Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 1.217
Filtrar
1.
Nat Protoc ; 15(10): 3264-3283, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32913232

RESUMO

We recently introduced Cleavage Under Targets & Tagmentation (CUT&Tag), an epigenomic profiling strategy in which antibodies are bound to chromatin proteins in situ in permeabilized nuclei. These antibodies are then used to tether the cut-and-paste transposase Tn5. Activation of the transposase simultaneously cleaves DNA and adds adapters ('tagmentation') for paired-end DNA sequencing. Here, we introduce a streamlined CUT&Tag protocol that suppresses DNA accessibility artefacts to ensure high-fidelity mapping of the antibody-targeted protein and improves the signal-to-noise ratio over current chromatin profiling methods. Streamlined CUT&Tag can be performed in a single PCR tube, from cells to amplified libraries, providing low-cost genome-wide chromatin maps. By simplifying library preparation CUT&Tag requires less than a day at the bench, from live cells to sequencing-ready barcoded libraries. As a result of low background levels, barcoded and pooled CUT&Tag libraries can be sequenced for as little as $25 per sample. This enables routine genome-wide profiling of chromatin proteins and modifications and requires no special skills or equipment.


Assuntos
Cromatina/genética , Mapeamento Cromossômico/métodos , Epigenômica/métodos , Sequência de Bases , DNA/genética , Biblioteca Gênica , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Histonas/metabolismo , Análise de Sequência de DNA/métodos , Análise de Célula Única/métodos , Transposases/genética , Transposases/metabolismo
2.
Nat Commun ; 11(1): 3446, 2020 07 10.
Artigo em Inglês | MEDLINE | ID: mdl-32651359

RESUMO

The piggyBac DNA transposon is used widely in genome engineering applications. Unlike other transposons, its excision site can be precisely repaired without leaving footprints and it integrates specifically at TTAA tetranucleotides. We present cryo-EM structures of piggyBac transpososomes: a synaptic complex with hairpin DNA intermediates and a strand transfer complex capturing the integration step. The results show that the excised TTAA hairpin intermediate and the TTAA target adopt essentially identical conformations, providing a mechanistic link connecting the two unique properties of piggyBac. The transposase forms an asymmetric dimer in which the two central domains synapse the ends while two C-terminal domains form a separate dimer that contacts only one transposon end. In the strand transfer structure, target DNA is severely bent and the TTAA target is unpaired. In-cell data suggest that asymmetry promotes synaptic complex formation, and modifying ends with additional transposase binding sites stimulates activity.


Assuntos
Elementos de DNA Transponíveis/genética , Transposases/metabolismo , Microscopia Crioeletrônica , Humanos , Espectroscopia de Ressonância Magnética , Ligação Proteica , Estrutura Secundária de Proteína , Transposases/genética
3.
PLoS Genet ; 16(7): e1008949, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32702045

RESUMO

In Paramecium tetraurelia, a large proportion of the germline genome is reproducibly removed from the somatic genome after sexual events via a process involving small (s)RNA-directed heterochromatin formation and DNA excision and repair. How germline limited DNA sequences are specifically recognized in the context of chromatin remains elusive. Here, we use a reverse genetics approach to identify factors involved in programmed genome rearrangements. We have identified a P. tetraurelia homolog of the highly conserved histone chaperone Spt16 subunit of the FACT complex, Spt16-1, and show its expression is developmentally regulated. A functional GFP-Spt16-1 fusion protein localized exclusively in the nuclei where genome rearrangements take place. Gene silencing of Spt16-1 showed it is required for the elimination of all germline-limited sequences, for the survival of sexual progeny, and for the accumulation of internal eliminated sequence (ies)RNAs, an sRNA population produced when elimination occurs. Normal accumulation of 25 nt scanRNAs and deposition of silent histone marks H3K9me3 and H3K27me3 indicated that Spt16-1 does not regulate the scanRNA-directed heterochromatin pathway involved in the early steps of DNA elimination. We further show that Spt16-1 is required for the correct nuclear localization of the PiggyMac (Pgm) endonuclease, which generates the DNA double-strand breaks required for DNA elimination. Thus, Spt16-1 is essential for Pgm function during programmed genome rearrangements. We propose a model in which Spt16-1 mediates interactions between the excision machinery and chromatin, facilitating endonuclease access to DNA cleavage sites during genome rearrangements.


