Publications by authors named "Ulrich Elling"

27 Publications

  • Page 1 of 1

A human tissue screen identifies a regulator of ER secretion as a brain-size determinant.

Science 2020 11 29;370(6519):935-941. Epub 2020 Oct 29.

Institute of Molecular Biotechnology of the Austrian Academy of Science (IMBA), Vienna BioCenter (VBC), Vienna, Austria.

Loss-of-function (LOF) screens provide a powerful approach to identify regulators in biological processes. Pioneered in laboratory animals, LOF screens of human genes are currently restricted to two-dimensional cell cultures, which hinders the testing of gene functions requiring tissue context. Here, we present CRISPR-lineage tracing at cellular resolution in heterogeneous tissue (CRISPR-LICHT), which enables parallel LOF studies in human cerebral organoid tissue. We used CRISPR-LICHT to test 173 microcephaly candidate genes, revealing 25 to be involved in known and uncharacterized microcephaly-associated pathways. We characterized , which regulates the endoplasmic reticulum (ER) function and extracellular matrix protein secretion crucial for tissue integrity, the dysregulation of which results in microcephaly. Our human tissue screening technology identifies microcephaly genes and mechanisms involved in brain-size control.
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http://dx.doi.org/10.1126/science.abb5390DOI Listing
November 2020

Multilayered VBC score predicts sgRNAs that efficiently generate loss-of-function alleles.

Nat Methods 2020 07 8;17(7):708-716. Epub 2020 Jun 8.

Institute of Molecular Biotechnology of the Austrian Academy of Science (IMBA), Vienna BioCenter (VBC), Vienna, Austria.

CRISPR-Cas9 screens have emerged as a transformative approach to systematically probe gene functions. The quality and success of these screens depends on the frequencies of loss-of-function alleles, particularly in negative-selection screens widely applied for probing essential genes. Using optimized screening workflows, we performed essentialome screens in cancer cell lines and embryonic stem cells and achieved dropout efficiencies that could not be explained by common frameshift frequencies. We find that these superior effect sizes are mainly determined by the impact of in-frame mutations on protein function, which can be predicted based on amino acid composition and conservation. We integrate protein features into a 'Bioscore' and fuse it with improved predictors of single-guide RNA activity and indel formation to establish a score that captures all relevant processes in CRISPR-Cas9 mutagenesis. This Vienna Bioactivity CRISPR score (www.vbc-score.org) outperforms previous prediction tools and enables the selection of sgRNAs that effectively produce loss-of-function alleles.
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http://dx.doi.org/10.1038/s41592-020-0850-8DOI Listing
July 2020

CRISPR-Switch regulates sgRNA activity by Cre recombination for sequential editing of two loci.

Nat Commun 2019 11 29;10(1):5454. Epub 2019 Nov 29.

Institute of Molecular Biotechnology of the Austrian Academy of Science (IMBA), Vienna Biocenter (VBC), Dr. Bohr Gasse 3, 1030, Vienna, Austria.

CRISPR-Cas9 is an efficient and versatile tool for genome engineering in many species. However, inducible CRISPR-Cas9 editing systems that regulate Cas9 activity or sgRNA expression often suffer from significant limitations, including reduced editing capacity, off-target effects, or leaky expression. Here, we develop a precisely controlled sgRNA expression cassette that can be combined with widely-used Cre systems, termed CRISPR-Switch (SgRNA With Induction/Termination by Cre Homologous recombination). Switch-ON facilitates controlled, rapid induction of sgRNA activity. In turn, Switch-OFF-mediated termination of editing improves generation of heterozygous genotypes and can limit off-target effects. Furthermore, we design sequential CRISPR-Switch-based editing of two loci in a strictly programmable manner and determined the order of mutagenic events that leads to development of glioblastoma in mice. Thus, CRISPR-Switch substantially increases the versatility of gene editing through precise and rapid switching ON or OFF sgRNA activity, as well as switching OVER to secondary sgRNAs.
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http://dx.doi.org/10.1038/s41467-019-13403-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6884486PMC
November 2019

Derivation and maintenance of mouse haploid embryonic stem cells.

Nat Protoc 2019 07 3;14(7):1991-2014. Epub 2019 Jun 3.

UK Dementia Research Institute at University of Cambridge and Department of Clinical Neurosciences, University of Cambridge, Cambridge, UK.

Ploidy represents the number of chromosome sets in a cell. Although gametes have a haploid genome (n), most mammalian cells have diploid genomes (2n). The diploid status of most cells correlates with the number of probable alleles for each autosomal gene and makes it difficult to target these genes via mutagenesis techniques. Here, we describe a 7-week protocol for the derivation of mouse haploid embryonic stem cells (hESCs) from female gametes that also outlines how to maintain the cells once derived. We detail additional procedures that can be used with cell lines obtained from the mouse Haplobank, a biobank of >100,000 individual mouse hESC lines with targeted mutations in 16,970 genes. hESCs can spontaneously diploidize and can be maintained in both haploid and diploid states. Mouse hESCs are genomically and karyotypically stable, are innately immortal and isogenic, and can be derived in an array of differentiated cell types; they are thus highly amenable to genetic screens and to defining molecular connectivity pathways.
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http://dx.doi.org/10.1038/s41596-019-0169-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6997032PMC
July 2019

Canonical PRC1 controls sequence-independent propagation of Polycomb-mediated gene silencing.

Nat Commun 2019 04 29;10(1):1931. Epub 2019 Apr 29.

Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna Biocenter (VBC), Dr. Bohr-Gasse 3, 1030, Vienna, Austria.

