RESUMO
Chromosome-scale genome assemblies based on ultralong-read sequencing technologies are able to illuminate previously intractable aspects of genome biology such as fine-scale centromere structure and large-scale variation in genome features such as heterochromatin, GC content, recombination rate, and gene content. We present here a new chromosome-scale genome of the Mongolian gerbil (Meriones unguiculatus), which includes the complete sequence of all centromeres. Gerbils are thus the one of the first vertebrates to have their centromeres completely sequenced. Gerbil centromeres are composed of four different repeats of length 6, 37, 127, or 1,747â bp, which occur in simple alternating arrays and span 1-6â Mb. Gerbil genomes have both an extensive set of GC-rich genes and chromosomes strikingly enriched for constitutive heterochromatin. We sought to determine if there was a link between these two phenomena and found that the two heterochromatic chromosomes of the Mongolian gerbil have distinct underpinnings: Chromosome 5 has a large block of intraarm heterochromatin as the result of a massive expansion of centromeric repeats, while chromosome 13 is comprised of extremely large (>150â kb) repeated sequences. In addition to characterizing centromeres, our results demonstrate the importance of including karyotypic features such as chromosome number and the locations of centromeres in the interpretation of genome sequence data and highlight novel patterns involved in the evolution of chromosomes.
Assuntos
Centrômero , Heterocromatina , Animais , Gerbillinae/genética , Heterocromatina/genética , Centrômero/genética , Genoma , Sequências Repetitivas de Ácido NucleicoRESUMO
DNA comprises molecular information stored in genetic and epigenetic bases, both of which are vital to our understanding of biology. Most DNA sequencing approaches address either genetics or epigenetics and thus capture incomplete information. Methods widely used to detect epigenetic DNA bases fail to capture common C-to-T mutations or distinguish 5-methylcytosine from 5-hydroxymethylcytosine. We present a single base-resolution sequencing methodology that sequences complete genetics and the two most common cytosine modifications in a single workflow. DNA is copied and bases are enzymatically converted. Coupled decoding of bases across the original and copy strand provides a phased digital readout. Methods are demonstrated on human genomic DNA and cell-free DNA from a blood sample of a patient with cancer. The approach is accurate, requires low DNA input and has a simple workflow and analysis pipeline. Simultaneous, phased reading of genetic and epigenetic bases provides a more complete picture of the information stored in genomes and has applications throughout biomedicine.
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BACKGROUND: HAP1, a near-haploid human leukemic cancer cell line is often used in combination with CRISPR-Cas9 gene editing technology for genetic screens. HAP1 carries the Philadelphia chromosome (Ph) and an additional ~ 30 Mb fragment of chromosome 15 inserted into chromosome 19. The potential use of an in vitro cell line as a model system in biomedical research studies depends on its ability to maintain genome stability. Being a cancer cell line with a near-haploid genome, HAP1 is prone to genetic instability, which is further compounded by its tendency to diploidise in culture spontaneously. Moreover, CRISPR-Cas9 gene editing coupled with prolonged in-vitro cell culturing has the potential to induce unintended 'off-target' cytogenetic mutations. To gain an insight into chromosomal instability (CIN) and karyotype heterogeneity, 19 HAP1 cell lines were cytogenetically characterised, 17 of which were near-haploids and two double-haploids, using multiplex fluorescence in situ hybridisation (M-FISH), at single cell resolution. We focused on novel numerical (N) and structural (S) CIN and discussed the potential causal factors for the observed instability. For each cell line we examined its ploidy, gene editing status and its length of in-vitro cell culturing. RESULTS: Sixteen of the 19 cell lines had been gene edited with passage numbers ranging from 10 to 35. Diploidisation in 17 near-haploid cell lines ranged from 4 to 35% and percentage of N- and S-CIN in [1n] and [2n] metaphases ranged from 7 to 50% with two cell lines showing no CIN. Percentage of cells with CIN in the two double-haploid cell lines were 96% and 100% respectively. The most common S-CIN observed was deletion followed by translocation of both types, non-reciprocal and Robertsonian. Interestingly, we observed a prevalence of S-CIN associated with chromosome 13 in both near-and double-haploid cell lines, with a high incidence of Robertsonian translocation involving chromosome 13. Furthermore, locus-specific BAC (bacterial artificial chromosome) FISH enabled us to show for the first time that the additional chromosome 15 fragment is inserted into the p-arm rather than the q-arm of chromosome 19 of the HAP1 genome. CONCLUSION: Our study revealed a high incidence of CIN leading to karyotype heterogeneity in majority of the HAP1 cell lines with the number of chromosomal aberrations varying between cell lines. A noteworthy observation was the high frequency of structural chromosomal aberrations associated with chromosome 13. We showed that CRISPR-Cas9 gene editing technology in combination with spontaneous diploidisation and prolonged in-vitro cell culturing is potentially instrumental in inducing further chromosomal rearrangements in the HAP1 cell lines with existing CIN. We highlight the importance of maintaining cell lines at low passage and the need for regular monitoring to prevent implications in downstream applications. Our study also established that the additional fragment of chromosome 15 in the HAP1 genome is inserted into chromosome 19p rather than 19q.