Assuntos
Núcleo Celular/genética , Chaperonas de Histonas/genética , Paramecium/genética , Transposases/genética , Sequência de Bases/genética , Quebras de DNA de Cadeia Dupla , Clivagem do DNA , DNA de Protozoário/genética , Endonucleases , Rearranjo Gênico/genética , Genoma de Protozoário/genética , Paramecium/crescimento & desenvolvimento
4.
Am J Physiol Heart Circ Physiol ; 319(2): H349-H358, 2020 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-32589443

RESUMO

Here, we report the generation of a Cre-recombinase (iCre) transgenic rat, where iCre is driven using a vascular endothelial-cadherin (CDH5) promoter. The CDH5 promoter was cloned from rat pulmonary microvascular endothelial cells and demonstrated ~60% similarity to the murine counterpart. The cloned rat promoter was 2,508 bp, it extended 79 bp beyond the transcription start site, and it was 22,923 bp upstream of the translation start site. The novel promoter was cloned upstream of codon-optimized iCre and subcloned into a Sleeping Beauty transposon vector for transpositional transgenesis in Sprague-Dawley rats. Transgenic founders were generated and selected for iCre expression. Crossing the CDH5-iCre rat with a tdTomato reporter rat resulted in progeny displaying endothelium-restricted fluorescence. tdTomato fluorescence was prominent in major arteries and veins, and it was similar in males and females. Quantitative analysis of the carotid artery and the jugular vein revealed that, on average, more than 50% of the vascular surface area exhibited strong fluorescence. tdTomato fluorescence was observed in the circulations of every tissue tested. The microcirculation in all tissues tested displayed homogenous fluorescence. Fluorescence was examined across young (6-7.5 mo), middle (14-16.5 mo), and old age (17-19.5 mo) groups. Although tdTomato fluorescence was seen in middle- and old-age animals, the intensity of the fluorescence was significantly reduced compared with that seen in the young rats. Thus, this endothelium-restricted transgenic rat offers a novel platform to test endothelial microheterogeneity within all vascular segments, and it provides exceptional resolution of endothelium within-organ microcirculation for application to translational disease models.NEW & NOTEWORTHY The use of transgenic mice has been instrumental in advancing molecular insight of physiological processes, yet these models oftentimes do not faithfully recapitulate human physiology and pathophysiology. Rat models better replicate some human conditions, like Group 1 pulmonary arterial hypertension. Here, we report the development of an endothelial cell-restricted transgenic reporter rat that has broad application to vascular biology. This first-in-kind model offers exceptional endothelium-restricted tdTomato expression, in both conduit vessels and the microcirculations of organs.


Assuntos
Antígenos CD/genética , Caderinas/genética , Células Endoteliais/metabolismo , Genes Reporter , Integrases/genética , Proteínas Luminescentes/genética , Regiões Promotoras Genéticas , Fatores Etários , Animais , Feminino , Regulação da Expressão Gênica , Integrases/metabolismo , Proteínas Luminescentes/biossíntese , Masculino , Microcirculação , Ratos Sprague-Dawley , Ratos Transgênicos , Distribuição Tecidual , Transposases/genética , Transposases/metabolismo
5.
Nat Biotechnol ; 38(7): 824-844, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32572269

RESUMO

The development of new CRISPR-Cas genome editing tools continues to drive major advances in the life sciences. Four classes of CRISPR-Cas-derived genome editing agents-nucleases, base editors, transposases/recombinases and prime editors-are currently available for modifying genomes in experimental systems. Some of these agents have also moved rapidly into the clinic. Each tool comes with its own capabilities and limitations, and major efforts have broadened their editing capabilities, expanded their targeting scope and improved editing specificity. We analyze key considerations when choosing genome editing agents and identify opportunities for future improvements and applications in basic research and therapeutics.