Polycomb group (PcG) proteins play critical roles in the epigenetic inheritance of cell fate. The Polycomb Repressive Complexes PRC1 and PRC2 catalyse distinct chromatin modifications to enforce gene silencing, but how transcriptional repression is propagated through mitotic cell divisions remains a key unresolved question. Using reversible tethering of PcG proteins to ectopic sites in mouse embryonic stem cells, here we show that PRC1 can trigger transcriptional repression and Polycomb-dependent chromatin modifications. We find that canonical PRC1 (cPRC1), but not variant PRC1, maintains gene silencing through cell division upon reversal of tethering. Propagation of gene repression is sustained by cis-acting histone modifications, PRC2-mediated H3K27me3 and cPRC1-mediated H2AK119ub1, promoting a sequence-independent feedback mechanism for PcG protein recruitment. Thus, the distinct PRC1 complexes present in vertebrates can differentially regulate epigenetic maintenance of gene silencing, potentially enabling dynamic heritable responses to complex stimuli. Our findings reveal how PcG repression is potentially inherited in vertebrates.
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http://dx.doi.org/10.1038/s41467-019-09628-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6488670PMC
April 2019

Using Functional Genetics in Haploid Cells for Drug Target Identification.

Methods Mol Biol 2019 ;1953:3-21

IMBA - Institute of Molecular Biotechnology of the Austrian Academy of Sciences, Vienna, Austria.

Pooled genetic screens are a powerful tool to identify targets for drug development as well as chemogenetic interactions. Various complementary methods for mutagenesis are available to generate highly complex cell populations, including mRNA knockdown, directed genome editing, as well as random genome mutagenesis. With the availability of a growing number of haploid mammalian cell lines, random mutagenesis is becoming increasingly powerful and represents an attractive alternative, e.g., to CRISPR-based screening. This chapter provides a step-by-step protocol for performing haploid gene trap screens.
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http://dx.doi.org/10.1007/978-1-4939-9145-7_1DOI Listing
July 2019

The novel lncRNA is pro-neurogenic and mutated in human neurodevelopmental disorders.

Elife 2019 01 10;8. Epub 2019 Jan 10.

Center for Personal Dynamic Regulomes, Stanford University, Stanford, United States.

Long noncoding RNAs (lncRNAs) have been shown to act as important cell biological regulators including cell fate decisions but are often ignored in human genetics. Combining differential lncRNA expression during neuronal lineage induction with copy number variation morbidity maps of a cohort of children with autism spectrum disorder/intellectual disability versus healthy controls revealed focal genomic mutations affecting several lncRNA candidate loci. Here we find that a t(5:12) chromosomal translocation in a family manifesting neurodevelopmental symptoms disrupts specifically . We further show that is an evolutionarily conserved lncRNA functionally enhances induced neuronal cell maturation and directly occupies and regulates transcription of neuronal genes including autism-associated genes. Thus, integrating human genetics and functional testing in neuronal lineage induction is a promising approach for discovering candidate lncRNAs involved in neurodevelopmental diseases.
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http://dx.doi.org/10.7554/eLife.41770DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6380841PMC
January 2019

Neutralizing Gatad2a-Chd4-Mbd3/NuRD Complex Facilitates Deterministic Induction of Naive Pluripotency.

Cell Stem Cell 2018 09 16;23(3):412-425.e10. Epub 2018 Aug 16.

Department of Molecular Genetics, Weizmann Institute of Science, 234 Herzl, Rehovot 76100, Israel. Electronic address:

Mbd3, a member of nucleosome remodeling and deacetylase (NuRD) co-repressor complex, was previously identified as an inhibitor for deterministic induced pluripotent stem cell (iPSC) reprogramming, where up to 100% of donor cells successfully complete the process. NuRD can assume multiple mutually exclusive conformations, and it remains unclear whether this deterministic phenotype can be attributed to a specific Mbd3/NuRD subcomplex. Moreover, since complete ablation of Mbd3 blocks somatic cell proliferation, we aimed to explore functionally relevant alternative ways to neutralize Mbd3-dependent NuRD activity. We identify Gatad2a, a NuRD-specific subunit, whose complete deletion specifically disrupts Mbd3/NuRD repressive activity on the pluripotency circuitry during iPSC differentiation and reprogramming without ablating somatic cell proliferation. Inhibition of Gatad2a facilitates deterministic murine iPSC reprogramming within 8 days. We validate a distinct molecular axis, Gatad2a-Chd4-Mbd3, within Mbd3/NuRD as being critical for blocking reestablishment of naive pluripotency and further highlight signaling-dependent and post-translational modifications of Mbd3/NuRD that influence its interactions and assembly.
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http://dx.doi.org/10.1016/j.stem.2018.07.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7116536PMC
September 2018

Unbiased compound-protein interface mapping and prediction of chemoresistance loci through forward genetics in haploid stem cells.

Oncotarget 2018 Feb 23;9(11):9838-9851. Epub 2018 Jan 23.

Max Planck Institute for Biology of Aging, Cologne D-50931, Germany.