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Mutations in the SETX gene, which encodes Senataxin, are associated with the progressive neurodegenerative diseases ataxia with oculomotor apraxia 2 (AOA2) and amyotrophic lateral sclerosis 4 (ALS4). To identify the causal defect in AOA2, patient-derived cells and SETX knockouts (human and mouse) were analyzed using integrated genomic and transcriptomic approaches. A genome-wide increase in chromosome instability (gains and losses) within genes and at chromosome fragile sites was observed, resulting in changes to gene-expression profiles. Transcription stress near promoters correlated with high GCskew and the accumulation of R-loops at promoter-proximal regions, which localized with chromosomal regions where gains and losses were observed. In the absence of Senataxin, the Cockayne syndrome protein CSB was required for the recruitment of the transcription-coupled repair endonucleases (XPG and XPF) and RAD52 recombination protein to target and resolve transcription bubbles containing R-loops, leading to genomic instability. These results show that transcription stress is an important contributor to SETX mutation-associated chromosome fragility and AOA2.
Assuntos
Instabilidade Cromossômica/genética , DNA Helicases/metabolismo , Enzimas Multifuncionais/metabolismo , RNA Helicases/metabolismo , Ataxias Espinocerebelares/congênito , Animais , Apraxias/genética , Ataxia/genética , Linhagem Celular , Ataxia Cerebelar/genética , DNA Helicases/genética , Reparo do DNA/genética , Perfilação da Expressão Gênica/métodos , Instabilidade Genômica/genética , Genômica/métodos , Humanos , Camundongos , Células-Tronco Embrionárias Murinas , Enzimas Multifuncionais/genética , Mutação/genética , Doenças Neurodegenerativas/genética , Cultura Primária de Células , Regiões Promotoras Genéticas/genética , RNA Helicases/genética , Ataxias Espinocerebelares/genética , Ataxias Espinocerebelares/fisiopatologia , Transcriptoma/genéticaRESUMO
Duchenne muscular dystrophy (DMD) is caused by dystrophin gene mutations leading to skeletal muscle weakness and wasting. Dystrophin is enriched at the neuromuscular junction (NMJ), but how NMJ abnormalities contribute to DMD pathogenesis remains unclear. Here, we combine transcriptome analysis and modeling of DMD patient-derived neuromuscular circuits with CRISPR-corrected isogenic controls in compartmentalized microdevices. We show that NMJ volumes and optogenetic motor neuronstimulated myofiber contraction are compromised in DMD neuromuscular circuits, which can be rescued by pharmacological inhibition of TGFß signaling, an observation validated in a 96-well human neuromuscular circuit coculture assay. These beneficial effects are associated with normalization of dysregulated gene expression in DMD myogenic transcriptomes affecting NMJ assembly (e.g., MUSK) and axon guidance (e.g., SLIT2 and SLIT3). Our study provides a new human microphysiological model for investigating NMJ defects in DMD and assessing candidate drugs and suggests that enhancing neuromuscular connectivity may be an effective therapeutic strategy.
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Genetic redundancy has evolved as a way for human cells to survive the loss of genes that are single copy and essential in other organisms, but also allows tumours to survive despite having highly rearranged genomes. In this study we CRISPR screen 1191 gene pairs, including paralogues and known and predicted synthetic lethal interactions to identify 105 gene combinations whose co-disruption results in a loss of cellular fitness. 27 pairs influence fitness across multiple cell lines including the paralogues FAM50A/FAM50B, two genes of unknown function. Silencing of FAM50B occurs across a range of tumour types and in this context disruption of FAM50A reduces cellular fitness whilst promoting micronucleus formation and extensive perturbation of transcriptional programmes. Our studies reveal the fitness effects of FAM50A/FAM50B in cancer cells.