Assuntos
Sistemas CRISPR-Cas/genética , Edição de Genes , Genoma/genética , Quebras de DNA de Cadeia Dupla , Endonucleases/genética , Edição de Genes/métodos , Humanos , Recombinases/genética , Transposases/genética
6.
PLoS Genet ; 16(5): e1008681, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32463832

RESUMO

A large fraction of plant genomes is composed of transposable elements (TE), which provide a potential source of novel genes through "domestication"-the process whereby the proteins encoded by TE diverge in sequence, lose their ability to catalyse transposition and instead acquire novel functions for their hosts. In Arabidopsis, ANTAGONIST OF LIKE HETEROCHROMATIN PROTEIN 1 (ALP1) arose by domestication of the nuclease component of Harbinger class TE and acquired a new function as a component of POLYCOMB REPRESSIVE COMPLEX 2 (PRC2), a histone H3K27me3 methyltransferase involved in regulation of host genes and in some cases TE. It was not clear how ALP1 associated with PRC2, nor what the functional consequence was. Here, we identify ALP2 genetically as a suppressor of Polycomb-group (PcG) mutant phenotypes and show that it arose from the second, DNA binding component of Harbinger transposases. Molecular analysis of PcG compromised backgrounds reveals that ALP genes oppose silencing and H3K27me3 deposition at key PcG target genes. Proteomic analysis reveals that ALP1 and ALP2 are components of a variant PRC2 complex that contains the four core components but lacks plant-specific accessory components such as the H3K27me3 reader LIKE HETEROCHROMATION PROTEIN 1 (LHP1). We show that the N-terminus of ALP2 interacts directly with ALP1, whereas the C-terminus of ALP2 interacts with MULTICOPY SUPPRESSOR OF IRA1 (MSI1), a core component of PRC2. Proteomic analysis reveals that in alp2 mutant backgrounds ALP1 protein no longer associates with PRC2, consistent with a role for ALP2 in recruitment of ALP1. We suggest that the propensity of Harbinger TE to insert in gene-rich regions of the genome, together with the modular two component nature of their transposases, has predisposed them for domestication and incorporation into chromatin modifying complexes.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis , Proteínas do Grupo Polycomb/metabolismo , Proteínas Repressoras/metabolismo , Transposases/fisiologia , Animais , Arabidopsis/enzimologia , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Domínio Catalítico/genética , Células Cultivadas , Domesticação , Regulação da Expressão Gênica de Plantas , Plantas Geneticamente Modificadas , Proteínas do Grupo Polycomb/genética , Ligação Proteica , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas Repressoras/química , Proteínas Repressoras/genética , Células Sf9 , Spodoptera , Transposases/genética
7.
PLoS One ; 15(4): e0230857, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32240216

RESUMO

The nucleotide sequences of plasmids pRC12 (12,342 bp; GC 43.99%) and pRC18 (18,664 bp; GC 34.33%), harbored by the bacteriocin-producer Lactobacillus curvatus CRL 705, were determined and analyzed. Plasmids pRC12 and pRC18 share a region with high DNA identity (> 83% identity between RepA, a Type II toxin-antitoxin system and a tyrosine integrase genes) and are stably maintained in their natural host L. curvatus CRL 705. Both plasmids are low copy number and belong to the theta-type replicating group. While pRC12 is a pUCL287-like plasmid that possesses iterons and the repA and repB genes for replication, pRC18 harbors a 168 amino acid replication protein affiliated to RepB, which was named RepB'. Plasmid pRC18 also possesses a pUCL287-like repA gene but it was disrupted by an 11 kb insertion element that contains RepB', several transposases/IS elements, and the lactocin Lac705 operon. An Escherichia coli / Lactobacillus shuttle vector, named plasmid p3B1, carrying the pRC18 replicon (i.e. repB' and replication origin), a chloramphenicol resistance gene and a pBluescript backbone, was constructed and used to define the host range of RepB'. Chloramphenicol-resistant transformants were obtained after electroporation of Lactobacillus plantarum CRL 691, Lactobacillus sakei 23K and a plasmid-cured derivative of L. curvatus CRL 705, but not of L. curvatus DSM 20019 or Lactococcus lactis NZ9000. Depending on the host, transformation efficiency ranged from 102 to 107 per µg of DNA; in the new hosts, the plasmid was relatively stable as 29-53% of recombinants kept it after cell growth for 100 generations in the absence of selective pressure. Plasmid p3B1 could therefore be used for cloning and functional studies in several Lactobacillus species.


