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1.
Nat Commun ; 11(1): 4915, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-33004811

RESUMO

A phenotype of Escherichia coli and Klebsiella pneumoniae, resistant to piperacillin/tazobactam (TZP) but susceptible to carbapenems and 3rd generation cephalosporins, has emerged. The resistance mechanism associated with this phenotype has been identified as hyperproduction of the ß-lactamase TEM. However, the mechanism of hyperproduction due to gene amplification is not well understood. Here, we report a mechanism of gene amplification due to a translocatable unit (TU) excising from an IS26-flanked pseudo-compound transposon, PTn6762, which harbours blaTEM-1B. The TU re-inserts into the chromosome adjacent to IS26 and forms a tandem array of TUs, which increases the copy number of blaTEM-1B, leading to TEM-1B hyperproduction and TZP resistance. Despite a significant increase in blaTEM-1B copy number, the TZP-resistant isolate does not incur a fitness cost compared to the TZP-susceptible ancestor. This mechanism of amplification of blaTEM-1B is an important consideration when using genomic data to predict susceptibility to TZP.


Assuntos
Antibacterianos/farmacologia , Farmacorresistência Bacteriana Múltipla/genética , Infecções por Escherichia coli/tratamento farmacológico , Proteínas de Escherichia coli/genética , Escherichia coli/genética , beta-Lactamases/genética , Antibacterianos/uso terapêutico , Cromossomos Bacterianos/genética , Elementos de DNA Transponíveis/genética , DNA Bacteriano/genética , Quimioterapia Combinada/métodos , Escherichia coli/efeitos dos fármacos , Escherichia coli/isolamento & purificação , Infecções por Escherichia coli/microbiologia , Amplificação de Genes , Regulação Bacteriana da Expressão Gênica , Genoma Bacteriano/genética , Humanos , Testes de Sensibilidade Microbiana , Piperacilina/farmacologia , Piperacilina/uso terapêutico , Polimorfismo de Fragmento de Restrição , RNA Ribossômico 16S/genética , Tazobactam/farmacologia , Tazobactam/uso terapêutico , Sequenciamento Completo do Genoma
2.
Nat Commun ; 11(1): 4940, 2020 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-33009411

RESUMO

The HUSH complex represses retroviruses, transposons and genes to maintain the integrity of vertebrate genomes. HUSH regulates deposition of the epigenetic mark H3K9me3, but how its three core subunits - TASOR, MPP8 and Periphilin - contribute to assembly and targeting of the complex remains unknown. Here, we define the biochemical basis of HUSH assembly and find that its modular architecture resembles the yeast RNA-induced transcriptional silencing complex. TASOR, the central HUSH subunit, associates with RNA processing components. TASOR is required for H3K9me3 deposition over LINE-1 repeats and repetitive exons in transcribed genes. In the context of previous studies, this suggests that an RNA intermediate is important for HUSH activity. We dissect the TASOR and MPP8 domains necessary for transgene repression. Structure-function analyses reveal TASOR bears a catalytically-inactive PARP domain necessary for targeted H3K9me3 deposition. We conclude that TASOR is a multifunctional pseudo-PARP that directs HUSH assembly and epigenetic regulation of repetitive genomic targets.


Assuntos
Elementos de DNA Transponíveis/genética , Epigênese Genética , Complexos Multiproteicos/metabolismo , Proteínas Nucleares/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Sequência de Aminoácidos , Antígenos de Neoplasias/metabolismo , Sítios de Ligação , Éxons/genética , Genoma , Células HEK293 , Células HeLa , Histonas/metabolismo , Humanos , Lisina/metabolismo , Espectroscopia de Ressonância Magnética , Metilação , NAD/metabolismo , Proteínas Nucleares/química , Fosfoproteínas/metabolismo , Ligação Proteica , Domínios Proteicos , RNA/metabolismo , Processamento Pós-Transcricional do RNA , Transcrição Genética
3.
BMC Infect Dis ; 20(1): 657, 2020 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-32894079