Assuntos
Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Genoma , Proteínas/genética , Animais , Apoptose , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/genética , Técnicas de Inativação de Genes , Xenoenxertos , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Proteínas de Ligação a RNA/genética , TranscriptomaRESUMO
Structural variants contribute substantially to genetic diversity and are important evolutionarily and medically, but they are still understudied. Here we present a comprehensive analysis of structural variation in the Human Genome Diversity panel, a high-coverage dataset of 911 samples from 54 diverse worldwide populations. We identify, in total, 126,018 variants, 78% of which were not identified in previous global sequencing projects. Some reach high frequency and are private to continental groups or even individual populations, including regionally restricted runaway duplications and putatively introgressed variants from archaic hominins. By de novo assembly of 25 genomes using linked-read sequencing, we discover 1,643 breakpoint-resolved unique insertions, in aggregate accounting for 1.9 Mb of sequence absent from the GRCh38 reference. Our results illustrate the limitation of a single human reference and the need for high-quality genomes from diverse populations to fully discover and understand human genetic variation.
Assuntos
Genética Populacional , Variação Estrutural do Genoma , Alelos , Bases de Dados Genéticas , Dosagem de Genes , Duplicação Gênica , Frequência do Gene/genética , Variação Genética , Genoma Humano , HumanosRESUMO
Dystroglycan, an extracellular matrix receptor, has essential functions in various tissues. Loss of α-dystroglycan-laminin interaction due to defective glycosylation of α-dystroglycan underlies a group of congenital muscular dystrophies often associated with brain malformations, referred to as dystroglycanopathies. The lack of isogenic human dystroglycanopathy cell models has limited our ability to test potential drugs in a human- and neural-specific context. Here, we generated induced pluripotent stem cells (iPSCs) from a severe dystroglycanopathy patient with homozygous FKRP (fukutin-related protein gene) mutation. We showed that CRISPR/Cas9-mediated gene correction of FKRP restored glycosylation of α-dystroglycan in iPSC-derived cortical neurons, whereas targeted gene mutation of FKRP in wild-type cells disrupted this glycosylation. In parallel, we screened 31,954 small molecule compounds using a mouse myoblast line for increased glycosylation of α-dystroglycan. Using human FKRP-iPSC-derived neural cells for hit validation, we demonstrated that compound 4-(4-bromophenyl)-6-ethylsulfanyl-2-oxo-3,4-dihydro-1H-pyridine-5-carbonitrile (4BPPNit) significantly augmented glycosylation of α-dystroglycan, in part through upregulation of LARGE1 glycosyltransferase gene expression. Together, isogenic human iPSC-derived cells represent a valuable platform for facilitating dystroglycanopathy drug discovery and therapeutic development.
Assuntos
Avaliação Pré-Clínica de Medicamentos , Distroglicanas/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Sequência de Bases , Sistemas CRISPR-Cas , Células Cultivadas , Avaliação Pré-Clínica de Medicamentos/métodos , Distroglicanas/genética , Edição de Genes , Marcação de Genes , Loci Gênicos , Glicosilação/efeitos dos fármacos , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Imagem Molecular , Distrofias Musculares/tratamento farmacológico , Distrofias Musculares/etiologia , Distrofias Musculares/metabolismo , Mutação , N-Acetilglucosaminiltransferases/genética , N-Acetilglucosaminiltransferases/metabolismo , Células-Tronco Neurais/metabolismo , Neurônios/metabolismo , Pentosiltransferases/genética , Pentosiltransferases/metabolismoRESUMO
Pluripotent stem cell (PSC) differentiation in vitro represents a powerful and tractable model to study mammalian development and an unlimited source of cells for regenerative medicine. Within hematology, in vitro PSC hematopoiesis affords novel insights into blood formation and represents an exciting potential approach to generate hematopoietic and immune cell types for transplantation and transfusion. Most studies to date have focused on in vitro hematopoiesis from mouse PSCs and human PSCs. However, differences in mouse and human PSC culture protocols have complicated the translation of discoveries between these systems. We recently developed a novel chemical media formulation, expanded potential stem cell medium (EPSCM), that maintains mouse PSCs in a unique cellular state and extraembryonic differentiation capacity. Herein, we describe how EPSCM can be directly used to stably maintain human PSCs. We further demonstrate that human PSCs maintained in EPSCM can spontaneously form embryoid bodies and undergo in vitro hematopoiesis using a simple differentiation protocol, similar to mouse PSC differentiation. EPSCM-maintained human PSCs generated at least two hematopoietic cell populations, which displayed distinct transcriptional profiles by RNA-sequencing (RNA-seq) analysis. EPSCM also supports gene targeting using homologous recombination, affording generation of an SPI1 (PU.1) reporter PSC line to study and track in vitro hematopoiesis. EPSCM therefore provides a useful tool not only to study pluripotency but also hematopoietic cell specification and developmental-lineage commitment.
