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1.
Nature ; 620(7975): 863-872, 2023 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-37587336

RESUMO

Cells undergo a major epigenome reconfiguration when reprogrammed to human induced pluripotent stem cells (hiPS cells). However, the epigenomes of hiPS cells and human embryonic stem (hES) cells differ significantly, which affects hiPS cell function1-8. These differences include epigenetic memory and aberrations that emerge during reprogramming, for which the mechanisms remain unknown. Here we characterized the persistence and emergence of these epigenetic differences by performing genome-wide DNA methylation profiling throughout primed and naive reprogramming of human somatic cells to hiPS cells. We found that reprogramming-induced epigenetic aberrations emerge midway through primed reprogramming, whereas DNA demethylation begins early in naive reprogramming. Using this knowledge, we developed a transient-naive-treatment (TNT) reprogramming strategy that emulates the embryonic epigenetic reset. We show that the epigenetic memory in hiPS cells is concentrated in cell of origin-dependent repressive chromatin marked by H3K9me3, lamin-B1 and aberrant CpH methylation. TNT reprogramming reconfigures these domains to a hES cell-like state and does not disrupt genomic imprinting. Using an isogenic system, we demonstrate that TNT reprogramming can correct the transposable element overexpression and differential gene expression seen in conventional hiPS cells, and that TNT-reprogrammed hiPS and hES cells show similar differentiation efficiencies. Moreover, TNT reprogramming enhances the differentiation of hiPS cells derived from multiple cell types. Thus, TNT reprogramming corrects epigenetic memory and aberrations, producing hiPS cells that are molecularly and functionally more similar to hES cells than conventional hiPS cells. We foresee TNT reprogramming becoming a new standard for biomedical and therapeutic applications and providing a novel system for studying epigenetic memory.


Assuntos
Reprogramação Celular , Epigênese Genética , Células-Tronco Pluripotentes Induzidas , Humanos , Cromatina/genética , Cromatina/metabolismo , Desmetilação do DNA , Metilação de DNA , Elementos de DNA Transponíveis , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Embrionárias Humanas/citologia , Células-Tronco Embrionárias Humanas/metabolismo , Lamina Tipo B
2.
Mol Cell ; 80(5): 915-928.e5, 2020 12 03.
Artigo em Inglês | MEDLINE | ID: mdl-33186547

RESUMO

Transposable elements (TEs) drive genome evolution and are a notable source of pathogenesis, including cancer. While CpG methylation regulates TE activity, the locus-specific methylation landscape of mobile human TEs has to date proven largely inaccessible. Here, we apply new computational tools and long-read nanopore sequencing to directly infer CpG methylation of novel and extant TE insertions in hippocampus, heart, and liver, as well as paired tumor and non-tumor liver. As opposed to an indiscriminate stochastic process, we find pronounced demethylation of young long interspersed element 1 (LINE-1) retrotransposons in cancer, often distinct to the adjacent genome and other TEs. SINE-VNTR-Alu (SVA) retrotransposons, including their internal tandem repeat-associated CpG island, are near-universally methylated. We encounter allele-specific TE methylation and demethylation of aberrantly expressed young LINE-1s in normal tissues. Finally, we recover the complete sequences of tumor-specific LINE-1 insertions and their retrotransposition hallmarks, demonstrating how long-read sequencing can simultaneously survey the epigenome and detect somatic TE mobilization.


Assuntos
Metilação de DNA , Elementos de DNA Transponíveis , DNA de Neoplasias , Epigênese Genética , Epigenoma , Regulação Neoplásica da Expressão Gênica , Elementos Nucleotídeos Longos e Dispersos , Sequenciamento por Nanoporos , Neoplasias , DNA de Neoplasias/genética , DNA de Neoplasias/metabolismo , Feminino , Perfilação da Expressão Gênica , Humanos , Pessoa de Meia-Idade , Neoplasias/genética , Neoplasias/metabolismo , Especificidade de Órgãos
3.
Methods Mol Biol ; 2607: 151-171, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36449163

RESUMO

Over the past 20 years, high-throughput genomic assays have fundamentally changed how transposable elements (TEs) are studied. While short-read DNA sequencing has been at the heart of these efforts, novel technologies that generate longer reads are driving a shift in the field. Long-read sequencing now permits locus-specific approaches to locate individual TE insertions and understand their epigenetic and transcriptional regulation, while still profiling TE activity genome-wide. Here we provide detailed guidelines to implement Oxford Nanopore Technologies (ONT) sequencing to identify polymorphic TE insertions and profile TE epigenetic landscapes. Using human long interspersed element-1 (LINE-1, L1) as an example, we explain the procedures involved, including final visualization, and potential bottlenecks and pitfalls. ONT sequencing will be, in our view, a workhorse technology for the foreseeable future in the TE field.