RESUMO

BACKGROUND: Tuberculosis is still a significant diagnostic and therapeutic challenge with high proportion of smear- and culture- negative incidences worldwide. The conventional diagnostic tests are time-consuming and have a low sensitivity. Digital PCR is a novel technology which can detect target sequences with relatively low abundance and obtain the absolute copy numbers of the targets. METHODS: We assessed the accuracy of dPCR in TB diagnosis using more than 250 specimens, and for the first time, we selected M.tuberculosis-specific IS1081 in addition to widely used IS6110 as the amplification targets for dPCR. The quantification of target DNA was calculated using QuantaSoft Version 1.7.4.0917 (BioRad), and SPSS version 13.0 software (SPSS Inc., Chicago, IL, USA) was used for statistical analyses. RESULTS: IS6110-dPCR was more sensitive than IS1081, with the sensitivity and specificity accounting for 40.6 and 93.4% respectively. When we classified the TB patients by personal factors for high copy number of M.tuberculosis derived DNA in plasma: bilateral TB, extrapulmonary TB and disseminated TB, the sensitivity of both IS6110- and IS1081- dPCR was the highest in patients with disseminated TB (IS6110, 100%; IS1081, 68.8%), while their sensitivity was a bit higher in patients with extrapulmonary TB (IS6110, 50.0%; IS1081, 39.3%) than that in bilateral TB (IS6110, 43.3%; IS1081, 33.3%). Compared with traditional TB diagnostic tests, joint detection IS6110 & IS1081-dPCR was not as sensitive as smear microscope or mycobacterial culture, but it was higher than IS6110 qPCR (p < 0.05) and was able to detect 47.4% of smear-negative TB patients. CONCLUSION: Our study suggested that plasma IS6110-dPCR is a rapid, moderate accurate and less-invasive method to detect M.tuberculosis DNA in plasma of TB patients and IS6110 & IS1081-dPCR has a potential to aid diagnosis of smear-negative TB.


Assuntos
Elementos de DNA Transponíveis/genética , DNA Bacteriano/genética , Mycobacterium tuberculosis/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , Tuberculose Pulmonar/diagnóstico , Adolescente , Adulto , Idoso , DNA Bacteriano/sangue , Confiabilidade dos Dados , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Sensibilidade e Especificidade , Adulto Jovem
4.
Nat Commun ; 11(1): 4515, 2020 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-32908127

RESUMO

The discovery of ancestral RAG transposons in early deuterostomia reveals the origin of vertebrate V(D)J recombination. Here, we analyze the functional regulation of a RAG transposon, ProtoRAG, in lancelet. We find that a specific interaction between the cis-acting element within the TIR sequences of ProtoRAG and a trans-acting factor, lancelet YY1-like (bbYY1), is important for the transcriptional regulation of lancelet RAG-like genes (bbRAG1L and bbRAG2L). Mechanistically, bbYY1 suppresses the transposition of ProtoRAG; meanwhile, bbYY1 promotes host DNA rejoins (HDJ) and TIR-TIR joints (TTJ) after TIR-dependent excision by facilitating the binding of bbRAG1L/2 L to TIR-containing DNA, and by interacting with the bbRAG1L/2 L complex. Our data thus suggest that bbYY1 has dual functions in fine-tuning the activity of ProtoRAG and maintaining the genome stability of the host.


Assuntos
Elementos de DNA Transponíveis/genética , Proteínas de Ligação a DNA/metabolismo , Anfioxos/genética , Recombinação V(D)J , Fator de Transcrição YY1/metabolismo , Animais , Técnicas de Silenciamento de Genes , Genes RAG-1 , Instabilidade Genômica , Células HEK293 , Células HeLa , Humanos , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Fator de Transcrição YY1/genética , Fator de Transcrição YY1/isolamento & purificação
5.
Nat Commun ; 11(1): 4522, 2020 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-32908144

RESUMO

A unique, protective cell envelope contributes to the broad drug resistance of the nosocomial pathogen Acinetobacter baumannii. Here we use transposon insertion sequencing to identify A. baumannii mutants displaying altered susceptibility to a panel of diverse antibiotics. By examining mutants with antibiotic susceptibility profiles that parallel mutations in characterized genes, we infer the function of multiple uncharacterized envelope proteins, some of which have roles in cell division or cell elongation. Remarkably, mutations affecting a predicted cell wall hydrolase lead to alterations in lipooligosaccharide synthesis. In addition, the analysis of altered susceptibility signatures and antibiotic-induced morphology patterns allows us to predict drug synergies; for example, certain beta-lactams appear to work cooperatively due to their preferential targeting of specific cell wall assembly machineries. Our results indicate that the pathogen may be effectively inhibited by the combined targeting of multiple pathways critical for envelope growth.