Assuntos
Lactobacillus/genética , Plasmídeos/genética , Sequência de Aminoácidos/genética , Proteínas de Bactérias/genética , Sequência de Bases/genética , Replicação do DNA/genética , DNA Bacteriano/genética , Vetores Genéticos/genética , Origem de Replicação/genética , Replicon/genética , Análise de Sequência de DNA/métodos , Transposases/genética
8.
Sci Rep ; 10(1): 2610, 2020 02 13.
Artigo em Inglês | MEDLINE | ID: mdl-32054918

RESUMO

Mboumar-9 is an active mariner-transposable element previously isolated in the ant Messor bouvieri. In this work, a mariner-like element, Mboumar, isolated from 22 species of ants, is analyzed. These species belong to nine different subfamilies, including Leptanillinae, the most primitive ant subfamily, and Myrmicinae and Formicidae, the most derived ones. Consequently, Mboumar-like elements seem to be well-represented in ant genomes. The phylogenetic tree drawn for mariner elements is highly inconsistent with the phylogeny of host ants, with almost identical elements found in clearly distant species and, on the contrary, more variable elements in closely related species. The inconsistency between the two phylogenetic trees indicates that these transposable elements have evolved independently from the speciation events of the ants that host them. Besides, we found closer genetic relationships among elements than among their host ants. We also found potential coding copies with an uninterrupted open reading frame of 345 aa in 11 species. The putative transposase codified by them showed a high sequence identity with the active Mboumar-9 transposase. The results of selection tests suggest the intervention of purifying selection in the evolution of these elements. Overall, our study suggests a complex evolutionary history of the Mboumar-like mariner in ants, with important participation of horizontal transfer events. We also suggest that the evolutionary dynamics of Mboumar-like elements can be influenced by the genetic system of their host ants, which are eusocial insects with a haplodiploid genetic system.


Assuntos
Formigas/genética , Elementos de DNA Transponíveis , Animais , Evolução Molecular , Genoma de Inseto , Proteínas de Insetos/genética , Filogenia , Transposases/genética
9.
Int J Mol Sci ; 21(4)2020 Feb 13.
Artigo em Inglês | MEDLINE | ID: mdl-32070024

RESUMO

NCAPG is a subunit of condensin I that plays a crucial role in chromatin condensation during mitosis. NCAPG has been demonstrated to be associated with farm animal growth traits. However, its role in regulating myoblast differentiation is still unclear. We used myoblasts derived from fetal bovine tissue as an in vitro model and found that NCAPG was expressed during myogenic differentiation in the cytoplasm and nucleus. Silencing NCAPG prolonged the mitosis and impaired the differentiation due to increased myoblast apoptosis. After 1.5 days of differentiation, silencing NCAPG enhanced muscle-specific gene expression. An assay for transposase-accessible chromatin- high throughput sequencing (ATAC-seq) revealed that silencing NCAPG altered chromatin accessibility to activating protein 1 (AP-1) and its subunits. Knocking down the expression of the AP-1 subunits fos-related antigen 2 (FOSL2) or junB proto-oncogene (JUNB) enhanced part of the muscle-specific gene expression. In conclusion, our data provide valuable evidence about NCAPG's function in myogenesis, as well as its potential role in gene expression.


Assuntos
Proteínas de Ciclo Celular/genética , Desenvolvimento Fetal/genética , Antígeno 2 Relacionado a Fos/genética , Desenvolvimento Muscular/genética , Fator de Transcrição AP-1/genética , Fatores de Transcrição/genética , Animais , Bovinos , Diferenciação Celular/genética , Cromatina/genética , Feto/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Mitose/genética , Mioblastos/metabolismo , Transposases/genética
10.
Cell ; 180(4): 703-716.e18, 2020 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-32059782

RESUMO

The three-dimensional structures of chromosomes are increasingly being recognized as playing a major role in cellular regulatory states. The efficiency and promiscuity of phage Mu transposition was exploited to directly measure in vivo interactions between genomic loci in E. coli. Two global organizing principles have emerged: first, the chromosome is well-mixed and uncompartmentalized, with transpositions occurring freely between all measured loci; second, several gene families/regions show "clustering": strong three-dimensional co-localization regardless of linear genomic distance. The activities of the SMC/condensin protein MukB and nucleoid-compacting protein subunit HU-α are essential for the well-mixed state; HU-α is also needed for clustering of 6/7 ribosomal RNA-encoding loci. The data are explained by a model in which the chromosomal structure is driven by dynamic competition between DNA replication and chromosomal relaxation, providing a foundation for determining how region-specific properties contribute to both chromosomal structure and gene regulation.


