell infiltration was detected in Evi-LOF skin. Interestingly, an age-dependent depletion of dendritic epidermal T cells (DETCs) and an infiltration of γδlow T cells in Evi mutant epidermis was observed. Collectively, the described inflammatory skin phenotype in Evi-deficient mice revealed an essential role of Wnt secretion in maintaining normal skin homeostasis by enabling a balanced epidermal-dermal cross talk, which affects immune cell recruitment and DETC survival.
doi:10.1084/jem.20121871
PMCID: PMC3754868  PMID: 23918954
9.  A novel phenotypic dissimilarity method for image-based high-throughput screens 
BMC Bioinformatics  2013;14:336.
Background
Discovering functional relationships of genes through cell-based phenotyping has become an important approach in functional genomics. High-throughput imaging offers the ability to quantitatively assess complex phenotypes after perturbation by RNA interference (RNAi). Such image-based high-throughput RNAi screening studies have facilitated the discovery of novel components of gene networks and their interactions. Images generated by automated microscopy are typically analyzed by extracting quantitative features of individual cells, resulting in large multidimensional data sets. Robust and sensitive methods to interpret these data sets and to derive biologically relevant information in a high-throughput and unbiased manner remain to be developed.
Results
Here we propose a new analysis method, PhenoDissim, which computes the phenotypic dissimilarity between cell populations via Support Vector Machine classification and cross validation. Applying this method to a kinome RNAi screening data set, we demonstrate that the proposed method shows a good replicate reproducibility, separation of controls and clustering quality, and we are able to identify siRNA phenotypes and discover potential functional links between genes.
Conclusions
PhenoDissim is a novel analysis method for image-based high-throughput screen, relying on two parameters which can be automatically optimized without a priori knowledge. PhenoDissim is freely available as an R package.
doi:10.1186/1471-2105-14-336
PMCID: PMC4225524  PMID: 24256072
Phenotypic dissimilarity; Image-based high-throughput screening; High-content screening; RNAi; Gene networks
10.  Wnt secretion is required to maintain high levels of Wnt activity in colon cancer cells 
Nature Communications  2013;4:2610.
Aberrant regulation of the Wnt/β-catenin pathway has an important role during the onset and progression of colorectal cancer, with over 90% of cases of sporadic colon cancer featuring mutations in APC or β-catenin. However, it has remained a point of controversy whether these mutations are sufficient to activate the pathway or require additional upstream signals. Here we show that colorectal tumours express elevated levels of Wnt3 and Evi/Wls/GPR177. We found that in colon cancer cells, even in the presence of mutations in APC or β-catenin, downstream signalling remains responsive to Wnt ligands and receptor proximal signalling. Furthermore, we demonstrate that truncated APC proteins bind β-catenin and key components of the destruction complex. These results indicate that cells with mutations in APC or β-catenin depend on Wnt ligands and their secretion for a sufficient level of β-catenin signalling, which potentially opens new avenues for therapeutic interventions by targeting Wnt secretion via Evi/Wls.
Activating mutations in the Wnt signalling pathway are associated with colon cancer. Here the authors show that tumour cells carrying mutations in APC and β-catenin are still regulated by Wnt ligands, suggesting that Wnt secretion and receptor signalling remains important to control downstream signalling.
doi:10.1038/ncomms3610
PMCID: PMC3826636  PMID: 24162018
11.  Control of Pro-Inflammatory Gene Programs by Regulated Trimethylation and Demethylation of Histone H4K20 
Molecular cell  2012;48(1):28-38.
Summary
Regulation of genes that initiate and amplify inflammatory programs of gene expression is achieved by signal-dependent exchange of co-regulator complexes that function to read, write and erase specific histone modifications linked to transcriptional activation or repression. Here, we provide evidence for the role of trimethylated histone H4 lysine 20 (H4K20me3) as a repression checkpoint that restricts expression of toll like receptor 4 (TLR4) target genes in macrophages. H4K20me3 is deposited at the promoters of a subset of these genes by the SMYD5 histone methyltransferase through its association with NCoR corepressor complexes. Signal-dependent erasure of H4K20me3 is required for effective gene activation and is achieved by NF-κB-dependent delivery of the histone demethylase PHF2. Liver X receptors antagonize TLR4-dependent gene activation by maintaining NCoR/SMYD5-mediated repression. These findings reveal a histone H4K20 tri-methylation/de-methylation strategy that integrates positive and negative signaling inputs that control immunity and homeostasis.
doi:10.1016/j.molcel.2012.07.020
PMCID: PMC3472359  PMID: 22921934
12.  Robust RNAi enhancement via human Argonaute-2 overexpression from plasmids, viral vectors and cell lines 
Nucleic Acids Research  2013;41(21):e199.