Assuntos
Infecções por Acinetobacter/tratamento farmacológico , Acinetobacter baumannii/genética , Antibacterianos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Infecção Hospitalar/tratamento farmacológico , Farmacorresistência Bacteriana Múltipla/genética , Infecções por Acinetobacter/microbiologia , Acinetobacter baumannii/efeitos dos fármacos , Antibacterianos/uso terapêutico , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Parede Celular/efeitos dos fármacos , Parede Celular/genética , Parede Celular/metabolismo , Infecção Hospitalar/microbiologia , Análise Mutacional de DNA , Elementos de DNA Transponíveis/genética , DNA Bacteriano/genética , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Sinergismo Farmacológico , Humanos , Testes de Sensibilidade Microbiana , Mutação
6.
PLoS Biol ; 18(9): e3000636, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32991578

RESUMO

The Myriapoda, composed of millipedes and centipedes, is a fascinating but poorly understood branch of life, including species with a highly unusual body plan and a range of unique adaptations to their environment. Here, we sequenced and assembled 2 chromosomal-level genomes of the millipedes Helicorthomorpha holstii (assembly size = 182 Mb; shortest scaffold/contig length needed to cover 50% of the genome [N50] = 18.11 Mb mainly on 8 pseudomolecules) and Trigoniulus corallinus (assembly size = 449 Mb, N50 = 26.78 Mb mainly on 17 pseudomolecules). Unique genomic features, patterns of gene regulation, and defence systems in millipedes, not observed in other arthropods, are revealed. Both repeat content and intron size are major contributors to the observed differences in millipede genome size. Tight Hox and the first loose ecdysozoan ParaHox homeobox clusters are identified, and a myriapod-specific genomic rearrangement including Hox3 is also observed. The Argonaute (AGO) proteins for loading small RNAs are duplicated in both millipedes, but unlike in insects, an AGO duplicate has become a pseudogene. Evidence of post-transcriptional modification in small RNAs-including species-specific microRNA arm switching-providing differential gene regulation is also obtained. Millipedes possesses a unique ozadene defensive gland unlike the venomous forcipules found in centipedes. We identify sets of genes associated with the ozadene that play roles in chemical defence as well as antimicrobial activity. Macro-synteny analyses revealed highly conserved genomic blocks between the 2 millipedes and deuterostomes. Collectively, our analyses of millipede genomes reveal that a series of unique adaptations have occurred in this major lineage of arthropod diversity. The 2 high-quality millipede genomes provided here shed new light on the conserved and lineage-specific features of millipedes and centipedes. These findings demonstrate the importance of the consideration of both centipede and millipede genomes-and in particular the reconstruction of the myriapod ancestral situation-for future research to improve understanding of arthropod evolution, and animal evolutionary genomics more widely.


Assuntos
Adaptação Biológica/genética , Artrópodes , Evolução Molecular , Genoma/genética , Animais , Artrópodes/classificação , Artrópodes/genética , Sequência de Bases , Elementos de DNA Transponíveis/genética , Genes Homeobox , Genoma de Inseto , Insetos/classificação , Insetos/genética , MicroRNAs/genética , Filogenia , Sintenia
7.
PLoS One ; 15(8): e0232994, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32866155

RESUMO

Transposable elements (TEs) are mobile genetic elements in eukaryotic genomes. Recent research highlights the important role of TEs in the embryogenesis, neurodevelopment, and immune functions. However, there is a lack of a one-stop and easy to use computational pipeline for expression analysis of both genes and locus-specific TEs from RNA-Seq data. Here, we present GeneTEFlow, a fully automated, reproducible and platform-independent workflow, for the comprehensive analysis of gene and locus-specific TEs expression from RNA-Seq data employing Nextflow and Docker technologies. This application will help researchers more easily perform integrated analysis of both gene and TEs expression, leading to a better understanding of roles of gene and TEs regulation in human diseases. GeneTEFlow is freely available at https://github.com/zhongw2/GeneTEFlow.