Forward genetic screens in haploid mammalian cells have recently emerged as powerful tools for the discovery and investigation of recessive traits. Use of the haploid system provides unique genetic tractability and resolution. Upon positive selection, these screens typically employ analysis of loss-of-function (LOF) alleles and are thus limited to non-essential genes. Many relevant compounds, including anti-cancer therapeutics, however, target essential genes, precluding positive selection of LOF alleles. Here, we asked whether the use of random and saturating chemical mutagenesis might enable screens that identify essential biological targets of toxic compounds. We compare and contrast chemical mutagenesis with insertional mutagenesis. Selecting mutagenized cells with thapsigargin, an inhibitor of the essential Ca pump SERCA2, insertional mutagenesis retrieved cell clones overexpressing SERCA2. With chemical mutagenesis, we identify six single amino acid substitutions in the known SERCA2-thapsigargin binding interface that confer drug resistance. In a second screen, we used the anti-cancer drug MG132/bortezomib (Velcade), which inhibits proteasome activity. Using chemical mutagenesis, we found 7 point mutations in the essential subunit Psmb5 that map to the bortezomib binding surface. Importantly, 4 of these had previously been identified in human tumors with acquired bortezomib resistance. Insertional mutagenesis did not identify Psmb5 in this screen, demonstrating the unique ability of chemical mutagenesis to identify relevant point mutations in essential genes. Thus, chemical mutagenesis in haploid embryonic stem cells can define the interaction of toxic small molecules with essential proteins at amino acid resolution, fully mapping small molecule-protein binding interfaces.
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http://dx.doi.org/10.18632/oncotarget.24305DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5839405PMC
February 2018

CRISPR-UMI: single-cell lineage tracing of pooled CRISPR-Cas9 screens.

Nat Methods 2017 Dec 16;14(12):1191-1197. Epub 2017 Oct 16.

Institute of Molecular Biotechnology of the Austrian Academy of Science (IMBA), Vienna Biocenter (VBC), Vienna, Austria.

Pooled CRISPR screens are a powerful tool for assessments of gene function. However, conventional analysis is based exclusively on the relative abundance of integrated single guide RNAs (sgRNAs) between populations, which does not discern distinct phenotypes and editing outcomes generated by identical sgRNAs. Here we present CRISPR-UMI, a single-cell lineage-tracing methodology for pooled screening to account for cell heterogeneity. We generated complex sgRNA libraries with unique molecular identifiers (UMIs) that allowed for screening of clonally expanded, individually tagged cells. A proof-of-principle CRISPR-UMI negative-selection screen provided increased sensitivity and robustness compared with conventional analysis by accounting for underlying cellular and editing-outcome heterogeneity and detection of outlier clones. Furthermore, a CRISPR-UMI positive-selection screen uncovered new roadblocks in reprogramming mouse embryonic fibroblasts as pluripotent stem cells, distinguishing reprogramming frequency and speed (i.e., effect size and probability). CRISPR-UMI boosts the predictive power, sensitivity, and information content of pooled CRISPR screens.
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http://dx.doi.org/10.1038/nmeth.4466DOI Listing
December 2017

Comparative glycoproteomics of stem cells identifies new players in ricin toxicity.

Nature 2017 09 20;549(7673):538-542. Epub 2017 Sep 20.

IMBA, Institute of Molecular Biotechnology of the Austrian Academy of Sciences, Dr. Bohr Gasse 3, A-1030 Vienna, Austria.

Glycosylation, the covalent attachment of carbohydrate structures onto proteins, is the most abundant post-translational modification. Over 50% of human proteins are glycosylated, which alters their activities in diverse fundamental biological processes. Despite the importance of glycosylation in biology, the identification and functional validation of complex glycoproteins has remained largely unexplored. Here we develop a novel quantitative approach to identify intact glycopeptides from comparative proteomic data sets, allowing us not only to infer complex glycan structures but also to directly map them to sites within the associated proteins at the proteome scale. We apply this method to human and mouse embryonic stem cells to illuminate the stem cell glycoproteome. This analysis nearly doubles the number of experimentally confirmed glycoproteins, identifies previously unknown glycosylation sites and multiple glycosylated stemness factors, and uncovers evolutionarily conserved as well as species-specific glycoproteins in embryonic stem cells. The specificity of our method is confirmed using sister stem cells carrying repairable mutations in enzymes required for fucosylation, Fut9 and Slc35c1. Ablation of fucosylation confers resistance to the bioweapon ricin, and we discover proteins that carry a fucosylation-dependent sugar code for ricin toxicity. Mutations disrupting a subset of these proteins render cells ricin resistant, revealing new players that orchestrate ricin toxicity. Our comparative glycoproteomics platform, SugarQb, enables genome-wide insights into protein glycosylation and glycan modifications in complex biological systems.
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http://dx.doi.org/10.1038/nature24015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6003595PMC
September 2017

A reversible haploid mouse embryonic stem cell biobank resource for functional genomics.

Nature 2017 10 27;550(7674):114-118. Epub 2017 Sep 27.

Institute of Molecular Biotechnology of the Austrian Academy of Science (IMBA), Vienna Biocenter (VBC), Dr. Bohr Gasse 3, Vienna, Austria.

The ability to directly uncover the contributions of genes to a given phenotype is fundamental for biology research. However, ostensibly homogeneous cell populations exhibit large clonal variance that can confound analyses and undermine reproducibility. Here we used genome-saturated mutagenesis to create a biobank of over 100,000 individual haploid mouse embryonic stem (mES) cell lines targeting 16,970 genes with genetically barcoded, conditional and reversible mutations. This Haplobank is, to our knowledge, the largest resource of hemi/homozygous mutant mES cells to date and is available to all researchers. Reversible mutagenesis overcomes clonal variance by permitting functional annotation of the genome directly in sister cells. We use the Haplobank in reverse genetic screens to investigate the temporal resolution of essential genes in mES cells, and to identify novel genes that control sprouting angiogenesis and lineage specification of blood vessels. Furthermore, a genome-wide forward screen with Haplobank identified PLA2G16 as a host factor that is required for cytotoxicity by rhinoviruses, which cause the common cold. Therefore, clones from the Haplobank combined with the use of reversible technologies enable high-throughput, reproducible, functional annotation of the genome.
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http://dx.doi.org/10.1038/nature24027DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6235111PMC
October 2017

A vital sugar code for ricin toxicity.

Cell Res 2017 Nov 19;27(11):1351-1364. Epub 2017 Sep 19.

IMBA, Institute of Molecular Biotechnology of the Austrian Academy of Sciences, VBC - Vienna BioCenter Campus, Dr. Bohr-Gasse 3, 1030 Vienna, Austria.

Ricin is one of the most feared bioweapons in the world due to its extreme toxicity and easy access. Since no antidote exists, it is of paramount importance to identify the pathways underlying ricin toxicity. Here, we demonstrate that the Golgi GDP-fucose transporter Slc35c1 and fucosyltransferase Fut9 are key regulators of ricin toxicity. Genetic and pharmacological inhibition of fucosylation renders diverse cell types resistant to ricin via deregulated intracellular trafficking. Importantly, cells from a patient with SLC35C1 deficiency are also resistant to ricin. Mechanistically, we confirm that reduced fucosylation leads to increased sialylation of Lewis X structures and thus masking of ricin-binding sites. Inactivation of the sialyltransferase responsible for modifications of Lewis X (St3Gal4) increases the sensitivity of cells to ricin, whereas its overexpression renders cells more resistant to the toxin. Thus, we have provided unprecedented insights into an evolutionary conserved modular sugar code that can be manipulated to control ricin toxicity.
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http://dx.doi.org/10.1038/cr.2017.116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5674155PMC
November 2017

DUSP9 Modulates DNA Hypomethylation in Female Mouse Pluripotent Stem Cells.

Cell Stem Cell 2017 05 30;20(5):706-719.e7. Epub 2017 Mar 30.

Department of Molecular Biology, Cancer Center and Center for Regenerative Medicine, Massachusetts General Hospital, 185 Cambridge Street, Boston, MA 02114, USA; Harvard Stem Cell Institute, 1350 Massachusetts Avenue, Cambridge, MA 02138, USA; Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA 02138, USA. Electronic address:

Blastocyst-derived embryonic stem cells (ESCs) and gonad-derived embryonic germ cells (EGCs) represent two classic types of pluripotent cell lines, yet their molecular equivalence remains incompletely understood. Here, we compare genome-wide methylation patterns between isogenic ESC and EGC lines to define epigenetic similarities and differences. Surprisingly, we find that sex rather than cell type drives methylation patterns in ESCs and EGCs. Cell fusion experiments further reveal that the ratio of X chromosomes to autosomes dictates methylation levels, with female hybrids being hypomethylated and male hybrids being hypermethylated. We show that the X-linked MAPK phosphatase DUSP9 is upregulated in female compared to male ESCs, and its heterozygous loss in female ESCs leads to male-like methylation levels. However, male and female blastocysts are similarly hypomethylated, indicating that sex-specific methylation differences arise in culture. Collectively, our data demonstrate the epigenetic similarity of sex-matched ESCs and EGCs and identify DUSP9 as a regulator of female-specific hypomethylation.
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http://dx.doi.org/10.1016/j.stem.2017.03.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5524993PMC
May 2017

The mevalonate pathway regulates primitive streak formation via protein farnesylation.

Sci Rep 2016 11 24;6:37697. Epub 2016 Nov 24.

Department of Developmental and Regenerative Biology, Medical Research Institute, Tokyo Medical and Dental University (TMDU), 1-5-45 Yushima, Bunkyo-ku, Tokyo, Japan.

The primitive streak in peri-implantation embryos forms the mesoderm and endoderm and controls cell differentiation. The metabolic cues regulating primitive streak formation remain largely unknown. Here we utilised a mouse embryonic stem (ES) cell differentiation system and a library of well-characterised drugs to identify these metabolic factors. We found that statins, which inhibit the mevalonate metabolic pathway, suppressed primitive streak formation in vitro and in vivo. Using metabolomics and pharmacologic approaches we identified the downstream signalling pathway of mevalonate and revealed that primitive streak formation requires protein farnesylation but not cholesterol synthesis. A tagging-via-substrate approach revealed that nuclear lamin B1 and small G proteins were farnesylated in embryoid bodies and important for primitive streak gene expression. In conclusion, protein farnesylation driven by the mevalonate pathway is a metabolic cue essential for primitive streak formation.
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http://dx.doi.org/10.1038/srep37697DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5121603PMC
November 2016

Mutations in the murine homologue of TUBB5 cause microcephaly by perturbing cell cycle progression and inducing p53-associated apoptosis.

Development 2016 Apr 22;143(7):1126-33. Epub 2016 Feb 22.

Research Institute of Molecular Pathology (IMP), Vienna Biocenter (VBC), Dr. Bohr-Gasse 7, Vienna 1030, Austria

Microtubules play a crucial role in the generation, migration and differentiation of nascent neurons in the developing vertebrate brain. Mutations in the constituents of microtubules, the tubulins, are known to cause an array of neurological disorders, including lissencephaly, polymicrogyria and microcephaly. In this study we explore the genetic and cellular mechanisms that cause TUBB5-associated microcephaly by exploiting two new mouse models: a conditional E401K knock-in, and a conditional knockout animal. These mice present with profound microcephaly due to a loss of upper-layer neurons that correlates with massive apoptosis and upregulation of p53. This phenotype is associated with a delay in cell cycle progression and ectopic DNA elements in progenitors, which is dependent on the dosage of functional Tubb5. Strikingly, we report ectopic Sox2-positive progenitors and defects in spindle orientation in our knock-in mouse line, which are absent in knockout animals. This work sheds light on the functional repertoire of Tubb5, reveals that the E401K mutation acts by a complex mechanism, and demonstrates that the cellular pathology driving TUBB5-associated microcephaly is cell death.
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http://dx.doi.org/10.1242/dev.131516DOI Listing
April 2016

The histone chaperone CAF-1 safeguards somatic cell identity.

Nature 2015 Dec;528(7581):218-24

Department of Molecular Biology, Cancer Center and Center for Regenerative Medicine, Massachusetts General Hospital, Boston, Massachusetts 02114, USA.

Cellular differentiation involves profound remodelling of chromatic landscapes, yet the mechanisms by which somatic cell identity is subsequently maintained remain incompletely understood. To further elucidate regulatory pathways that safeguard the somatic state, we performed two comprehensive RNA interference (RNAi) screens targeting chromatin factors during transcription-factor-mediated reprogramming of mouse fibroblasts to induced pluripotent stem cells (iPS cells). Subunits of the chromatin assembly factor-1 (CAF-1) complex, including Chaf1a and Chaf1b, emerged as the most prominent hits from both screens, followed by modulators of lysine sumoylation and heterochromatin maintenance. Optimal modulation of both CAF-1 and transcription factor levels increased reprogramming efficiency by several orders of magnitude and facilitated iPS cell formation in as little as 4 days. Mechanistically, CAF-1 suppression led to a more accessible chromatin structure at enhancer elements early during reprogramming. These changes were accompanied by a decrease in somatic heterochromatin domains, increased binding of Sox2 to pluripotency-specific targets and activation of associated genes. Notably, suppression of CAF-1 also enhanced the direct conversion of B cells into macrophages and fibroblasts into neurons. Together, our findings reveal the histone chaperone CAF-1 to be a novel regulator of somatic cell identity during transcription-factor-induced cell-fate transitions and provide a potential strategy to modulate cellular plasticity in a regenerative setting.
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http://dx.doi.org/10.1038/nature15749DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4866648PMC
December 2015

Jagunal homolog 1 is a critical regulator of neutrophil function in fungal host defense.

Nat Genet 2014 Sep 17;46(9):1028-33. Epub 2014 Aug 17.

Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna, Austria.

Neutrophils are key innate immune effector cells that are essential to fighting bacterial and fungal pathogens. Here we report that mice carrying a hematopoietic lineage-specific deletion of Jagn1 (encoding Jagunal homolog 1) cannot mount an efficient neutrophil-dependent immune response to the human fungal pathogen Candida albicans. Global glycobiome analysis identified marked alterations in the glycosylation of proteins involved in cell adhesion and cytotoxicity in Jagn1-deficient neutrophils. Functional analysis confirmed marked defects in neutrophil migration in response to Candida albicans infection and impaired formation of cytotoxic granules, as well as defective myeloperoxidase release and killing of Candida albicans. Treatment with granulocyte/macrophage colony-stimulating factor (GM-CSF) protected mutant mice from increased weight loss and accelerated mortality after Candida albicans challenge. Notably, GM-CSF also restored the defective fungicidal activity of bone marrow cells from humans with JAGN1 mutations. These data directly identify Jagn1 (JAGN1 in humans) as a new regulator of neutrophil function in microbial pathogenesis and uncover a potential treatment option for humans.
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http://dx.doi.org/10.1038/ng.3070DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6245568PMC
September 2014

JAGN1 deficiency causes aberrant myeloid cell homeostasis and congenital neutropenia.

Nat Genet 2014 Sep 17;46(9):1021-7. Epub 2014 Aug 17.

Department of Pediatrics, Dr. von Hauner Children's Hospital, Ludwig Maximilians University, Munich, Germany.

The analysis of individuals with severe congenital neutropenia (SCN) may shed light on the delicate balance of factors controlling the differentiation, maintenance and decay of neutrophils. We identify 9 distinct homozygous mutations in the JAGN1 gene encoding Jagunal homolog 1 in 14 individuals with SCN. JAGN1-mutant granulocytes are characterized by ultrastructural defects, a paucity of granules, aberrant N-glycosylation of multiple proteins and increased incidence of apoptosis. JAGN1 participates in the secretory pathway and is required for granulocyte colony-stimulating factor receptor-mediated signaling. JAGN1 emerges as a factor that is necessary in the differentiation and survival of neutrophils.
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http://dx.doi.org/10.1038/ng.3069DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4829076PMC
September 2014

Genome wide functional genetics in haploid cells.

FEBS Lett 2014 Aug 17;588(15):2415-21. Epub 2014 Jun 17.

IMBA, Institute of Molecular Biotechnology of the Austrian Academy of Sciences, Dr. Bohr Gasse 3, 1030 Vienna, Austria. Electronic address:

Some organisms such as yeast or males of social insects are haploid, i.e. they carry a single set of chromosomes, while haploidy in mammals is exclusively restricted to mature germ cells. A single copy of the genome provides the basis for genetic analyses where any recessive mutation of essential genes will show a clear phenotype due to the absence of a second gene copy. Most prominently, haploidy in yeast has been utilized for recessive genetic screens that have markedly contributed to our understanding of development, basic physiology, and disease. Somatic mammalian cells carry two copies of chromosomes (diploidy) that obscure genetic analysis. Near haploid human leukemic cells however have been developed as a high throughput screening tool. Although deemed impossible, we and others have generated mammalian haploid embryonic stem cells from parthenogenetic mouse embryos. Haploid stem cells open the possibility of combining the power of a haploid genome with pluripotency of embryonic stem cells to uncover fundamental biological processes in defined cell types at a genomic scale. Haploid genetics has thus become a powerful alternative to RNAi or CRISPR based screens.
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http://dx.doi.org/10.1016/j.febslet.2014.06.032DOI Listing
August 2014

Seventy-five genetic loci influencing the human red blood cell.