As the only mammalian Argonaute protein capable of directly cleaving mRNAs in a small RNA-guided manner, Argonaute-2 (Ago2) is a keyplayer in RNA interference (RNAi) silencing via small interfering (si) or short hairpin (sh) RNAs. It is also a rate-limiting factor whose saturation by si/shRNAs limits RNAi efficiency and causes numerous adverse side effects. Here, we report a set of versatile tools and widely applicable strategies for transient or stable Ago2 co-expression, which overcome these concerns. Specifically, we engineered plasmids and viral vectors to co-encode a codon-optimized human Ago2 cDNA along with custom shRNAs. Furthermore, we stably integrated this Ago2 cDNA into a panel of standard human cell lines via plasmid transfection or lentiviral transduction. Using various endo- or exogenous targets, we demonstrate the potential of all three strategies to boost mRNA silencing efficiencies in cell culture by up to 10-fold, and to facilitate combinatorial knockdowns. Importantly, these robust improvements were reflected by augmented RNAi phenotypes and accompanied by reduced off-targeting effects. We moreover show that Ago2/shRNA-co-encoding vectors can enhance and prolong transgene silencing in livers of adult mice, while concurrently alleviating hepatotoxicity. Our customizable reagents and avenues should broadly improve future in vitro and in vivo RNAi experiments in mammalian systems.
doi:10.1093/nar/gkt836
PMCID: PMC3834839  PMID: 24049077
13.  E-TALEN: a web tool to design TALENs for genome engineering 
Nucleic Acids Research  2013;41(20):e190.
Use of transcription activator-like effector nucleases (TALENs) is a promising new technique in the field of targeted genome engineering, editing and reverse genetics. Its applications span from introducing knockout mutations to endogenous tagging of proteins and targeted excision repair. Owing to this wide range of possible applications, there is a need for fast and user-friendly TALEN design tools. We developed E-TALEN (http://www.e-talen.org), a web-based tool to design TALENs for experiments of varying scale. E-TALEN enables the design of TALENs against a single target or a large number of target genes. We significantly extended previously published design concepts to consider genomic context and different applications. E-TALEN guides the user through an end-to-end design process of de novo TALEN pairs, which are specific to a certain sequence or genomic locus. Furthermore, E-TALEN offers a functionality to predict targeting and specificity for existing TALENs. Owing to the computational complexity of many of the steps in the design of TALENs, particular emphasis has been put on the implementation of fast yet accurate algorithms. We implemented a user-friendly interface, from the input parameters to the presentation of results. An additional feature of E-TALEN is the in-built sequence and annotation database available for many organisms, including human, mouse, zebrafish, Drosophila and Arabidopsis, which can be extended in the future.
doi:10.1093/nar/gkt789
PMCID: PMC3814377  PMID: 24003033
14.  The microtubule affinity regulating kinase MARK4 promotes axoneme extension during early ciliogenesis 
The Journal of Cell Biology  2013;200(4):505-522.
A functional screen identified MARK4 as a positive regulator of axonemal extension and ciliogenesis via its interaction with the mother centriolar protein ODF2.
Despite the critical contributions of cilia to embryonic development and human health, key regulators of cilia formation await identification. In this paper, a functional RNA interference–based screen linked 30 novel protein kinases with ciliogenesis. Of them, we have studied the role of the microtubule (MT)-associated protein/MT affinity regulating kinase 4 (MARK4) in depth. MARK4 associated with the basal body and ciliary axoneme in human and murine cell lines. Ultrastructural and functional analyses established that MARK4 kinase activity was required for initiation of axoneme extension. We identified the mother centriolar protein ODF2 as an interaction partner of MARK4 and showed that ODF2 localization to the centriole partially depended on MARK4. Our data indicated that, upon MARK4 or ODF2 knockdown, the ciliary program arrested before the complete removal of the CP110–Cep97 inhibitory complex from the mother centriole, suggesting that these proteins act at this level of axonemal extension. We propose that MARK4 is a critical positive regulator of early steps in ciliogenesis.
doi:10.1083/jcb.201206013
PMCID: PMC3575539  PMID: 23400999
15.  GenomeRNAi: a database for cell-based and in vivo RNAi phenotypes, 2013 update 
Nucleic Acids Research  2012;41(Database issue):D1021-D1026.