Assuntos
Elementos de DNA Transponíveis , RNA-Seq/estatística & dados numéricos , Software , Biologia Computacional , Bases de Dados de Ácidos Nucleicos/estatística & dados numéricos , Perfilação da Expressão Gênica/estatística & dados numéricos , Genoma Humano , Humanos , Fluxo de Trabalho
8.
Nat Commun ; 11(1): 4079, 2020 08 14.
Artigo em Inglês | MEDLINE | ID: mdl-32796936

RESUMO

DNA methylation is an epigenetic modification that specifies the basic state of pluripotent stem cells and regulates the developmental transition from stem cells to various cell types. In flowering plants, the shoot apical meristem (SAM) contains a pluripotent stem cell population which generates the aerial part of plants including the germ cells. Under appropriate conditions, the SAM undergoes a developmental transition from a leaf-forming vegetative SAM to an inflorescence- and flower-forming reproductive SAM. While SAM characteristics are largely altered in this transition, the complete picture of DNA methylation remains elusive. Here, by analyzing whole-genome DNA methylation of isolated rice SAMs in the vegetative and reproductive stages, we show that methylation at CHH sites is kept high, particularly at transposable elements (TEs), in the vegetative SAM relative to the differentiated leaf, and increases in the reproductive SAM via the RNA-dependent DNA methylation pathway. We also show that half of the TEs that were highly methylated in gametes had already undergone CHH hypermethylation in the SAM. Our results indicate that changes in DNA methylation begin in the SAM long before germ cell differentiation to protect the genome from harmful TEs.


Assuntos
Metilação de DNA , Meristema/crescimento & desenvolvimento , Meristema/genética , Oryza/genética , Brotos de Planta/crescimento & desenvolvimento , Brotos de Planta/genética , Elementos de DNA Transponíveis , Biologia do Desenvolvimento , Epigenômica , Flores , Regulação da Expressão Gênica de Plantas , Inflorescência , Folhas de Planta/metabolismo , Proteínas de Plantas/genética
9.
Mol Cell ; 79(5): 758-767.e6, 2020 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-32755596

RESUMO

During proteotoxic stress, bacteria maintain critical processes like DNA replication while removing misfolded proteins, which are degraded by the Lon protease. Here, we show that in Caulobacter crescentus Lon controls deoxyribonucleoside triphosphate (dNTP) pools during stress through degradation of the transcription factor CcrM. Elevated dNTP/nucleotide triphosphate (NTP) ratios in Δlon cells protects them from deletion of otherwise essential deoxythymidine triphosphate (dTTP)-producing pathways and shields them from hydroxyurea-induced loss of dNTPs. Increased dNTP production in Δlon results from higher expression of ribonucleotide reductase driven by increased CcrM. We show that misfolded proteins can stabilize CcrM by competing for limited protease and that Lon-dependent control of dNTPs improves fitness during protein misfolding conditions. We propose that linking dNTP production with availability of Lon allows Caulobacter to maintain replication capacity when misfolded protein burden increases, such as during rapid growth. Because Lon recognizes misfolded proteins regardless of the stress, this mechanism allows for response to a variety of unanticipated conditions.


Assuntos
Caulobacter crescentus/metabolismo , Nucleotídeos/metabolismo , Protease La/metabolismo , Dobramento de Proteína , Proteínas de Bactérias/metabolismo , Caulobacter crescentus/enzimologia , Elementos de DNA Transponíveis , Didesoxinucleosídeos/metabolismo , Regulação Bacteriana da Expressão Gênica , Nucleotídeo Desaminases/genética , Nucleotídeo Desaminases/metabolismo , Ribonucleotídeo Redutases/metabolismo , Estresse Fisiológico , Fatores de Transcrição/metabolismo , Regulação para Cima
10.
Science ; 370(6513): 241-247, 2020 10 09.
Artigo em Inglês | MEDLINE | ID: mdl-32855215