Assuntos
Bacteriófago mu/genética , Cromossomos Bacterianos/genética , Elementos de DNA Transponíveis , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Cromossomos Bacterianos/química , DNA Bacteriano/química , DNA Bacteriano/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Escherichia coli , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Genoma Bacteriano , Conformação de Ácido Nucleico , Transposases/genética , Transposases/metabolismo
11.
Nat Commun ; 11(1): 859, 2020 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-32103003

RESUMO

Pogo transposable element derived with ZNF domain (POGZ) has been identified as one of the most recurrently de novo mutated genes in patients with neurodevelopmental disorders (NDDs), including autism spectrum disorder (ASD), intellectual disability and White-Sutton syndrome; however, the neurobiological basis behind these disorders remains unknown. Here, we show that POGZ regulates neuronal development and that ASD-related de novo mutations impair neuronal development in the developing mouse brain and induced pluripotent cell lines from an ASD patient. We also develop the first mouse model heterozygous for a de novo POGZ mutation identified in a patient with ASD, and we identify ASD-like abnormalities in the mice. Importantly, social deficits can be treated by compensatory inhibition of elevated cell excitability in the mice. Our results provide insight into how de novo mutations on high-confidence ASD genes lead to impaired mature cortical network function, which underlies the cellular pathogenesis of NDDs, including ASD.


Assuntos
Transtorno Autístico/genética , Predisposição Genética para Doença/genética , Malformações do Desenvolvimento Cortical/genética , Mutação , Fenótipo , Transposases/genética , Adolescente , Animais , Comportamento Animal , Encéfalo/patologia , Diferenciação Celular , Linhagem Celular , Proliferação de Células , Feminino , Edição de Genes , Técnicas de Silenciamento de Genes , Heterozigoto , Humanos , Deficiência Intelectual , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Transtornos do Neurodesenvolvimento/genética , Neurogênese , Neurônios/metabolismo
12.
Gene ; 730: 144318, 2020 Mar 10.
Artigo em Inglês | MEDLINE | ID: mdl-31917231

RESUMO

Although the chicken embryo has been a classical model for developmental studies, the lack of straightforward technologies for chicken transgenesis limited the usefulness of this animal model. Here, we assessed electroporation and lipofection approaches for in ovo transfection of Sleeping Beauty transposon system in stage X-XII chicken embryos. Electroporation of chicken embryos could transfect the trophectodermal cells. Then, a mixture of transposon lipoplexes and high concentrated carboxymethylcellulose (HCC) solution was injected into the subgerminal cavity of day 0 embryos. The lipoplex-HCC mixture substantially increased the number of trophectodermal cells expressing the reporter. Importantly, the fluorescent reporter was detected in cells inside of the embryos as well as circulation cells in the bloodstream during days 3-4 of incubation. This study provided evidence for direct in ovo transfection of early chicken embryos, though the long-term outcome of this approach warrants further studies.


Assuntos
Eletroporação/métodos , Transfecção/métodos , Transposases/genética , Animais , Animais Geneticamente Modificados , Carboximetilcelulose Sódica , Embrião de Galinha , Galinhas/genética , Elementos de DNA Transponíveis/genética , Embrião de Mamíferos/embriologia , Regulação da Expressão Gênica no Desenvolvimento/genética , Técnicas de Transferência de Genes
13.
Mol Genet Genomics ; 295(3): 621-633, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-31975241