RNA interference (RNAi) represents a powerful method to systematically study loss-of-function phenotypes on a large scale with a wide variety of biological assays, constituting a rich source for the assignment of gene function. The GenomeRNAi database (http://www.genomernai.org) makes available RNAi phenotype data extracted from the literature for human and Drosophila. It also provides RNAi reagent information, along with an assessment as to their efficiency and specificity. This manuscript describes an update of the database previously featured in the NAR Database Issue. The new version has undergone a complete re-design of the user interface, providing an intuitive, flexible framework for additional functionalities. Screen information and gene-reagent-phenotype associations are now available for download. The integration with other resources has been improved by allowing in-links via GenomeRNAi screen IDs, or external gene or reagent identifiers. A distributed annotation system (DAS) server enables the visualization of the phenotypes and reagents in the context of a genome browser. We have added a page listing ‘frequent hitters’, i.e. genes that show a phenotype in many screens, which might guide on-going RNAi studies. Structured annotation guidelines have been established to facilitate consistent curation, and a submission template for direct submission by data producers is available for download.
doi:10.1093/nar/gks1170
PMCID: PMC3531141  PMID: 23193271
16.  A Genome-Wide RNA Interference Screen Identifies Caspase 4 as a Factor Required for Tumor Necrosis Factor Alpha Signaling 
Molecular and Cellular Biology  2012;32(17):3372-3381.
Tumor necrosis factor alpha (TNF-α) is a potent inflammatory cytokine secreted upon cellular stress as well as immunological stimuli and is implicated in the pathology of inflammatory diseases and cancer. The therapeutic potential of modifying TNF-α pathway activity has been realized in several diseases, and antagonists of TNF-α have reached clinical applications. While much progress in the understanding of signaling downstream of the TNF-α receptor complex has been made, the compendium of factors required for signal transduction is still not complete. In order to find novel regulators of proinflammatory signaling induced by TNF-α, we conducted a genome-wide small interfering RNA screen in human cells. We identified several new candidate modulators of TNF-α signaling, which were confirmed in independent experiments. Specifically, we show that caspase 4 is required for the induction of NF-κB activity, while it appears to be dispensable for the activation of the Jun N-terminal protein kinase signaling branch. Taken together, our experiments identify caspase 4 as a novel regulator of TNF-α-induced NF-κB signaling that is required for the activation of IκB kinase. We further provide the genome-wide RNA interference data set as a compendium in a format compliant with minimum information about an interfering RNA experiment (MAIRE).
doi:10.1128/MCB.06739-11
PMCID: PMC3422000  PMID: 22733992
17.  Identification of Human Proteins That Modify Misfolding and Proteotoxicity of Pathogenic Ataxin-1 
PLoS Genetics  2012;8(8):e1002897.
Proteins with long, pathogenic polyglutamine (polyQ) sequences have an enhanced propensity to spontaneously misfold and self-assemble into insoluble protein aggregates. Here, we have identified 21 human proteins that influence polyQ-induced ataxin-1 misfolding and proteotoxicity in cell model systems. By analyzing the protein sequences of these modifiers, we discovered a recurrent presence of coiled-coil (CC) domains in ataxin-1 toxicity enhancers, while such domains were not present in suppressors. This suggests that CC domains contribute to the aggregation- and toxicity-promoting effects of modifiers in mammalian cells. We found that the ataxin-1–interacting protein MED15, computationally predicted to possess an N-terminal CC domain, enhances spontaneous ataxin-1 aggregation in cell-based assays, while no such effect was observed with the truncated protein MED15ΔCC, lacking such a domain. Studies with recombinant proteins confirmed these results and demonstrated that the N-terminal CC domain of MED15 (MED15CC) per se is sufficient to promote spontaneous ataxin-1 aggregation in vitro. Moreover, we observed that a hybrid Pum1 protein harboring the MED15CC domain promotes ataxin-1 aggregation in cell model systems. In strong contrast, wild-type Pum1 lacking a CC domain did not stimulate ataxin-1 polymerization. These results suggest that proteins with CC domains are potent enhancers of polyQ-mediated protein misfolding and aggregation in vitro and in vivo.