RESUMO

Recent outbreaks of Ebola virus (EBOV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have exposed our limited therapeutic options for such diseases and our poor understanding of the cellular mechanisms that block viral infections. Using a transposon-mediated gene-activation screen in human cells, we identify that the major histocompatibility complex (MHC) class II transactivator (CIITA) has antiviral activity against EBOV. CIITA induces resistance by activating expression of the p41 isoform of invariant chain CD74, which inhibits viral entry by blocking cathepsin-mediated processing of the Ebola glycoprotein. We further show that CD74 p41 can block the endosomal entry pathway of coronaviruses, including SARS-CoV-2. These data therefore implicate CIITA and CD74 in host defense against a range of viruses, and they identify an additional function of these proteins beyond their canonical roles in antigen presentation.


Assuntos
Antígenos de Diferenciação de Linfócitos B/fisiologia , Betacoronavirus/fisiologia , Infecções por Coronavirus/imunologia , Ebolavirus/fisiologia , Doença pelo Vírus Ebola/imunologia , Antígenos de Histocompatibilidade Classe II/fisiologia , Interações Hospedeiro-Patógeno/imunologia , Proteínas Nucleares/fisiologia , Pneumonia Viral/imunologia , Transativadores/fisiologia , Internalização do Vírus , Antígenos de Diferenciação de Linfócitos B/genética , Linhagem Celular Tumoral , Infecções por Coronavirus/virologia , Elementos de DNA Transponíveis , Endossomos/virologia , Testes Genéticos , Doença pelo Vírus Ebola/virologia , Antígenos de Histocompatibilidade Classe II/genética , Interações Hospedeiro-Patógeno/genética , Humanos , Proteínas Nucleares/genética , Pandemias , Pneumonia Viral/virologia , Transativadores/genética , Transcrição Genética
11.
Nat Commun ; 11(1): 4058, 2020 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-32792480

RESUMO

Tomatoes come in a multitude of shapes and flavors despite a narrow genetic pool. Here, we leverage whole-genome resequencing data available for 602 cultivated and wild accessions to determine the contribution of transposable elements (TEs) to tomato diversity. We identify 6,906 TE insertions polymorphisms (TIPs), which result from the mobilization of 337 distinct TE families. Most TIPs are low frequency variants and TIPs are disproportionately located within or adjacent to genes involved in environmental responses. In addition, genic TE insertions tend to have strong transcriptional effects and they can notably lead to the generation of multiple transcript isoforms. Using genome-wide association studies (GWAS), we identify at least 40 TIPs robustly associated with extreme variation in major agronomic traits or secondary metabolites and in most cases, no SNP tags the TE insertion allele. Collectively, these findings highlight the unique role of TE mobilization in tomato diversification, with important implications for breeding.


Assuntos
Elementos de DNA Transponíveis/genética , Lycopersicon esculentum/genética , Evolução Molecular , Genoma de Planta/genética , Estudo de Associação Genômica Ampla , Polimorfismo Genético/genética
12.
Nat Protoc ; 15(8): 2705-2727, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32681154

RESUMO

Invasive fungal infections caused by Candida species are life threatening with high mortality, posing a severe public health threat. New technologies for rapid, genome-wide identification of virulence genes and therapeutic targets are urgently needed. Our recent engineering of a piggyBac (PB) transposon-mediated mutagenesis system in haploid Candida albicans provides a powerful discovery tool, which we anticipate should be adaptable to other haploid Candida species. In this protocol, we use haploid C. albicans as an example to present an improved version of the mutagenesis system and provide a detailed description of the protocol for constructing high-quality mutant libraries. We also describe a method for quantitative PB insertion site sequencing, PBISeq. The PBISeq library preparation procedure exploits tagmentation to quickly and efficiently construct sequencing libraries. Finally, we present a pipeline to analyze PB insertion sites in a de novo assembled genome of our engineered haploid C. albicans strain. The entire protocol takes ~7 d from transposition induction to having a final library ready for sequencing. This protocol is highly efficient and less labor intensive than alternative approaches and significantly accelerates genetic studies of Candida.