RESUMO

Transposable elements (TEs) are DNA sequences capable of transpositions within the genome and thus exerting a considerable influence on the genome functioning and structure and serving as a source of new genes. TE biodiversity studies in previously unexplored species are important for the fundamental understanding of the TE influence on eukaryotic genomes. TEs are classified into retrotransposons and DNA transposons. IS630/Tc1/mariner (ITm) superfamily of DNA transposons is one of the most diverse groups broadly represented among the eukaryotes. The study of 19 mollusk genomes revealed a new group of ITm superfamily elements, which we henceforth refer to as TLEWI. These TEs are characterized by the low copy number, the lack of terminal inverted repeats, the catalytic domain with DD36E signature and the presence of spliceosomal introns in transposase coding sequence. Their prevalence among the mollusks is limited to the class Bivalvia. Since TLEWI possess the features of domesticated TE and structures similar to the eukaryotic genes which are not typical for the DNA transposons, we consider the hypothesis of co-optation of TLEWI gene by the bivalves. The results of our study will fill the gap of knowledge about the prevalence, activity, and evolution of the ITm DNA transposons in multicellular genomes and will facilitate our understanding of the mechanisms of TE domestication by the host genome.


Assuntos
Elementos de DNA Transponíveis , Genoma , Íntrons , Moluscos/genética , Filogenia , Processamento de RNA/genética , Transposases/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Evolução Molecular , Homologia de Sequência
14.
Nucleic Acids Res ; 48(1): 316-331, 2020 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-31777924

RESUMO

The Sleeping Beauty (SB) transposon is an advanced tool for genetic engineering and a useful model to investigate cut-and-paste DNA transposition in vertebrate cells. Here, we identify novel SB transposase mutants that display efficient and canonical excision but practically unmeasurable genomic re-integration. Based on phylogenetic analyses, we establish compensating amino acid replacements that fully rescue the integration defect of these mutants, suggesting epistasis between these amino acid residues. We further show that the transposons excised by the exc+/int- transposase mutants form extrachromosomal circles that cannot undergo a further round of transposition, thereby representing dead-end products of the excision reaction. Finally, we demonstrate the utility of the exc+/int- transposase in cassette removal for the generation of reprogramming factor-free induced pluripotent stem cells. Lack of genomic integration and formation of transposon circles following excision is reminiscent of signal sequence removal during V(D)J recombination, and implies that cut-and-paste DNA transposition can be converted to a unidirectional process by a single amino acid change.


Assuntos
Reprogramação Celular , Elementos de DNA Transponíveis , Células-Tronco Pluripotentes Induzidas/metabolismo , Transposases/genética , Substituição de Aminoácidos , Animais , Epistasia Genética , Engenharia Genética/métodos , Células HeLa , Células Hep G2 , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Camundongos , Mutação , Transposases/metabolismo
15.
Genome Biol Evol ; 11(12): 3353-3371, 2019 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-31702783

RESUMO

The genus Rhododendron (Ericaceae), which includes horticulturally important plants such as azaleas, is a highly diverse and widely distributed genus of >1,000 species. Here, we report the chromosome-scale de novo assembly and genome annotation of Rhododendron williamsianum as a basis for continued study of this large genus. We created multiple short fragment genomic libraries, which were assembled using ALLPATHS-LG. This was followed by contiguity preserving transposase sequencing (CPT-seq) and fragScaff scaffolding of a large fragment library, which improved the assembly by decreasing the number of scaffolds and increasing scaffold length. Chromosome-scale scaffolding was performed by proximity-guided assembly (LACHESIS) using chromatin conformation capture (Hi-C) data. Chromosome-scale scaffolding was further refined and linkage groups defined by restriction-site associated DNA (RAD) sequencing of the parents and progeny of a genetic cross. The resulting linkage map confirmed the LACHESIS clustering and ordering of scaffolds onto chromosomes and rectified large-scale inversions. Assessments of the R. williamsianum genome assembly and gene annotation estimate them to be 89% and 79% complete, respectively. Predicted coding sequences from genome annotation were used in syntenic analyses and for generating age distributions of synonymous substitutions/site between paralgous gene pairs, which identified whole-genome duplications (WGDs) in R. williamsianum. We then analyzed other publicly available Ericaceae genomes for shared WGDs. Based on our spatial and temporal analyses of paralogous gene pairs, we find evidence for two shared, ancient WGDs in Rhododendron and Vaccinium (cranberry/blueberry) members that predate the Ericaceae family and, in one case, the Ericales order.