Assuntos
Meios de Cultura/farmacologia , Hematopoese/efeitos dos fármacos , Células-Tronco Embrionárias Humanas/efeitos dos fármacos , Células-Tronco Pluripotentes/efeitos dos fármacos , Animais , Técnicas de Cultura de Células/métodos , Ciclo Celular , Linhagem da Célula , Células Cultivadas , Técnicas de Reprogramação Celular , Corpos Embrioides/efeitos dos fármacos , Fibroblastos/citologia , Genes Reporter , Células-Tronco Embrionárias Humanas/citologia , Humanos , Camundongos , Células-Tronco Neurais/citologia , Células-Tronco Neurais/efeitos dos fármacos , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/transplante , Análise de Sequência de RNA , Especificidade da Espécie , Transplante de Células-Tronco/efeitos adversos , Teratoma/etiologiaRESUMO
We recently derived mouse expanded potential stem cells (EPSCs) from individual blastomeres by inhibiting the critical molecular pathways that predispose their differentiation. EPSCs had enriched molecular signatures of blastomeres and possessed developmental potency for all embryonic and extra-embryonic cell lineages. Here, we report the derivation of porcine EPSCs, which express key pluripotency genes, are genetically stable, permit genome editing, differentiate to derivatives of the three germ layers in chimeras and produce primordial germ cell-like cells in vitro. Under similar conditions, human embryonic stem cells and induced pluripotent stem cells can be converted, or somatic cells directly reprogrammed, to EPSCs that display the molecular and functional attributes reminiscent of porcine EPSCs. Importantly, trophoblast stem-cell-like cells can be generated from both human and porcine EPSCs. Our pathway-inhibition paradigm thus opens an avenue for generating mammalian pluripotent stem cells, and EPSCs present a unique cellular platform for translational research in biotechnology and regenerative medicine.
Assuntos
Diferenciação Celular/genética , Reprogramação Celular/genética , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes/citologia , Animais , Blastômeros/citologia , Blastômeros/metabolismo , Linhagem da Célula/genética , Células-Tronco Embrionárias/citologia , Camadas Germinativas/crescimento & desenvolvimento , Camadas Germinativas/metabolismo , Humanos , Camundongos , Medicina Regenerativa , Transdução de Sinais/genética , Suínos , Trofoblastos/citologia , Trofoblastos/metabolismoRESUMO
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.
Assuntos
Técnicas de Cultura de Células/métodos , Haploidia , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/fisiologia , Animais , Blastocisto/citologia , Linhagem Celular , Separação Celular/métodos , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , Fluxo de TrabalhoRESUMO
Many gene fusions are reported in tumours and for most their role remains unknown. As fusions are used for diagnostic and prognostic purposes, and are targets for treatment, it is crucial to assess their function in cancer. To systematically investigate the role of fusions in tumour cell fitness, we utilized RNA-sequencing data from 1011 human cancer cell lines to functionally link 8354 fusion events with genomic data, sensitivity to >350 anti-cancer drugs and CRISPR-Cas9 loss-of-fitness effects. Established clinically-relevant fusions were identified. Overall, detection of functional fusions was rare, including those involving cancer driver genes, suggesting that many fusions are dispensable for tumour fitness. Therapeutically actionable fusions involving RAF1, BRD4 and ROS1 were verified in new histologies. In addition, recurrent YAP1-MAML2 fusions were identified as activators of Hippo-pathway signaling in multiple cancer types. Our approach discriminates functional fusions, identifying new drivers of carcinogenesis and fusions that could have clinical implications.