Assuntos
Candida/genética , Elementos de DNA Transponíveis/genética , Genoma Fúngico/genética , Haploidia , Mutagênese Insercional/métodos
13.
PLoS Comput Biol ; 16(7): e1007980, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32678849

RESUMO

Bacteria need to survive in a wide range of environments. Currently, there is an incomplete understanding of the genetic basis for mechanisms underpinning survival in stressful conditions, such as the presence of anti-microbials. Transposon directed insertion-site sequencing (TraDIS) is a powerful tool to identify genes and networks which are involved in survival and fitness under a given condition by simultaneously assaying the fitness of millions of mutants, thereby relating genotype to phenotype and contributing to an understanding of bacterial cell biology. A recent refinement of this approach allows the roles of essential genes in conditional stress survival to be inferred by altering their expression. These advancements combined with the rapidly falling costs of sequencing now allows comparisons between multiple experiments to identify commonalities in stress responses to different conditions. This capacity however poses a new challenge for analysis of multiple data sets in conjunction. To address this analysis need, we have developed 'AlbaTraDIS'; a software application for rapid large-scale comparative analysis of TraDIS experiments that predicts the impact of transposon insertions on nearby genes. AlbaTraDIS can identify genes which are up or down regulated, or inactivated, between multiple conditions, producing a filtered list of genes for further experimental validation as well as several accompanying data visualisations. We demonstrate the utility of our new approach by applying it to identify genes used by Escherichia coli to survive in a wide range of different concentrations of the biocide Triclosan. AlbaTraDIS identified all well characterised Triclosan resistance genes, including the primary target, fabI. A number of new loci were also implicated in Triclosan resistance and the predicted phenotypes for a selection of these were validated experimentally with results being consistent with predictions. AlbaTraDIS provides a simple and rapid method to analyse multiple transposon mutagenesis data sets allowing this technology to be used at large scale. To our knowledge this is the only tool currently available that can perform these tasks. AlbaTraDIS is written in Python 3 and is available under the open source licence GNU GPL 3 from https://github.com/quadram-institute-bioscience/albatradis.


Assuntos
Biologia Computacional , Elementos de DNA Transponíveis , Escherichia coli/genética , Mutagênese Insercional , Software , Algoritmos , Anti-Infecciosos/farmacologia , Farmacorresistência Bacteriana , Biblioteca Gênica , Genes Essenciais , Genoma Bacteriano , Genótipo , Sequenciamento de Nucleotídeos em Larga Escala , Mutação , Fenótipo , Biossíntese de Proteínas , Triclosan/farmacologia
14.
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
15.
Nat Commun ; 11(1): 3506, 2020 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-32665538

RESUMO

Acute myeloid leukemia (AML) is characterised by a series of genetic and epigenetic alterations that result in deregulation of transcriptional networks. One understudied source of transcriptional regulators are transposable elements (TEs), whose aberrant usage could contribute to oncogenic transcriptional circuits. However, the regulatory influence of TEs and their links to AML pathogenesis remain unexplored. Here we identify six endogenous retrovirus (ERV) families with AML-associated enhancer chromatin signatures that are enriched in binding of key regulators of hematopoiesis and AML pathogenesis. Using both locus-specific genetic editing and simultaneous epigenetic silencing of multiple ERVs, we demonstrate that ERV deregulation directly alters the expression of adjacent genes in AML. Strikingly, deletion or epigenetic silencing of an ERV-derived enhancer suppresses cell growth by inducing apoptosis in leukemia cell lines. This work reveals that ERVs are a previously unappreciated source of AML enhancers that may be exploited by cancer cells to help drive tumour heterogeneity and evolution.