Authors:
Pim van der Harst Weihua Zhang Irene Mateo Leach Augusto Rendon Niek Verweij Joban Sehmi Dirk S Paul Ulrich Elling Hooman Allayee Xinzhong Li Aparna Radhakrishnan Sian-Tsung Tan Katrin Voss Christian X Weichenberger Cornelis A Albers Abtehale Al-Hussani Folkert W Asselbergs Marina Ciullo Fabrice Danjou Christian Dina Tõnu Esko David M Evans Lude Franke Martin Gögele Jaana Hartiala Micha Hersch Hilma Holm Jouke-Jan Hottenga Stavroula Kanoni Marcus E Kleber Vasiliki Lagou Claudia Langenberg Lorna M Lopez Leo-Pekka Lyytikäinen Olle Melander Federico Murgia Ilja M Nolte Paul F O'Reilly Sandosh Padmanabhan Afshin Parsa Nicola Pirastu Eleonora Porcu Laura Portas Inga Prokopenko Janina S Ried So-Youn Shin Clara S Tang Alexander Teumer Michela Traglia Sheila Ulivi Harm-Jan Westra Jian Yang Jing Hua Zhao Franco Anni Abdel Abdellaoui Antony Attwood Beverley Balkau Stefania Bandinelli François Bastardot Beben Benyamin Bernhard O Boehm William O Cookson Debashish Das Paul I W de Bakker Rudolf A de Boer Eco J C de Geus Marleen H de Moor Maria Dimitriou Francisco S Domingues Angela Döring Gunnar Engström Gudmundur Ingi Eyjolfsson Luigi Ferrucci Krista Fischer Renzo Galanello Stephen F Garner Bernd Genser Quince D Gibson Giorgia Girotto Daniel Fannar Gudbjartsson Sarah E Harris Anna-Liisa Hartikainen Claire E Hastie Bo Hedblad Thomas Illig Jennifer Jolley Mika Kähönen Ido P Kema John P Kemp Liming Liang Heather Lloyd-Jones Ruth J F Loos Stuart Meacham Sarah E Medland Christa Meisinger Yasin Memari Evelin Mihailov Kathy Miller Miriam F Moffatt Matthias Nauck Maria Novatchkova Teresa Nutile Isleifur Olafsson Pall T Onundarson Debora Parracciani Brenda W Penninx Lucia Perseu Antonio Piga Giorgio Pistis Anneli Pouta Ursula Puc Olli Raitakari Susan M Ring Antonietta Robino Daniela Ruggiero Aimo Ruokonen Aude Saint-Pierre Cinzia Sala Andres Salumets Jennifer Sambrook Hein Schepers Carsten Oliver Schmidt Herman H W Silljé Rob Sladek Johannes H Smit John M Starr Jonathan Stephens Patrick Sulem Toshiko Tanaka Unnur Thorsteinsdottir Vinicius Tragante Wiek H van Gilst L Joost van Pelt Dirk J van Veldhuisen Uwe Völker John B Whitfield Gonneke Willemsen Bernhard R Winkelmann Gerald Wirnsberger Ale Algra Francesco Cucca Adamo Pio d'Adamo John Danesh Ian J Deary Anna F Dominiczak Paul Elliott Paolo Fortina Philippe Froguel Paolo Gasparini Andreas Greinacher Stanley L Hazen Marjo-Riitta Jarvelin Kay Tee Khaw Terho Lehtimäki Winfried Maerz Nicholas G Martin Andres Metspalu Braxton D Mitchell Grant W Montgomery Carmel Moore Gerjan Navis Mario Pirastu Peter P Pramstaller Ramiro Ramirez-Solis Eric Schadt James Scott Alan R Shuldiner George Davey Smith J Gustav Smith Harold Snieder Rossella Sorice Tim D Spector Kari Stefansson Michael Stumvoll W H Wilson Tang Daniela Toniolo Anke Tönjes Peter M Visscher Peter Vollenweider Nicholas J Wareham Bruce H R Wolffenbuttel Dorret I Boomsma Jacques S Beckmann George V Dedoussis Panos Deloukas Manuel A Ferreira Serena Sanna Manuela Uda Andrew A Hicks Josef Martin Penninger Christian Gieger Jaspal S Kooner Willem H Ouwehand Nicole Soranzo John C Chambers

Nature 2012 Dec 5;492(7429):369-75. Epub 2012 Dec 5.

Department of Cardiology, University of Groningen, University Medical Center Groningen, 9700 RB Groningen, The Netherlands.

Anaemia is a chief determinant of global ill health, contributing to cognitive impairment, growth retardation and impaired physical capacity. To understand further the genetic factors influencing red blood cells, we carried out a genome-wide association study of haemoglobin concentration and related parameters in up to 135,367 individuals. Here we identify 75 independent genetic loci associated with one or more red blood cell phenotypes at P < 10(-8), which together explain 4-9% of the phenotypic variance per trait. Using expression quantitative trait loci and bioinformatic strategies, we identify 121 candidate genes enriched in functions relevant to red blood cell biology. The candidate genes are expressed preferentially in red blood cell precursors, and 43 have haematopoietic phenotypes in Mus musculus or Drosophila melanogaster. Through open-chromatin and coding-variant analyses we identify potential causal genetic variants at 41 loci. Our findings provide extensive new insights into genetic mechanisms and biological pathways controlling red blood cell formation and function.
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http://dx.doi.org/10.1038/nature11677DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3623669PMC
December 2012

New gene functions in megakaryopoiesis and platelet formation.

Authors:
Christian Gieger Aparna Radhakrishnan Ana Cvejic Weihong Tang Eleonora Porcu Giorgio Pistis Jovana Serbanovic-Canic Ulrich Elling Alison H Goodall Yann Labrune Lorna M Lopez Reedik Mägi Stuart Meacham Yukinori Okada Nicola Pirastu Rossella Sorice Alexander Teumer Katrin Voss Weihua Zhang Ramiro Ramirez-Solis Joshua C Bis David Ellinghaus Martin Gögele Jouke-Jan Hottenga Claudia Langenberg Peter Kovacs Paul F O'Reilly So-Youn Shin Tõnu Esko Jaana Hartiala Stavroula Kanoni Federico Murgia Afshin Parsa Jonathan Stephens Pim van der Harst C Ellen van der Schoot Hooman Allayee Antony Attwood Beverley Balkau François Bastardot Saonli Basu Sebastian E Baumeister Ginevra Biino Lorenzo Bomba Amélie Bonnefond François Cambien John C Chambers Francesco Cucca Pio D'Adamo Gail Davies Rudolf A de Boer Eco J C de Geus Angela Döring Paul Elliott Jeanette Erdmann David M Evans Mario Falchi Wei Feng Aaron R Folsom Ian H Frazer Quince D Gibson Nicole L Glazer Chris Hammond Anna-Liisa Hartikainen Susan R Heckbert Christian Hengstenberg Micha Hersch Thomas Illig Ruth J F Loos Jennifer Jolley Kay Tee Khaw Brigitte Kühnel Marie-Christine Kyrtsonis Vasiliki Lagou Heather Lloyd-Jones Thomas Lumley Massimo Mangino Andrea Maschio Irene Mateo Leach Barbara McKnight Yasin Memari Braxton D Mitchell Grant W Montgomery Yusuke Nakamura Matthias Nauck Gerjan Navis Ute Nöthlings Ilja M Nolte David J Porteous Anneli Pouta Peter P Pramstaller Janne Pullat Susan M Ring Jerome I Rotter Daniela Ruggiero Aimo Ruokonen Cinzia Sala Nilesh J Samani Jennifer Sambrook David Schlessinger Stefan Schreiber Heribert Schunkert James Scott Nicholas L Smith Harold Snieder John M Starr Michael Stumvoll Atsushi Takahashi W H Wilson Tang Kent Taylor Albert Tenesa Swee Lay Thein Anke Tönjes Manuela Uda Sheila Ulivi Dirk J van Veldhuisen Peter M Visscher Uwe Völker H-Erich Wichmann Kerri L Wiggins Gonneke Willemsen Tsun-Po Yang Jing Hua Zhao Paavo Zitting John R Bradley George V Dedoussis Paolo Gasparini Stanley L Hazen Andres Metspalu Mario Pirastu Alan R Shuldiner L Joost van Pelt Jaap-Jan Zwaginga Dorret I Boomsma Ian J Deary Andre Franke Philippe Froguel Santhi K Ganesh Marjo-Riitta Jarvelin Nicholas G Martin Christa Meisinger Bruce M Psaty Timothy D Spector Nicholas J Wareham Jan-Willem N Akkerman Marina Ciullo Panos Deloukas Andreas Greinacher Steve Jupe Naoyuki Kamatani Jyoti Khadake Jaspal S Kooner Josef Penninger Inga Prokopenko Derek Stemple Daniela Toniolo Lorenz Wernisch Serena Sanna Andrew A Hicks Augusto Rendon Manuel A Ferreira Willem H Ouwehand Nicole Soranzo

Nature 2011 Nov 30;480(7376):201-8. Epub 2011 Nov 30.

Institute of Genetic Epidemiology, Helmholtz Zentrum München, German Research Center for Environmental Health, Ingolstädter Landstr 1, 85764 Neuherberg, Germany.

Platelets are the second most abundant cell type in blood and are essential for maintaining haemostasis. Their count and volume are tightly controlled within narrow physiological ranges, but there is only limited understanding of the molecular processes controlling both traits. Here we carried out a high-powered meta-analysis of genome-wide association studies (GWAS) in up to 66,867 individuals of European ancestry, followed by extensive biological and functional assessment. We identified 68 genomic loci reliably associated with platelet count and volume mapping to established and putative novel regulators of megakaryopoiesis and platelet formation. These genes show megakaryocyte-specific gene expression patterns and extensive network connectivity. Using gene silencing in Danio rerio and Drosophila melanogaster, we identified 11 of the genes as novel regulators of blood cell formation. Taken together, our findings advance understanding of novel gene functions controlling fate-determining events during megakaryopoiesis and platelet formation, providing a new example of successful translation of GWAS to function.
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http://dx.doi.org/10.1038/nature10659DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3335296PMC
November 2011

Forward and reverse genetics through derivation of haploid mouse embryonic stem cells.

Cell Stem Cell 2011 Dec;9(6):563-74

IMBA, Institute of Molecular Biotechnology of the Austrian Academy of Sciences, 1030 Vienna, Austria.