Author Summary
Spinocerebellar ataxias (SCAs) are a group of inherited neurodegenerative diseases with around 30 subtypes, which are characterized by a progressive loss of cerebellar neurons. Neuronal death has been linked to the aggregation of mutated disease-causing proteins, such as ataxin-1 (ATXN1). Pathogenic ATXN1 contains an elongated glutamine stretch, which triggers spontaneous misfolding and self-assembly of the protein into aggregates. Earlier studies in lower organisms have discovered many non-human proteins that alter aggregation and/or toxicity of mutant ATXN1. Here, we combine an experimental screening approach with bioinformatics to find human proteins that modulate aggregation and toxicity of ATXN1. We identified 21 proteins affecting mutant ATXN1 in mammalian cells. Further characterization revealed that enhancers of ATXN1-mediated toxicity contain α-helical coiled-coil domains as structural motifs, while suppressors do not. Detailed studies with the ATXN1 interacting proteins MED15 and Pum1 finally demonstrated that coiled-coil domains are indeed critical for the aggregation and toxicity promoting effects of human proteins. Our study contributes to a deeper understanding of ATXN1 aggregation and SCA1 pathogenesis and highlights potential therapeutic targets for further investigations.
doi:10.1371/journal.pgen.1002897
PMCID: PMC3420947  PMID: 22916034
18.  Transmembrane Protein 198 Promotes LRP6 Phosphorylation and Wnt Signaling Activation▿ 
Molecular and Cellular Biology  2011;31(13):2577-2590.
Wnt/β-catenin signaling is fundamental in embryogenesis and tissue homeostasis in metazoans. Upon Wnt stimulation, cognate coreceptors LRP5 and LRP6 ([LRP5/6] low-density lipoprotein receptor-related proteins 5 and 6) are activated via phosphorylation at key residues. Although several kinases have been implicated, the LRP5/6 activation mechanism remains unclear. Here, we report that transmembrane protein 198 (TMEM198), a previously uncharacterized seven-transmembrane protein, is able to specifically activate LRP6 in transducing Wnt signaling. TMEM198 associates with LRP6 and recruits casein kinase family proteins, via the cytoplasmic domain, to phosphorylate key residues important for LRP6 activation. In mammalian cells, TMEM198 is required for Wnt signaling and casein kinase 1-induced LRP6 phosphorylation. During Xenopus embryogenesis, maternal and zygotic tmem198 mRNAs are widely distributed in the ectoderm and mesoderm. TMEM198 is required for Wnt-mediated neural crest formation, antero-posterior patterning, and particularly engrailed-2 expression in Xenopus embryos. Thus, our results identified TMEM198 as a membrane scaffold protein that promotes LRP6 phosphorylation and Wnt signaling activation.
doi:10.1128/MCB.05103-11
PMCID: PMC3133378  PMID: 21536646
19.  The Wnt secretion protein Evi/Gpr177 promotes glioma tumourigenesis 
EMBO Molecular Medicine  2012;4(1):38-51.
Malignant astrocytomas are highly aggressive brain tumours with poor prognosis. While a number of structural genomic changes and dysregulation of signalling pathways in gliomas have been described, the identification of biomarkers and druggable targets remains an important task for novel diagnostic and therapeutic approaches. Here, we show that the Wnt-specific secretory protein Evi (also known as GPR177/Wntless/Sprinter) is overexpressed in astrocytic gliomas. Evi/Wls is a core Wnt signalling component and a specific regulator of pan-Wnt protein secretion, affecting both canonical and non-canonical signalling. We demonstrate that its depletion in glioma and glioma-derived stem-like cells led to decreased cell proliferation and apoptosis. Furthermore, Evi/Wls silencing in glioma cells reduced cell migration and the capacity to form tumours in vivo. We further show that Evi/Wls overexpression is sufficient to promote downstream Wnt signalling. Taken together, our study identifies Evi/Wls as an essential regulator of glioma tumourigenesis, identifying a pathway-specific protein trafficking factor as an oncogene and offering novel therapeutic options to interfere with the aberrant regulation of growth factors at the site of production.
doi:10.1002/emmm.201100186
PMCID: PMC3306557  PMID: 22147553
cancer research; glioma; RNAi; Wnt secretion; Wnt signalling
20.  A Novel Multiplex Cell Viability Assay for High-Throughput RNAi Screening 
PLoS ONE  2011;6(12):e28338.