Assuntos
Cromatina/metabolismo , Leucemia Mieloide Aguda/genética , Animais , Linhagem Celular , Cromatina/genética , Elementos de DNA Transponíveis/genética , Retrovirus Endógenos/genética , Epigênese Genética/genética , Redes Reguladoras de Genes/genética , Redes Reguladoras de Genes/fisiologia , Genoma Humano/genética , Humanos , Sequências Repetitivas Dispersas/genética
16.
J Med Microbiol ; 69(8): 1089-1094, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32692646

RESUMO

Introduction. The bla CTX-M-3 gene has rarely been reported in Morganella morganii strains and its genetic environment has not yet been investigated.Aim. To identify the bla CTX-M-3 gene in M. morganii isolated from swine and characterize its genetic environment.Methodology. A M. morganii isolate (named MM1L5) from a deceased swine was identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and subjected to antimicrobial susceptibility testing. The bla genes were detected and then the genetic location and environment of bla CTX-M-3 were investigated by Southern blot and PCR mapping, respectively. The M. morganii bla CTX-M-3 gene was cloned and expressed in Escherichia coli.Results. Isolate MM1L5 harboured the bla CTX-M-3 and bla TEM-1 genes. The bla CTX-M-3 gene, located on the chromosome, was co-carried with an IS26 and bla TEM-1 gene by a novel 6361 bp IS26-flanked composite transposon, designated Tn6741. This transposon consisted of a novel bla CTX-M-3-containing module, IS26-ΔISEcp1-bla CTX-M-3-Δorf477-IS26 (named Tn6710), and a bla TEM-1-containing module, IS26-Δorf477-bla TEM-1-tnpR-IS26, differing from previous reports. Phylogenetic analysis showed a significant variation based on the sequence of Tn6741, as compared to those of other related transposons. Interestingly, although the cloned bla CTX-M-3 gene could confer resistance to ceftiofur, cefquinome, ceftriaxone and cefotaxime, one amino acid substitution (Ile-142-Thr) resulted in a significant reduction of resistance to these antimicrobials.Conclusion. This is the first time that bla CTX-M-3 has been identified on a chromosome from a M. morganii isolate. Furthermore, the bla CTX-M-3 gene was located with an IS26 element and bla TEM-1 gene on a novel IS26-flanked composite transposon, Tn6741, suggesting that Tn6741 might act as a reservoir for the bla CTX-M-3 and bla TEM-1 genes and may become an important vehicle for their dissemination among M. morganii.


Assuntos
Elementos de DNA Transponíveis/genética , Morganella morganii/genética , beta-Lactamases/genética , Animais , Anti-Infecciosos/farmacologia , Sequência de Bases , Clonagem Molecular , Morganella morganii/classificação , Morganella morganii/efeitos dos fármacos , Filogenia , Reação em Cadeia da Polimerase , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Suínos
17.
J Vis Exp ; (161)2020 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-32716393

RESUMO

Transposon sequencing (Tn-seq) is a powerful method that combines transposon mutagenesis and massive parallel sequencing to identify genes and pathways that contribute to bacterial fitness under a wide range of environmental conditions. Tn-seq applications are extensive and have not only enabled examination of genotype-phenotype relationships at an organism level but also at the population, community and systems levels. Gram-negative bacteria are highly associated with antimicrobial resistance phenotypes, which has increased incidents of antibiotic treatment failure. Antimicrobial resistance is defined as bacterial growth in the presence of otherwise lethal antibiotics. The "last-line" antimicrobial colistin is used to treat Gram-negative bacterial infections. However, several Gram-negative pathogens, including Acinetobacter baumannii can develop colistin resistance through a range of molecular mechanisms, some of which were characterized using Tn-seq. Furthermore, signal transduction pathways that regulate colistin resistance vary within Gram-negative bacteria. Here we propose an efficient method of transposon mutagenesis in A. baumannii that streamlines generation of a saturating transposon insertion library and amplicon library construction by eliminating the need for restriction enzymes, adapter ligation, and gel purification. The methods described herein will enable in-depth analysis of molecular determinants that contribute to A. baumannii fitness when challenged with colistin. The protocol is also applicable to other Gram-negative ESKAPE pathogens, which are primarily associated with drug resistant hospital-acquired infections.