Assuntos
Biomarcadores Tumorais/genética , Sistemas CRISPR-Cas/genética , Fusão Gênica/genética , Neoplasias/genética , Antineoplásicos/farmacologia , Carcinogênese/genética , Linhagem Celular Tumoral , Conjuntos de Dados como Assunto , Resistencia a Medicamentos Antineoplásicos/genética , Detecção Precoce de Câncer/métodos , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Genômica/métodos , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Neoplasias/diagnóstico , Análise de Sequência de RNARESUMO
The increasing prevalence of diabetes has resulted in a global epidemic1. Diabetes is a major cause of blindness, kidney failure, heart attacks, stroke and amputation of lower limbs. These are often caused by changes in blood vessels, such as the expansion of the basement membrane and a loss of vascular cells2-4. Diabetes also impairs the functions of endothelial cells5 and disturbs the communication between endothelial cells and pericytes6. How dysfunction of endothelial cells and/or pericytes leads to diabetic vasculopathy remains largely unknown. Here we report the development of self-organizing three-dimensional human blood vessel organoids from pluripotent stem cells. These human blood vessel organoids contain endothelial cells and pericytes that self-assemble into capillary networks that are enveloped by a basement membrane. Human blood vessel organoids transplanted into mice form a stable, perfused vascular tree, including arteries, arterioles and venules. Exposure of blood vessel organoids to hyperglycaemia and inflammatory cytokines in vitro induces thickening of the vascular basement membrane. Human blood vessels, exposed in vivo to a diabetic milieu in mice, also mimic the microvascular changes found in patients with diabetes. DLL4 and NOTCH3 were identified as key drivers of diabetic vasculopathy in human blood vessels. Therefore, organoids derived from human stem cells faithfully recapitulate the structure and function of human blood vessels and are amenable systems for modelling and identifying the regulators of diabetic vasculopathy, a disease that affects hundreds of millions of patients worldwide.
Assuntos
Membrana Basal/patologia , Vasos Sanguíneos/patologia , Angiopatias Diabéticas/patologia , Modelos Biológicos , Organoides/patologia , Organoides/transplante , Proteínas Adaptadoras de Transdução de Sinal , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Secretases da Proteína Precursora do Amiloide/metabolismo , Animais , Artérias/citologia , Artérias/efeitos dos fármacos , Arteríolas/citologia , Arteríolas/efeitos dos fármacos , Membrana Basal/citologia , Membrana Basal/efeitos dos fármacos , Vasos Sanguíneos/citologia , Vasos Sanguíneos/efeitos dos fármacos , Vasos Sanguíneos/crescimento & desenvolvimento , Proteínas de Ligação ao Cálcio , Angiopatias Diabéticas/enzimologia , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Humanos , Hiperglicemia/complicações , Técnicas In Vitro , Mediadores da Inflamação/farmacologia , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Camundongos , Organoides/citologia , Organoides/efeitos dos fármacos , Pericitos/citologia , Pericitos/efeitos dos fármacos , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/efeitos dos fármacos , Receptor Notch3/metabolismo , Transdução de Sinais , Vênulas/citologia , Vênulas/efeitos dos fármacosRESUMO
Mutations in the ATM tumor suppressor gene confer hypersensitivity to DNA-damaging chemotherapeutic agents. To explore genetic resistance mechanisms, we performed genome-wide CRISPR-Cas9 screens in cells treated with the DNA topoisomerase I inhibitor topotecan. Thus, we here establish that inactivating terminal components of the non-homologous end-joining (NHEJ) machinery or of the BRCA1-A complex specifically confer topotecan resistance to ATM-deficient cells. We show that hypersensitivity of ATM-mutant cells to topotecan or the poly-(ADP-ribose) polymerase (PARP) inhibitor olaparib reflects delayed engagement of homologous recombination at DNA-replication-fork associated single-ended double-strand breaks (DSBs), allowing some to be subject to toxic NHEJ. Preventing DSB ligation by NHEJ, or enhancing homologous recombination by BRCA1-A complex disruption, suppresses this toxicity, highlighting a crucial role for ATM in preventing toxic LIG4-mediated chromosome fusions. Notably, suppressor mutations in ATM-mutant backgrounds are different to those in BRCA1-mutant scenarios, suggesting new opportunities for patient stratification and additional therapeutic vulnerabilities for clinical exploitation.
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Antineoplásicos/farmacologia , Proteínas Mutadas de Ataxia Telangiectasia/genética , Reparo do DNA por Junção de Extremidades/genética , Resistencia a Medicamentos Antineoplásicos/genética , Animais , Antineoplásicos/uso terapêutico , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteína BRCA1/metabolismo , Sistemas CRISPR-Cas/genética , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , DNA Ligase Dependente de ATP/metabolismo , Replicação do DNA/efeitos dos fármacos , Replicação do DNA/genética , Feminino , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos Knockout , Células-Tronco Embrionárias Murinas , Mutação , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/genética , Neoplasias Experimentais/patologia , Ftalazinas/farmacologia , Ftalazinas/uso terapêutico , Piperazinas/farmacologia , Piperazinas/uso terapêutico , Topotecan/farmacologia , Topotecan/uso terapêuticoRESUMO
The use of the DNA dyes Hoechst (HO) and chromomycin A3 (CA3) has become the preferred combination for the bivariate analysis of chromosomes from both human and animals. This analysis requires a flow cytometer equipped with lasers of specific wavelength and of higher power than is typical on a conventional bench top flow cytometer. In this study, we have investigated the resolution of chromosome peaks in a human cell line with normal flow karyotype using different combinations of DNA dyes on a number of flow cytometers available in a flow cytometry core facility. Chromosomes were prepared from the human cell line using a modified polyamine isolation buffer. The bivariate flow karyotypes of different DNA dyes combination; 4'-6-diamidino-2-phenylindole (DAPI) or Hoechst with propidium iodide (PI), obtained from different flow cytometers were compared to the reference flow karyotype of DAPI or Hoechst with chromomycin A3, generated from a Mo-Flo cell sorter using laser power settings of 300 mW each of UV and 457 nm. Good chromosome separation was observed in most of the flow cytometers used in the study. This study demonstrates that chromosome analysis and sorting can also be performed on benchtop flow cytometers equipped with the standard solid state 488 and 355 nm lasers, using a DNA dye combination of DAPI or Hoechst with PI. © 2018 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.
Assuntos
Cromossomos/química , DNA/análise , Citometria de Fluxo/métodos , Cariotipagem/métodos , Linhagem Celular , DNA/química , Fluorescência , Corantes Fluorescentes/química , Humanos , Lasers , Masculino , PropídioRESUMO
We report full-length draft de novo genome assemblies for 16 widely used inbred mouse strains and find extensive strain-specific haplotype variation. We identify and characterize 2,567 regions on the current mouse reference genome exhibiting the greatest sequence diversity. These regions are enriched for genes involved in pathogen defence and immunity and exhibit enrichment of transposable elements and signatures of recent retrotransposition events. Combinations of alleles and genes unique to an individual strain are commonly observed at these loci, reflecting distinct strain phenotypes. We used these genomes to improve the mouse reference genome, resulting in the completion of 10 new gene structures. Also, 62 new coding loci were added to the reference genome annotation. These genomes identified a large, previously unannotated, gene (Efcab3-like) encoding 5,874 amino acids. Mutant Efcab3-like mice display anomalies in multiple brain regions, suggesting a possible role for this gene in the regulation of brain development.
Assuntos
Mapeamento Cromossômico , Loci Gênicos , Genoma , Haplótipos , Camundongos Endogâmicos/genética , Animais , Animais de Laboratório , Mapeamento Cromossômico/veterinária , Haplótipos/genética , Camundongos , Camundongos Endogâmicos BALB C/genética , Camundongos Endogâmicos C3H/genética , Camundongos Endogâmicos C57BL/genética , Camundongos Endogâmicos CBA/genética , Camundongos Endogâmicos DBA/genética , Camundongos Endogâmicos NOD/genética , Camundongos Endogâmicos/classificação , Anotação de Sequência Molecular , Filogenia , Polimorfismo de Nucleotídeo Único , Especificidade da EspécieRESUMO
BRCA1 deficiencies cause breast, ovarian, prostate and other cancers, and render tumours hypersensitive to poly(ADP-ribose) polymerase (PARP) inhibitors. To understand the resistance mechanisms, we conducted whole-genome CRISPR-Cas9 synthetic-viability/resistance screens in BRCA1-deficient breast cancer cells treated with PARP inhibitors. We identified two previously uncharacterized proteins, C20orf196 and FAM35A, whose inactivation confers strong PARP-inhibitor resistance. Mechanistically, we show that C20orf196 and FAM35A form a complex, 'Shieldin' (SHLD1/2), with FAM35A interacting with single-stranded DNA through its C-terminal oligonucleotide/oligosaccharide-binding fold region. We establish that Shieldin acts as the downstream effector of 53BP1/RIF1/MAD2L2 to promote DNA double-strand break (DSB) end-joining by restricting DSB resection and to counteract homologous recombination by antagonizing BRCA2/RAD51 loading in BRCA1-deficient cells. Notably, Shieldin inactivation further sensitizes BRCA1-deficient cells to cisplatin, suggesting how defining the SHLD1/2 status of BRCA1-deficient tumours might aid patient stratification and yield new treatment opportunities. Highlighting this potential, we document reduced SHLD1/2 expression in human breast cancers displaying intrinsic or acquired PARP-inhibitor resistance.
Assuntos
Proteína BRCA1/genética , Neoplasias Ósseas/tratamento farmacológico , Neoplasias da Mama/tratamento farmacológico , Reparo do DNA por Junção de Extremidades , Resistencia a Medicamentos Antineoplásicos , Osteossarcoma/tratamento farmacológico , Neoplasias Ovarianas/tratamento farmacológico , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Proteínas/metabolismo , Reparo de DNA por Recombinação , Animais , Proteína BRCA1/deficiência , Neoplasias Ósseas/genética , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/patologia , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Cisplatino/farmacologia , Quebras de DNA de Cadeia Dupla , Proteínas de Ligação a DNA , Relação Dose-Resposta a Droga , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Células HEK293 , Humanos , Proteínas Mad2/genética , Proteínas Mad2/metabolismo , Camundongos , Complexos Multiproteicos , Osteossarcoma/genética , Osteossarcoma/metabolismo , Osteossarcoma/patologia , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Proteínas/genética , Proteínas de Ligação a Telômeros/genética , Proteínas de Ligação a Telômeros/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/genética , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Transmissible cancers are clonal lineages that spread through populations via contagious cancer cells. Although rare in nature, two facial tumor clones affect Tasmanian devils. Here we perform comparative genetic and functional characterization of these lineages. The two cancers have similar patterns of mutation and show no evidence of exposure to exogenous mutagens or viruses. Genes encoding PDGF receptors have copy number gains and are present on extrachromosomal double minutes. Drug screening indicates causative roles for receptor tyrosine kinases and sensitivity to inhibitors of DNA repair. Y chromosome loss from a male clone infecting a female host suggests immunoediting. These results imply that Tasmanian devils may have inherent susceptibility to transmissible cancers and present a suite of therapeutic compounds for use in conservation.
Assuntos
Neoplasias Faciais/veterinária , Marsupiais/genética , Mutação , Receptores do Fator de Crescimento Derivado de Plaquetas/genética , Animais , Linhagem Celular Tumoral , Cromossomos de Mamíferos/genética , Células Clonais/imunologia , Células Clonais/patologia , Neoplasias Faciais/genética , Neoplasias Faciais/imunologia , Feminino , Dosagem de Genes , Edição de Genes , Imunidade , MasculinoRESUMO
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RESUMO
The poor correlation of mutational landscapes with phenotypes limits our understanding of the pathogenesis and metastasis of pancreatic ductal adenocarcinoma (PDAC). Here we show that oncogenic dosage-variation has a critical role in PDAC biology and phenotypic diversification. We find an increase in gene dosage of mutant KRAS in human PDAC precursors, which drives both early tumorigenesis and metastasis and thus rationalizes early PDAC dissemination. To overcome the limitations posed to gene dosage studies by the stromal richness of PDAC, we have developed large cell culture resources of metastatic mouse PDAC. Integration of cell culture genomes, transcriptomes and tumour phenotypes with functional studies and human data reveals additional widespread effects of oncogenic dosage variation on cell morphology and plasticity, histopathology and clinical outcome, with the highest KrasMUT levels underlying aggressive undifferentiated phenotypes. We also identify alternative oncogenic gains (Myc, Yap1 or Nfkb2), which collaborate with heterozygous KrasMUT in driving tumorigenesis, but have lower metastatic potential. Mechanistically, different oncogenic gains and dosages evolve along distinct evolutionary routes, licensed by defined allelic states and/or combinations of hallmark tumour suppressor alterations (Cdkn2a, Trp53, Tgfß-pathway). Thus, evolutionary constraints and contingencies direct oncogenic dosage gain and variation along defined routes to drive the early progression of PDAC and shape its downstream biology. Our study uncovers universal principles of Ras-driven oncogenesis that have potential relevance beyond pancreatic cancer.