Cell-based high-throughput RNAi screening has become a powerful research tool in addressing a variety of biological questions. In RNAi screening, one of the most commonly applied assay system is measuring the fitness of cells that is usually quantified using fluorescence, luminescence and absorption-based readouts. These methods, typically implemented and scaled to large-scale screening format, however often only yield limited information on the cell fitness phenotype due to evaluation of a single and indirect physiological indicator. To address this problem, we have established a cell fitness multiplexing assay which combines a biochemical approach and two fluorescence-based assaying methods. We applied this assay in a large-scale RNAi screening experiment with siRNA pools targeting the human kinome in different modified HEK293 cell lines. Subsequent analysis of ranked fitness phenotypes assessed by the different assaying methods revealed average phenotype intersections of 50.7±2.3%–58.7±14.4% when two indicators were combined and 40–48% when a third indicator was taken into account. From these observations we conclude that combination of multiple fitness measures may decrease false-positive rates and increases confidence for hit selection. Our robust experimental and analytical method improves the classical approach in terms of time, data comprehensiveness and cost.
doi:10.1371/journal.pone.0028338
PMCID: PMC3230607  PMID: 22162763
21.  p24 proteins are required for secretion of Wnt ligands 
EMBO Reports  2011;12(12):1265-1272.
p24 proteins are required for secretion of Wnt ligands
Wnt proteins are signalling molecules that follow a dedicated secretory pathway. The authors here identify novel components of this pathway, providing evidence that p24 proteins act as cargo receptors in an anterograde secretory route for Wnts.
During development and disease, the exocytosis of signalling molecules, such as Wnt ligands, is essential to orchestrate cellular programs in multicellular organisms. However, it remains a largely unresolved question whether signalling molecules follow specialized transport routes through the exocytic pathway. Here we identify several Drosophila p24 proteins that are required for Wnt signalling. We demonstrate that one of these p24 proteins, namely Opossum, shuttles in the early secretory pathway, and that the Drosophila Wnt proteins are retained in the absence of p24 proteins. Our results indicate that Wnt secretion relies on a specialized anterograde secretion route with p24 proteins functioning as conserved cargo receptors.
doi:10.1038/embor.2011.212
PMCID: PMC3245698  PMID: 22094269
Wnt secretion; p24 proteins; protein transporting; signalling
22.  Extracting quantitative genetic interaction phenotypes from matrix combinatorial RNAi 
BMC Bioinformatics  2011;12:342.
Background
Systematic measurement of genetic interactions by combinatorial RNAi (co-RNAi) is a powerful tool for mapping functional modules and discovering components. It also provides insights into the role of epistasis on the way from genotype to phenotype. The interpretation of co-RNAi data requires computational and statistical analysis in order to detect interactions reliably and sensitively.
Results
We present a comprehensive approach to the analysis of univariate phenotype measurements, such as cell growth. The method is based on a quantitative model and is demonstrated on two example Drosophila cell culture data sets. We discuss adjustments for technical variability, data quality assessment, model parameter fitting and fit diagnostics, choice of scale, and assessment of statistical significance.
Conclusions
As a result, we obtain quantitative genetic interactions and interaction networks reflecting known biological relationships between target genes. The reliable extraction of presence, absence, and strength of interactions provides insights into molecular mechanisms.
doi:10.1186/1471-2105-12-342
PMCID: PMC3230910  PMID: 21849035
23.  The head-regeneration transcriptome of the planarian Schmidtea mediterranea 
Genome Biology  2011;12(8):R76.
Background
Planarian flatworms can regenerate their head, including a functional brain, within less than a week. Despite the enormous potential of these animals for medical research and regenerative medicine, the mechanisms of regeneration and the molecules involved remain largely unknown.