Assuntos
Acinetobacter baumannii/genética , Elementos de DNA Transponíveis , DNA Bacteriano/genética , Biblioteca Gênica , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Mutagênese
18.
PLoS Genet ; 16(7): e1008931, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32644999

RESUMO

Shigella species are specialised lineages of Escherichia coli that have converged to become human-adapted and cause dysentery by invading human gut epithelial cells. Most studies of Shigella evolution have been restricted to comparisons of single representatives of each species; and population genomic studies of individual Shigella species have focused on genomic variation caused by single nucleotide variants and ignored the contribution of insertion sequences (IS) which are highly prevalent in Shigella genomes. Here, we investigate the distribution and evolutionary dynamics of IS within populations of Shigella dysenteriae Sd1, Shigella sonnei and Shigella flexneri. We find that five IS (IS1, IS2, IS4, IS600 and IS911) have undergone expansion in all Shigella species, creating substantial strain-to-strain variation within each population and contributing to convergent patterns of functional gene loss within and between species. We find that IS expansion and genome degradation are most advanced in S. dysenteriae and least advanced in S. sonnei; and using genome-scale models of metabolism we show that Shigella species display convergent loss of core E. coli metabolic capabilities, with S. sonnei and S. flexneri following a similar trajectory of metabolic streamlining to that of S. dysenteriae. This study highlights the importance of IS to the evolution of Shigella and provides a framework for the investigation of IS dynamics and metabolic reduction in other bacterial species.


Assuntos
Elementos de DNA Transponíveis/genética , Disenteria/genética , Evolução Molecular , Shigella dysenteriae/genética , DNA Bacteriano/genética , Disenteria/microbiologia , Escherichia coli/genética , Escherichia coli/patogenicidade , Genoma Bacteriano/genética , Humanos , Shigella dysenteriae/patogenicidade
19.
PLoS Genet ; 16(7): e1008872, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32673310

RESUMO

Transposable elements (TEs) are genomic parasites that selfishly replicate at the expense of host fitness. Fifty years of evolutionary studies of TEs have concentrated on the deleterious genetic effects of TEs, such as their effects on disrupting genes and regulatory sequences. However, a flurry of recent work suggests that there is another important source of TEs' harmful effects-epigenetic silencing. Host genomes typically silence TEs by the deposition of repressive epigenetic marks. While this silencing reduces the selfish replication of TEs and should benefit hosts, a picture is emerging that the epigenetic silencing of TEs triggers inadvertent spreading of repressive marks to otherwise expressed neighboring genes, ultimately jeopardizing host fitness. In this Review, we provide a long-overdue overview of the recent genome-wide evidence for the presence and prevalence of TEs' epigenetic effects, highlighting both the similarities and differences across mammals, insects, and plants. We lay out the current understanding of the functional and fitness consequences of TEs' epigenetic effects, and propose possible influences of such effects on the evolution of both hosts and TEs themselves. These unique evolutionary consequences indicate that TEs' epigenetic effect is not only a crucial component of TE biology but could also be a significant contributor to genome function and evolution.


Assuntos
Elementos de DNA Transponíveis/genética , Epigênese Genética , Evolução Molecular , Inativação Gênica , Animais , Repressão Epigenética/genética , Regulação da Expressão Gênica/genética , Insetos/genética , Mamíferos/genética , Plantas/genética
20.
Nat Commun ; 11(1): 3739, 2020 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-32719317

RESUMO

The PIWI protein MIWI2 and its associated PIWI-interacting RNAs (piRNAs) instruct DNA methylation of young active transposable elements (TEs) in the male germline. piRNAs are proposed to recruit MIWI2 to the transcriptionally active TE loci by base pairing to nascent transcripts, however the downstream mechanisms and effector proteins utilized by MIWI2 in directing de novo TE methylation remain incompletely understood. Here, we show that MIWI2 associates with TEX15 in foetal gonocytes. TEX15 is predominantly a nuclear protein that is not required for piRNA biogenesis but is essential for piRNA-directed TE de novo methylation and silencing. In summary, TEX15 is an essential executor of mammalian piRNA-directed DNA methylation.


Assuntos
Proteínas Argonauta/metabolismo , Proteínas de Ciclo Celular/metabolismo , Metilação de DNA/genética , Elementos de DNA Transponíveis/genética , Inativação Gênica , Animais , Proteínas Argonauta/genética , Feminino , Feto/citologia , Genoma , Células Germinativas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ligação Proteica , Testículo/metabolismo
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