All somatic mammalian cells carry two copies of chromosomes (diploidy), whereas organisms with a single copy of their genome, such as yeast, provide a basis for recessive genetics. Here we report the generation of haploid mouse ESC lines from parthenogenetic embryos. These cells carry 20 chromosomes, express stem cell markers, and develop into all germ layers in vitro and in vivo. We also developed a reversible mutagenesis protocol that allows saturated genetic recessive screens and results in homozygous alleles. This system allowed us to generate a knockout cell line for the microRNA processing enzyme Drosha. In a forward genetic screen, we identified Gpr107 as a molecule essential for killing by ricin, a toxin being used as a bioweapon. Our results open the possibility of combining the power of a haploid genome with pluripotency of embryonic stem cells to uncover fundamental biological processes in defined cell types at a genomic scale.
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http://dx.doi.org/10.1016/j.stem.2011.10.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4008724PMC
December 2011

The stress kinase MKK7 couples oncogenic stress to p53 stability and tumor suppression.

Nat Genet 2011 Mar 13;43(3):212-9. Epub 2011 Feb 13.

Institute of Molecular Biotechnology of Austrian Academy of Sciences, Vienna, Austria.

Most preneoplastic lesions are quiescent and do not progress to form overt tumors. It has been proposed that oncogenic stress activates the DNA damage response and the key tumor suppressor p53, which prohibits tumor growth. However, the molecular pathways by which cells sense a premalignant state in vivo are largely unknown. Here we report that tissue-specific inactivation of the stress signaling kinase MKK7 in KRas(G12D)-driven lung carcinomas and NeuT-driven mammary tumors markedly accelerates tumor onset and reduces overall survival. Mechanistically, MKK7 acts through the kinases JNK1 and JNK2, and this signaling pathway directly couples oncogenic and genotoxic stress to the stability of p53, which is required for cell cycle arrest and suppression of epithelial cancers. These results show that MKK7 functions as a major tumor suppressor in lung and mammary cancer in mouse and identify MKK7 as a vital molecular sensor to set a cellular anti-cancer barrier.
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http://dx.doi.org/10.1038/ng.767DOI Listing
March 2011

A global in vivo Drosophila RNAi screen identifies NOT3 as a conserved regulator of heart function.

Cell 2010 Apr;141(1):142-53

Institute of Molecular Biotechnology of Austrian Academy of Sciences, Dr. Bohr Gasse 3-5, A-1030 Vienna, Austria.

Heart diseases are the most common causes of morbidity and death in humans. Using cardiac-specific RNAi-silencing in Drosophila, we knocked down 7061 evolutionarily conserved genes under conditions of stress. We present a first global roadmap of pathways potentially playing conserved roles in the cardiovascular system. One critical pathway identified was the CCR4-Not complex implicated in transcriptional and posttranscriptional regulatory mechanisms. Silencing of CCR4-Not components in adult Drosophila resulted in myofibrillar disarray and dilated cardiomyopathy. Heterozygous not3 knockout mice showed spontaneous impairment of cardiac contractility and increased susceptibility to heart failure. These heart defects were reversed via inhibition of HDACs, suggesting a mechanistic link to epigenetic chromatin remodeling. In humans, we show that a common NOT3 SNP correlates with altered cardiac QT intervals, a known cause of potentially lethal ventricular tachyarrhythmias. Thus, our functional genome-wide screen in Drosophila can identify candidates that directly translate into conserved mammalian genes involved in heart function.
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http://dx.doi.org/10.1016/j.cell.2010.02.023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2855221PMC
April 2010

Murine inner cell mass-derived lineages depend on Sall4 function.

Proc Natl Acad Sci U S A 2006 Oct 23;103(44):16319-24. Epub 2006 Oct 23.

Developmental Biology Unit, European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany.

Sall4 is a mammalian Spalt transcription factor expressed by cells of the early embryo and germ cells, an expression pattern similar to that of both Oct4 and Sox2, which play essential roles during early murine development. We show that the activity of Sall4 is cell-autonomously required for the development of the epiblast and primitive endoderm from the inner cell mass. Furthermore, no embryonic or extraembryonic endoderm stem cell lines could be established from Sall4-deficient blastocysts. In contrast, neither the development of the trophoblast lineage nor the ability to generate trophoblast cell lines from murine blastocysts was impaired in the absence of Sall4. These data establish Sall4 as an essential transcription factor required for the early development of inner cell mass-derived cell lineages.
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http://dx.doi.org/10.1073/pnas.0607884103DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1637580PMC
October 2006

Roles of the Homothorax/Meis/Prep homolog UNC-62 and the Exd/Pbx homologs CEH-20 and CEH-40 in C. elegans embryogenesis.

Development 2002 Nov;129(22):5255-68

Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, CO 80309-0347, USA.

Co-factor homeodomain proteins such as Drosophila Homothorax (Hth) and Extradenticle (Exd) and their respective vertebrate homologs, the Meis/Prep and Pbx proteins, can increase the DNA-binding specificity of Hox protein transcription factors and appear to be required for many of their developmental functions. We show that the unc-62 gene encodes the C. elegans ortholog of Hth, and that maternal-effect unc-62 mutations can cause severe posterior disorganization during embryogenesis (Nob phenotype), superficially similar to that seen in embryos lacking function of either the two posterior-group Hox genes nob-1 and php-3 or the caudal homolog pal-1. Other zygotically acting unc-62 alleles cause earlier embryonic arrest or incompletely penetrant larval lethality with variable morphogenetic defects among the survivors, suggesting that unc-62 functions are required at several stages of development. The differential accumulation of four unc-62 transcripts is consistent with multiple functions. The C. elegans exd homologs ceh-20 and ceh-40 interact genetically with unc-62 and may have overlapping roles in embryogenesis: neither CEH-20 nor CEH-40 appears to be required when the other is present, but loss of both functions causes incompletely penetrant embryonic lethality in the presence of unc-62(+) and complete embryonic lethality in the presence of an unc-62 hypomorphic allele.
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November 2002