Results
To identify genes that are differentially expressed during early stages of planarian head regeneration, we generated a de novo transcriptome assembly from more than 300 million paired-end reads from planarian fragments regenerating the head at 16 different time points. The assembly yielded 26,018 putative transcripts, including very long transcripts spanning multiple genomic supercontigs, and thousands of isoforms. Using short-read data from two platforms, we analyzed dynamic gene regulation during the first three days of head regeneration. We identified at least five different temporal synexpression classes, including genes specifically induced within a few hours after injury. Furthermore, we characterized the role of a conserved Runx transcription factor, smed-runt-like1. RNA interference (RNAi) knockdown and immunofluorescence analysis of the regenerating visual system indicated that smed-runt-like1 encodes a transcriptional regulator of eye morphology and photoreceptor patterning.
Conclusions
Transcriptome sequencing of short reads allowed for the simultaneous de novo assembly and differential expression analysis of transcripts, demonstrating highly dynamic regulation during head regeneration in planarians.
doi:10.1186/gb-2011-12-8-r76
PMCID: PMC3245616  PMID: 21846378
24.  Identification of ER Proteins Involved in the Functional Organisation of the Early Secretory Pathway in Drosophila Cells by a Targeted RNAi Screen 
PLoS ONE  2011;6(2):e17173.
Background
In Drosophila, the early secretory apparatus comprises discrete paired Golgi stacks in close proximity to exit sites from the endoplasmic reticulum (tER sites), thus forming tER-Golgi units. Although many components involved in secretion have been identified, the structural components sustaining its organisation are less known. Here we set out to identify novel ER resident proteins involved in the of tER-Golgi unit organisation.
Results
To do so, we designed a novel screening strategy combining a bioinformatics pre-selection with an RNAi screen. We first selected 156 proteins exhibiting known or related ER retention/retrieval signals from a list of proteins predicted to have a signal sequence. We then performed a microscopy-based primary and confirmation RNAi screen in Drosophila S2 cells directly scoring the organisation of the tER-Golgi units. We identified 49 hits, most of which leading to an increased number of smaller tER-Golgi units (MG for “more and smaller Golgi”) upon depletion. 16 of them were validated and characterised, showing that this phenotype was not due to an inhibition in secretion, a block in G2, or ER stress. Interestingly, the MG phenotype was often accompanied by an increase in the cell volume. Out of 6 proteins, 4 were localised to the ER.
Conclusions
This work has identified novel proteins involved in the organisation of the Drosophila early secretory pathway. It contributes to the effort of assigning protein functions to gene annotation in the secretory pathway, and analysis of the MG hits revealed an enrichment of ER proteins. These results suggest a link between ER localisation, aspects of cell metabolism and tER-Golgi structural organisation.
doi:10.1371/journal.pone.0017173
PMCID: PMC3044168  PMID: 21383842
25.  A Genome-Wide RNA Interference Screen Identifies a Differential Role of the Mediator CDK8 Module Subunits for GATA/ RUNX-Activated Transcription in Drosophila▿ §  
Molecular and Cellular Biology  2010;30(11):2837-2848.
Transcription factors of the RUNX and GATA families play key roles in the control of cell fate choice and differentiation, notably in the hematopoietic system. During Drosophila hematopoiesis, the RUNX factor Lozenge and the GATA factor Serpent cooperate to induce crystal cell differentiation. We used Serpent/Lozenge-activated transcription as a paradigm to identify modulators of GATA/RUNX activity by a genome-wide RNA interference screen in cultured Drosophila blood cells. Among the 129 factors identified, several belong to the Mediator complex. Mediator is organized in three modules plus a regulatory “CDK8 module,” composed of Med12, Med13, CycC, and Cdk8, which has long been thought to behave as a single functional entity. Interestingly, our data demonstrate that Med12 and Med13 but not CycC or Cdk8 are essential for Serpent/Lozenge-induced transactivation in cell culture. Furthermore, our in vivo analysis of crystal cell development show that, while the four CDK8 module subunits control the emergence and the proliferation of this lineage, only Med12 and Med13 regulate its differentiation. We thus propose that Med12/Med13 acts as a coactivator for Serpent/Lozenge during crystal cell differentiation independently of CycC/Cdk8. More generally, we suggest that the set of conserved factors identified herein may regulate GATA/RUNX activity in mammals.
doi:10.1128/MCB.01625-09
PMCID: PMC2876525  PMID: 20368357

Results 1-25 (43)