Assuntos
Sequenciamento por Nanoporos , Humanos , Elementos de DNA Transponíveis/genética , Epigênese Genética , Epigenômica , Elementos Nucleotídeos Longos e Dispersos/genética
4.
Pharmaceuticals (Basel) ; 16(8)2023 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-37631079

RESUMO

Intravenous (IV) ketamine and FDA-approved intranasal (IN) esketamine are increasingly used for treatment-resistant depression (TRD). Preliminary studies have suggested a synergistic effect of ketamine and lamotrigine, although the data are inconclusive. Herein, we report the response to serial ketamine/esketamine treatment among patients with TRD with or without lamotrigine therapy. In this historical cohort study, we included adult patients with TRD who received serial IV racemic ketamine (0.5 mg/kg over 40-100 min) or IN esketamine (56/84 mg) treatments. A change in depressive symptoms was assessed using the 16-item Quick Inventory of Depressive Symptomatology self-report (QIDS-SR) scale. There were no significant differences in response or remission rates among the patients on or not on lamotrigine during the ketamine/esketamine treatments. For a percent change in the QIDS-SR from baseline, no interaction was found between the lamotrigine groups and treatment number (p = 0.70), nor the overall effect of the group (p = 0.38). There was a trend towards lower dissociation (based on the CADSS score) among current lamotrigine users, especially in patients who received IV ketamine. A major limitation is the limited number of patients taking lamotrigine (n = 13). This preliminary study provides insufficient evidence that continuing lamotrigine therapy attenuates the antidepressant effect of repeated ketamine/esketamine; however, there seems to be a signal toward attenuating dissociation with lamotrigine in patients receiving serial ketamine treatments. Further observational studies or randomized controlled trials are needed to replicate these findings.

6.
Cell Rep ; 36(7): 109530, 2021 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-34380018

RESUMO

A recent study proposed that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) hijacks the LINE-1 (L1) retrotransposition machinery to integrate into the DNA of infected cells. If confirmed, this finding could have significant clinical implications. Here, we apply deep (>50×) long-read Oxford Nanopore Technologies (ONT) sequencing to HEK293T cells infected with SARS-CoV-2 and do not find the virus integrated into the genome. By examining ONT data from separate HEK293T cultivars, we completely resolve 78 L1 insertions arising in vitro in the absence of L1 overexpression systems. ONT sequencing applied to hepatitis B virus (HBV)-positive liver cancer tissues located a single HBV insertion. These experiments demonstrate reliable resolution of retrotransposon and exogenous virus insertions by ONT sequencing. That we find no evidence of SARS-CoV-2 integration suggests that such events are, at most, extremely rare in vivo and therefore are unlikely to drive oncogenesis or explain post-recovery detection of the virus.


Assuntos
COVID-19/virologia , DNA Viral/genética , Genoma Humano , SARS-CoV-2/genética , Análise de Sequência de DNA , Integração Viral , Idoso , Animais , COVID-19/diagnóstico , Carcinoma Hepatocelular/virologia , Chlorocebus aethiops , Células HEK293 , Vírus da Hepatite B/genética , Interações Hospedeiro-Patógeno , Humanos , Neoplasias Hepáticas/virologia , Elementos Nucleotídeos Longos e Dispersos , Masculino , Sequenciamento por Nanoporos , Células Vero
7.
FEMS Microbiol Lett ; 365(9)2018 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-29579234

RESUMO

Pseudomonas baetica strain a390T is the type strain of this recently described species and here we present its high-contiguity draft genome. To celebrate the 16th International Conference on Pseudomonas, the genome of P. baetica strain a390T was sequenced using a unique combination of Ion Torrent semiconductor and Oxford Nanopore methods as part of a collaborative community-led project. The use of high-quality Ion Torrent sequences with long Nanopore reads gave rapid, high-contiguity and -quality, 16-contig genome sequence. Whole genome phylogenetic analysis places P. baetica within the P. koreensis clade of the P. fluorescens group. Comparison of the main genomic features of P. baetica with a variety of other Pseudomonas spp. suggests that it is a highly adaptable organism, typical of the genus. This strain was originally isolated from the liver of a diseased wedge sole fish, and genotypic and phenotypic analyses show that it is tolerant to osmotic stress and to oxytetracycline.


Assuntos
Doenças dos Peixes/microbiologia , Genômica/métodos , Infecções por Pseudomonas/veterinária , Pseudomonas/genética , Análise de Sequência de DNA/métodos , Animais , Genoma Bacteriano , Genômica/instrumentação , Nanoporos , Fenótipo , Filogenia , Pseudomonas/classificação , Pseudomonas/isolamento & purificação , Infecções por Pseudomonas/microbiologia , Semicondutores , Análise de Sequência de DNA/instrumentação
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