Assuntos
Cromossomos de Plantas/genética , Ericaceae/genética , Genoma de Planta/genética , Rhododendron/genética , Sintenia , Sequência de Bases , Cromatina/genética , Mapeamento Cromossômico , Ligação Genética , Biblioteca Genômica , Anotação de Sequência Molecular , Transposases/genética
16.
Nat Biotechnol ; 37(12): 1502-1512, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31685959

RESUMO

The Sleeping Beauty (SB) transposon system is an efficient non-viral gene transfer tool in mammalian cells, but its broad use has been hampered by uncontrolled transposase gene activity from DNA vectors, posing a risk of genome instability, and by the inability to use the transposase protein directly. In this study, we used rational protein design based on the crystal structure of the hyperactive SB100X variant to create an SB transposase (high-solubility SB, hsSB) with enhanced solubility and stability. We demonstrate that hsSB can be delivered with transposon DNA to genetically modify cell lines and embryonic, hematopoietic and induced pluripotent stem cells (iPSCs), overcoming uncontrolled transposase activity. We used hsSB to generate chimeric antigen receptor (CAR) T cells, which exhibit potent antitumor activity in vitro and in xenograft mice. We found that hsSB spontaneously penetrates cells, enabling modification of iPSCs and generation of CAR T cells without the use of transfection reagents. Titration of hsSB to modulate genomic integration frequency achieved as few as two integrations per genome.


Assuntos
Engenharia Genética/métodos , Mutagênese Insercional/genética , Transposases/genética , Engenharia Celular/métodos , Linhagem Celular , Células Cultivadas , Células HeLa , Humanos , Proteínas Recombinantes de Fusão/genética , Células-Tronco
17.
Genome Biol Evol ; 11(11): 3181-3193, 2019 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-31633753

RESUMO

Transposable elements represent the largest components of many eukaryotic genomes and different genomes harbor different combinations of elements. Here, we discovered a novel DNA transposon in the genome of the clubmoss Selaginella lepidophylla. Further searching for related sequences to the conserved DDE region uncovered the presence of this superfamily of elements in fish, coral, sea anemone, and other animal species. However, this element appears restricted to Bryophytes and Lycophytes in plants. This transposon, named GingerRoot, is associated with a 6 bp (base pair) target site duplication, and 100-150 bp terminal inverted repeats. Analysis of transposase sequences identified the DDE motif, a catalytic domain, which shows similarity to the integrase of Gypsy-like long terminal repeat retrotransposons, the most abundant component in plant genomes. A total of 77 intact and several hundred truncated copies of GingerRoot elements were identified in S. lepidophylla. Like Gypsy retrotransposons, GingerRoots show a lack of insertion preference near genes, which contrasts to the compact genome size of about 100 Mb. Nevertheless, a considerable portion of GingerRoot elements was found to carry gene fragments, suggesting the capacity of duplicating gene sequences is unlikely attributed to the proximity to genes. Elements carrying gene fragments appear to be less methylated, more diverged, and more distal to genes than those without gene fragments, indicating they are preferentially retained in gene-poor regions. This study has identified a broadly dispersed, novel DNA transposon, and the first plant DNA transposon with an integrase-related transposase, suggesting the possibility of de novo formation of Gypsy-like elements in plants.


Assuntos
Briófitas/genética , Elementos de DNA Transponíveis , Evolução Molecular , Lycopodiaceae/genética , Transposases/genética , Animais , Integrases , Filogenia , Plantas
18.
Nat Biotechnol ; 37(11): 1302-1313, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31548728

RESUMO

Targeting membrane proteins could improve the efficacy of T cell-based immunotherapies. To facilitate the identification of T cell targets, we developed a hybrid genetic screening system where the Sleeping Beauty (SB) transposon and single guide RNA cassette are nested in an adeno-associated virus (AAV). SB-mediated genomic integration of the single guide RNA cassette enables efficient gene editing in primary murine T cells as well as a screen readout. We performed in vivo AAV-SB-CRISPR screens for membrane protein targets in CD8+ T cells in mouse models of glioblastoma (GBM). We validated screen hits by demonstrating that adoptive transfer of CD8+ T cells with Pdia3, Mgat5, Emp1 or Lag3 gene editing enhances the survival of GBM-bearing mice in both syngeneic and T-cell receptor transgenic models. Transcriptome profiling, single cell sequencing, cytokine assays and T cell signaling analysis showed that Pdia3 editing in T cells enhances effector functions. Engineered PDIA3 mutant EGFRvIII chimeric antigen T cells are more potent in antigen-specific killing of human GBM cells.


