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1.
Nat Protoc ; 14(7): 1991-2014, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31160788

RESUMO

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.

2.
Nat Commun ; 10(1): 1931, 2019 04 29.
Artigo em Inglês | MEDLINE | ID: mdl-31036804

RESUMO

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.


Assuntos
Cromatina/metabolismo , Epigênese Genética , Inativação Gênica , Complexo Repressor Polycomb 1/genética , Complexo Repressor Polycomb 2/genética , Processamento de Proteína Pós-Traducional , Animais , Linhagem Celular , Cromatina/química , Retroalimentação Fisiológica , Histonas/genética , Histonas/metabolismo , Padrões de Herança , Camundongos , Mitose , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , Complexo Repressor Polycomb 1/metabolismo , Complexo Repressor Polycomb 2/metabolismo , Transcrição Genética
3.
Methods Mol Biol ; 1953: 3-21, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30912012

RESUMO

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.


Assuntos
Descoberta de Drogas/métodos , Avaliação Pré-Clínica de Medicamentos/métodos , Mutagênese , Células-Tronco/metabolismo , Animais , Técnicas de Cultura de Células/métodos , Linhagem Celular , Citometria de Fluxo/métodos , Testes Genéticos , Haploidia , Humanos , Camundongos , Reação em Cadeia da Polimerase/métodos , Células-Tronco/efeitos dos fármacos
4.
Elife ; 82019 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-30628890

RESUMO

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 lnc-NR2F1. We further show that lnc-NR2F1 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.

5.
Cell Stem Cell ; 23(3): 412-425.e10, 2018 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-30122475

RESUMO

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.

6.
Oncotarget ; 9(11): 9838-9851, 2018 Feb 09.
Artigo em Inglês | MEDLINE | ID: mdl-29515774

RESUMO

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 Ca2+ 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.

7.
Nat Methods ; 14(12): 1191-1197, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-29039415

RESUMO

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.


Assuntos
Sistemas CRISPR-Cas/genética , Linhagem da Célula/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Edição de Genes , RNA Guia , Análise de Célula Única/métodos , Animais , Células Cultivadas , Fibroblastos/citologia , Técnicas de Inativação de Genes , Vetores Genéticos , Camundongos , Células-Tronco Pluripotentes/citologia , Retroviridae/genética , Razão Sinal-Ruído
8.
Cell Res ; 27(11): 1351-1364, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-28925387

RESUMO

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.


Assuntos
Fucosiltransferases/genética , Proteínas de Membrana Transportadoras/genética , Ricina/toxicidade , Animais , Deleção de Genes , Complexo de Golgi/metabolismo , Humanos , Camundongos , Proteínas de Transporte de Monossacarídeos/genética , Proteínas de Transporte de Monossacarídeos/fisiologia , Mutação , Ricina/metabolismo , Sialiltransferases/genética
9.
Nature ; 550(7674): 114-118, 2017 10 05.
Artigo em Inglês | MEDLINE | ID: mdl-28953874

RESUMO

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.


Assuntos
Bancos de Espécimes Biológicos , Genômica/métodos , Haploidia , Células-Tronco Embrionárias Murinas/metabolismo , Mutação , Animais , Vasos Sanguíneos/citologia , Linhagem da Célula/genética , Resfriado Comum/genética , Resfriado Comum/virologia , Genes Essenciais/genética , Testes Genéticos , Células HEK293 , Homozigoto , Humanos , Camundongos , Células-Tronco Embrionárias Murinas/citologia , Neovascularização Fisiológica/genética , Fosfolipases A2 Independentes de Cálcio/genética , Fosfolipases A2 Independentes de Cálcio/metabolismo , Rhinovirus/patogenicidade
10.
Nature ; 549(7673): 538-542, 2017 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-28959962

RESUMO

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.


Assuntos
Células-Tronco Embrionárias/química , Células-Tronco Embrionárias/efeitos dos fármacos , Glicopeptídeos/análise , Glicoproteínas/análise , Proteoma/análise , Proteômica , Ricina/toxicidade , Animais , Células-Tronco Embrionárias/enzimologia , Células-Tronco Embrionárias/metabolismo , Fucosiltransferases/genética , Glicopeptídeos/química , Glicopeptídeos/metabolismo , Glicoproteínas/química , Glicoproteínas/metabolismo , Glicosilação , Humanos , Proteínas de Membrana Transportadoras/genética , Camundongos , Processamento de Proteína Pós-Traducional/genética , Proteoma/química , Proteoma/genética , Proteoma/metabolismo
11.
Cell Stem Cell ; 20(5): 706-719.e7, 2017 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-28366588

RESUMO

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.


Assuntos
Fosfatases de Especificidade Dupla/metabolismo , Células-Tronco Pluripotentes/metabolismo , Cromossomo X/metabolismo , Animais , Blastocisto/citologia , Blastocisto/metabolismo , Metilação de DNA/genética , Metilação de DNA/fisiologia , Fosfatases de Especificidade Dupla/genética , Células Germinativas Embrionárias/efeitos dos fármacos , Células Germinativas Embrionárias/metabolismo , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Feminino , Impressão Genômica/genética , Masculino , Camundongos , Modelos Biológicos , Células-Tronco Pluripotentes/citologia
12.
Sci Rep ; 6: 37697, 2016 11 24.
Artigo em Inglês | MEDLINE | ID: mdl-27883036

RESUMO

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.


Assuntos
Redes e Vias Metabólicas , Ácido Mevalônico/metabolismo , Linha Primitiva/embriologia , Linha Primitiva/metabolismo , Prenilação de Proteína , Animais , Diferenciação Celular , Regulação para Baixo/genética , Corpos Embrioides , Regulação da Expressão Gênica no Desenvolvimento , Metaboloma , Metabolômica , Camundongos Endogâmicos ICR , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Neurogênese , Análise de Sequência com Séries de Oligonucleotídeos , Organogênese , Peixe-Zebra
13.
Development ; 143(7): 1126-33, 2016 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-26903504

RESUMO

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.


Assuntos
Apoptose/genética , Ciclo Celular/genética , Microcefalia/genética , Microtúbulos/genética , Tubulina (Proteína)/genética , Proteína Supressora de Tumor p53/metabolismo , Animais , Encéfalo/anormalidades , Encéfalo/embriologia , Diferenciação Celular , Modelos Animais de Doenças , Embrião de Mamíferos/embriologia , Técnicas de Introdução de Genes , Imagem por Ressonância Magnética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microtúbulos/metabolismo , Células-Tronco Neurais/citologia , Fatores de Transcrição SOXB1/metabolismo , Fuso Acromático/genética , Células-Tronco/citologia , Proteína Supressora de Tumor p53/biossíntese
14.
Nature ; 528(7581): 218-24, 2015 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-26659182

RESUMO

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.


Assuntos
Reprogramação Celular/genética , Fator 1 de Modelagem da Cromatina/metabolismo , Animais , Células Cultivadas , Cromatina/metabolismo , Fator 1 de Modelagem da Cromatina/antagonistas & inibidores , Fator 1 de Modelagem da Cromatina/genética , Regulação da Expressão Gênica/genética , Heterocromatina/metabolismo , Camundongos , Nucleossomos/metabolismo , Interferência de RNA , Transdução Genética
15.
Nat Genet ; 46(9): 1021-7, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25129144

RESUMO

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.


Assuntos
Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Células Mieloides/metabolismo , Neutropenia/congênito , Adolescente , Adulto , Apoptose/genética , Diferenciação Celular/genética , Sobrevivência Celular/genética , Criança , Pré-Escolar , Feminino , Glicosilação , Homeostase/genética , Humanos , Lactente , Recém-Nascido , Masculino , Proteínas de Membrana/metabolismo , Mutação , Neutropenia/genética , Neutropenia/metabolismo , Neutropenia/patologia , Neutrófilos/metabolismo , Receptores de Fator Estimulador de Colônias de Granulócitos/genética , Receptores de Fator Estimulador de Colônias de Granulócitos/metabolismo , Transdução de Sinais , Adulto Jovem
16.
Nat Genet ; 46(9): 1028-33, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25129145

RESUMO

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.


Assuntos
Candidíase/imunologia , Proteínas de Membrana/imunologia , Neutrófilos/imunologia , Animais , Células da Medula Óssea/imunologia , Células da Medula Óssea/metabolismo , Células da Medula Óssea/microbiologia , Candida albicans , Candidíase/tratamento farmacológico , Candidíase/metabolismo , Candidíase/microbiologia , Glicosilação , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Humanos , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Neutrófilos/microbiologia
17.
FEBS Lett ; 588(15): 2415-21, 2014 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-24950427

RESUMO

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.


Assuntos
Técnicas Genéticas , Genoma , Haploidia , Animais , Camundongos
18.
Nature ; 492(7429): 369-75, 2012 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-23222517

RESUMO

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.


Assuntos
Eritrócitos/metabolismo , Loci Gênicos , Estudo de Associação Genômica Ampla , Fenótipo , Animais , Ciclo Celular/genética , Citocinas/metabolismo , Drosophila melanogaster/genética , Eritrócitos/citologia , Feminino , Regulação da Expressão Gênica/genética , Hematopoese/genética , Hemoglobinas/genética , Humanos , Masculino , Camundongos , Especificidade de Órgãos , Polimorfismo de Nucleotídeo Único/genética , Interferência de RNA , Transdução de Sinais/genética
19.
Nature ; 480(7376): 201-8, 2011 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-22139419

RESUMO

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.


Assuntos
Plaquetas/citologia , Hematopoese/genética , Megacariócitos/citologia , Animais , Plaquetas/metabolismo , Tamanho Celular , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Europa (Continente) , Perfilação da Expressão Gênica , Inativação Gênica , Genoma Humano/genética , Estudo de Associação Genômica Ampla , Humanos , Megacariócitos/metabolismo , Contagem de Plaquetas , Mapas de Interação de Proteínas , Transcrição Genética/genética , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética
20.
Cell Stem Cell ; 9(6): 563-74, 2011 Dec 02.
Artigo em Inglês | MEDLINE | ID: mdl-22136931

RESUMO

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.


Assuntos
Células-Tronco Embrionárias/fisiologia , Haploidia , Genética Reversa/métodos , Animais , Biomarcadores/metabolismo , Diferenciação Celular/genética , Linhagem Celular , Embrião de Mamíferos/citologia , Embrião de Mamíferos/fisiologia , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/efeitos dos fármacos , Estudo de Associação Genômica Ampla , Camundongos , Camundongos Endogâmicos C57BL , Partenogênese/genética , Ricina/toxicidade
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