Assuntos
Linfócitos T CD8-Positivos/transplante , Edição de Genes/métodos , Glioblastoma/terapia , Proteínas de Membrana/genética , Transposases/genética , Animais , Antígenos CD/genética , Linfócitos T CD8-Positivos/metabolismo , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Dependovirus/genética , Feminino , Glioblastoma/genética , Glioblastoma/imunologia , Humanos , Imunoterapia Adotiva , Masculino , Camundongos , N-Acetilglucosaminiltransferases/genética , Proteínas de Neoplasias/genética , Isomerases de Dissulfetos de Proteínas/genética , RNA Guia/genética , Receptores de Superfície Celular/genética , Transposases/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
19.
Nucleic Acids Res ; 47(15): 8126-8135, 2019 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-31429873

RESUMO

Homology-directed genome engineering is limited by transgene size. Although DNA transposons are more efficient with large transgenes, random integrations are potentially mutagenic. Here we present an in vitro mechanistic study that demonstrates efficient Cas9 targeting of the mariner transposon Hsmar1. Integrations were unidirectional and tightly constrained to one side of the sgRNA binding site. Further analysis of the nucleoprotein intermediates demonstrated that the transposase and Cas9 moieties can bind their respective substrates independently or in concert. Kinetic analysis of the reaction in the presence of the Cas9 target-DNA revealed a delay between first and second strand cleavage at the transposon end. This step involves a significant conformational change that may be hindered by the properties of the interdomainal linker. Otherwise, the transposase moiety behaved normally and was proficient for integration in vitro and in Escherichia coli. Specific integration into the lacZ gene in E. coli was obscured by a high background of random integrations. Nevertheless, Cas9 is an attractive candidate for transposon-targeting because it has a high affinity and long dwell-time at its target site. This will facilitate a future optogenetic strategy for the temporal control of integration, which will increase the ratio of targeted to untargeted events.


Assuntos
Proteína 9 Associada à CRISPR/genética , Elementos de DNA Transponíveis/genética , Proteínas Recombinantes de Fusão/genética , Transposases/genética , Sequência de Bases , Sítios de Ligação/genética , Proteína 9 Associada à CRISPR/metabolismo , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Cinética , Mutagênese Insercional , Proteínas Recombinantes de Fusão/metabolismo , Transgenes/genética , Transposases/metabolismo
20.
Nat Commun ; 10(1): 3747, 2019 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-31431618

RESUMO

Modern next-generation sequencing-based methods have empowered researchers to assay the epigenetic states of individual cells. Existing techniques for profiling epigenetic marks in single cells often require the use and optimization of time-intensive procedures such as drop fluidics, chromatin fragmentation, and end repair. Here we describe ACT-seq, a streamlined method for mapping genome-wide distributions of histone tail modifications, histone variants, and chromatin-binding proteins in a small number of or single cells. ACT-seq utilizes a fusion of Tn5 transposase to Protein A that is targeted to chromatin by a specific antibody, allowing chromatin fragmentation and sequence tag insertion specifically at genomic sites presenting the relevant antigen. The Tn5 transposase enables the use of an index multiplexing strategy (iACT-seq), which enables construction of thousands of single-cell libraries in one day by a single researcher without the need for drop-based fluidics or visual sorting. We conclude that ACT-seq present an attractive alternative to existing techniques for mapping epigenetic marks in single cells.


Assuntos
Mapeamento Cromossômico/métodos , Código das Histonas , Histonas/genética , Análise de Célula Única/métodos , Anticorpos/imunologia , Anticorpos/metabolismo , Cromatina/imunologia , Cromatina/metabolismo , Imunoprecipitação da Cromatina , Epigenômica/métodos , Células HEK293 , Histonas/metabolismo , Humanos , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Análise de Sequência de DNA/métodos , Coloração e Rotulagem/métodos , Proteína Estafilocócica A/genética , Proteína Estafilocócica A/metabolismo , Transposases/genética , Transposases/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA