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Understanding the impact of DNA methylation within different disease contexts often requires accurate assessment of these modifications in a genome-wide fashion. Frequently, patient-derived tissues stored in long-term hospital tissue banks have been preserved using formalin-fixation paraffin-embedding (FFPE). While these samples can comprise valuable resources for studying disease, the fixation process ultimately compromises the DNA's integrity and leads to degradation. Degraded DNA can complicate CpG methylome profiling using traditional techniques, particularly when performing methylation-sensitive restriction enzyme sequencing (MRE-seq), yielding high backgrounds and resulting in lowered library complexity. Here, we describe Capture MRE-seq, a new MRE-seq protocol tailored to preserving unmethylated CpG information when using samples with highly degraded DNA. The results using Capture MRE-seq correlate well (0.92) with traditional MRE-seq calls when profiling non-degraded samples, and can recover unmethylated regions in highly degraded samples when traditional MRE-seq fails, which we validate using bisulfite sequencing-based data (WGBS) as well as methylated DNA immunoprecipitation followed by sequencing (MeDIP-seq).
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Metilação de DNA , DNA , Humanos , Ilhas de CpG , DNA/genética , Análise de Sequência de DNA/métodos , GenomaRESUMO
PURPOSE: We hypothesized that resistance to hypomethylating agents (HMA) among patients with myelodysplastic syndrome (MDS) and chronic myelomonocytic leukemia (CMML) would be overcome by combining a programmed death-ligand 1 antibody with an HMA. PATIENTS AND METHODS: We conducted a Phase I/II, multicenter clinical trial for patients with MDS not achieving an International Working Group response after at least 4 cycles of an HMA ("refractory") or progressing after a response ("relapsed") with 3+ or higher risk MDS by the revised International Prognostic Scoring System (IPSS-R) and CMML-1 or -2. Phase I consisted of a 3+3 dose-escalation design beginning with guadecitabine at 30 mg/m2 and escalating to 60 mg/m2 Days 1 to 5 with fixed-dose atezolizumab: 840 mg intravenously Days 8 and 22 of a 28-day cycle. Primary endpoints were safety and tolerability; secondary endpoints were overall response rate (ORR) and survival. RESULTS: Thirty-three patients, median age 73 (range 54-85), were treated. Thirty patients had MDS and 3 had CMML, with 30% relapsed and 70% refractory. No dose-limiting toxicities were observed in Phase I. There were 3 (9%) deaths in ≤ 30 days. Five patients (16%) came off study for drug-related toxicity. Immune-related adverse events (IRAE) occurred in 12 (36%) patients (4 grade 3, 3 grade 2, and 5 grade1). ORR was 33% [95% confidence interval (CI), 19%-52%] with 2 complete remission (CR), 3 hematologic improvement, 5 marrow CR, and 1 partial remission. Median overall survival was 15.1 (95% CI, 8.5-25.3) months. CONCLUSIONS: Guadecitabine with atezolizumab has modest efficacy with manageable IRAEs and typical cytopenia-related safety concerns for patients with relapsed or refractory MDS and CMML.
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Leucemia Mielomonocítica Crônica , Síndromes Mielodisplásicas , Humanos , Idoso , Leucemia Mielomonocítica Crônica/tratamento farmacológico , Resultado do Tratamento , Linfócitos T , Síndromes Mielodisplásicas/tratamento farmacológicoRESUMO
Non-small cell lung cancer (NSCLC) is one of the most commonly diagnosed and deadliest cancers worldwide, with roughly half of all patients initially presenting with both primary and metastatic disease. While the major events in the metastatic cascade have been identified, a mechanistic understanding of how NSCLC routinely and successfully colonizes the brain is largely unknown. Recent studies have begun demonstrating the role of epigenetic misregulation during tumorigenesis and metastasis, including widespread changes in DNA methylation and histone modifications. To better understand the role of altered DNA methylation in NSCLC metastasis to the brain, we measured DNA methylation during disease progression for 12 patients, globally profiling the methylation status of normal lung, primary lung tumor, and brain metastasis samples. The variation in methylation was similar during metastatic spread and primary tumorigenesis but less coordinated across genomic features during metastasis. The greatest recurrent changes during metastatic progression were methylation gains in DNA methylation valleys (DMV) harboring the constitutive heterochromatin mark H3K9me3 as well as bivalent marks H3K27me3 and H3K4me1. In a lymph node-derived cancer cell line, EZH2 binding within DMVs was lost, accompanied by an increase in DNA methylation, exemplifying epigenetic switching. The vast majority of the differentially methylated region-associated DMVs harbored developmental genes, suggesting that altered epigenetic regulation of developmentally important genes may confer a selective advantage during metastatic progression. The characterization of epigenetic changes during NSCLC brain metastasis identified recurrent methylation patterns that may be prognostic biomarkers and contributors to disease progression. SIGNIFICANCE: Altered DNA methylation in lung cancer brain metastases corresponds with loss of EZH2 occupancy at developmental genes, which could promote stem-like phenotypes permissive of dissemination and survival in different microenvironments.
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Neoplasias Encefálicas , Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Neoplasias Encefálicas/genética , Carcinogênese/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Metilação de DNA , Progressão da Doença , Epigênese Genética , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Microambiente TumoralRESUMO
BACKGROUND: Zebrafish pigment cell differentiation provides an attractive model for studying cell fate progression as a neural crest progenitor engenders diverse cell types, including two morphologically distinct pigment cells: black melanophores and reflective iridophores. Nontrivial classical genetic and transcriptomic approaches have revealed essential molecular mechanisms and gene regulatory circuits that drive neural crest-derived cell fate decisions. However, how the epigenetic landscape contributes to pigment cell differentiation, especially in the context of iridophore cell fate, is poorly understood. RESULTS: We chart the global changes in the epigenetic landscape, including DNA methylation and chromatin accessibility, during neural crest differentiation into melanophores and iridophores to identify epigenetic determinants shaping cell type-specific gene expression. Motif enrichment in the epigenetically dynamic regions reveals putative transcription factors that might be responsible for driving pigment cell identity. Through this effort, in the relatively uncharacterized iridophores, we validate alx4a as a necessary and sufficient transcription factor for iridophore differentiation and present evidence on alx4a's potential regulatory role in guanine synthesis pathway. CONCLUSIONS: Pigment cell fate is marked by substantial DNA demethylation events coupled with dynamic chromatin accessibility to potentiate gene regulation through cis-regulatory control. Here, we provide a multi-omic resource for neural crest differentiation into melanophores and iridophores. This work led to the discovery and validation of iridophore-specific alx4a transcription factor.
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Diferenciação Celular/genética , Cromatóforos/metabolismo , Epigênese Genética , Melanóforos/metabolismo , Peixe-Zebra/genética , Animais , Cromatina/metabolismo , Ilhas de CpG , Metilação de DNA , Redes Reguladoras de Genes , Crista Neural/citologia , Crista Neural/metabolismo , Sequências Reguladoras de Ácido Nucleico , Fatores de Transcrição/metabolismo , Fatores de Transcrição/fisiologia , Transcrição Gênica , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/fisiologiaRESUMO
Increased appreciation of 5-hydroxymethylcytosine (5hmC) as a stable epigenetic mark, which defines cell identity and disease progress, has engendered a need for cost-effective, but high-resolution, 5hmC mapping technology. Current enrichment-based technologies provide cheap but low-resolution and relative enrichment of 5hmC levels, while single-base resolution methods can be prohibitively expensive to scale up to large experiments. To address this problem, we developed a deep learning-based method, "DeepH&M," which integrates enrichment and restriction enzyme sequencing methods to simultaneously estimate absolute hydroxymethylation and methylation levels at single-CpG resolution. Using 7-week-old mouse cerebellum data for training the DeepH&M model, we demonstrated that the 5hmC and 5mC levels predicted by DeepH&M were in high concordance with whole-genome bisulfite-based approaches. The DeepH&M model can be applied to 7-week-old frontal cortex and 79-week-old cerebellum, revealing the robust generalizability of this method to other tissues from various biological time points.
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Metilação de DNA , Epigenômica , Animais , Cerebelo , Epigênese Genética , Genoma , CamundongosRESUMO
BACKGROUND: Zebrafish can faithfully regenerate injured fins through the formation of a blastema, a mass of proliferative cells that can grow and develop into the lost body part. After amputation, various cell types contribute to blastema formation, where each cell type retains fate restriction and exclusively contributes to regeneration of its own lineage. Epigenetic changes that are associated with lineage restriction during regeneration remain underexplored. RESULTS: We produce epigenome maps, including DNA methylation and chromatin accessibility, as well as transcriptomes, of osteoblasts and other cells in uninjured and regenerating fins. This effort reveals regeneration as a process of highly dynamic and orchestrated transcriptomic and chromatin accessibility changes, coupled with stably maintained lineage-specific DNA methylation. The epigenetic signatures also reveal many novel regeneration-specific enhancers, which are experimentally validated. Regulatory networks important for regeneration are constructed through integrative analysis of the epigenome map, and a knockout of a predicted upstream regulator disrupts normal regeneration, validating our prediction. CONCLUSION: Our study shows that lineage-specific DNA methylation signatures are stably maintained during regeneration, and regeneration enhancers are preset as hypomethylated before injury. In contrast, chromatin accessibility is dynamically changed during regeneration. Many enhancers driving regeneration gene expression as well as upstream regulators of regeneration are identified and validated through integrative epigenome analysis.
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Nadadeiras de Animais/metabolismo , Linhagem da Célula , Metilação de DNA , Epigenoma , Regeneração , Nadadeiras de Animais/citologia , Nadadeiras de Animais/fisiologia , Animais , Montagem e Desmontagem da Cromatina , Redes Reguladoras de Genes , Osteoblastos/citologia , Osteoblastos/metabolismo , Peixe-ZebraRESUMO
Following publication of the original paper [1], an error was reported in the processing of Fig. 2. The correct Fig. 2 is supplied below and the original article [1] has been corrected. The publishers apologize for the error.
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BACKGROUND: Transposable elements (TEs) make up half of mammalian genomes and shape genome regulation by harboring binding sites for regulatory factors. These include binding sites for architectural proteins, such as CTCF, RAD21, and SMC3, that are involved in tethering chromatin loops and marking domain boundaries. The 3D organization of the mammalian genome is intimately linked to its function and is remarkably conserved. However, the mechanisms by which these structural intricacies emerge and evolve have not been thoroughly probed. RESULTS: Here, we show that TEs contribute extensively to both the formation of species-specific loops in humans and mice through deposition of novel anchoring motifs, as well as to the maintenance of conserved loops across both species through CTCF binding site turnover. The latter function demonstrates the ability of TEs to contribute to genome plasticity and reinforce conserved genome architecture as redundant loop anchors. Deleting such candidate TEs in human cells leads to the collapse of conserved loop and domain structures. These TEs are also marked by reduced DNA methylation and bear mutational signatures of hypomethylation through evolutionary time. CONCLUSIONS: TEs have long been considered a source of genetic innovation. By examining their contribution to genome topology, we show that TEs can contribute to regulatory plasticity by inducing redundancy and potentiating genetic drift locally while conserving genome architecture globally, revealing a paradigm for defining regulatory conservation in the noncoding genome beyond classic sequence-level conservation.
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Cromossomos de Mamíferos/química , Sequências Repetitivas Dispersas , Animais , Sítios de Ligação , Fator de Ligação a CCCTC/metabolismo , Linhagem Celular , Cromatina/química , Humanos , CamundongosRESUMO
In the version of this article initially published, grant PF-17-201-01-TBG from the American Cancer Society to author Erica C. Pehrsson was not included in the Acknowledgements. The error has been corrected in the HTML and PDF versions of the article.
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Transposable elements (TEs) are an abundant and rich genetic resource of regulatory sequences1-3. Cryptic regulatory elements within TEs can be epigenetically reactivated in cancer to influence oncogenesis in a process termed onco-exaptation4. However, the prevalence and impact of TE onco-exaptation events across cancer types are poorly characterized. Here, we analyzed 7,769 tumors and 625 normal datasets from 15 cancer types, identifying 129 TE cryptic promoter-activation events involving 106 oncogenes across 3,864 tumors. Furthermore, we interrogated the AluJb-LIN28B candidate: the genetic deletion of the TE eliminated oncogene expression, while dynamic DNA methylation modulated promoter activity, illustrating the necessity and sufficiency of a TE for oncogene activation. Collectively, our results characterize the global profile of TE onco-exaptation and highlight this prevalent phenomenon as an important mechanism for promiscuous oncogene activation and ultimately tumorigenesis.
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Elementos de DNA Transponíveis/genética , Neoplasias/genética , Oncogenes/genética , Linhagem Celular , Linhagem Celular Tumoral , Metilação de DNA/genética , Evolução Molecular , Células HEK293 , Humanos , Células K562 , Regiões Promotoras Genéticas/genética , Sequências Reguladoras de Ácido Nucleico/genéticaRESUMO
This corrects the article DOI: 10.1038/ng.3889.
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BACKGROUND: Uncovering mechanisms of epigenome evolution is an essential step towards understanding the evolution of different cellular phenotypes. While studies have confirmed DNA methylation as a conserved epigenetic mechanism in mammalian development, little is known about the conservation of tissue-specific genome-wide DNA methylation patterns. RESULTS: Using a comparative epigenomics approach, we identified and compared the tissue-specific DNA methylation patterns of rat against those of mouse and human across three shared tissue types. We confirmed that tissue-specific differentially methylated regions are strongly associated with tissue-specific regulatory elements. Comparisons between species revealed that at a minimum 11-37% of tissue-specific DNA methylation patterns are conserved, a phenomenon that we define as epigenetic conservation. Conserved DNA methylation is accompanied by conservation of other epigenetic marks including histone modifications. Although a significant amount of locus-specific methylation is epigenetically conserved, the majority of tissue-specific DNA methylation is not conserved across the species and tissue types that we investigated. Examination of the genetic underpinning of epigenetic conservation suggests that primary sequence conservation is a driving force behind epigenetic conservation. In contrast, evolutionary dynamics of tissue-specific DNA methylation are best explained by the maintenance or turnover of binding sites for important transcription factors. CONCLUSIONS: Our study extends the limited literature of comparative epigenomics and suggests a new paradigm for epigenetic conservation without genetic conservation through analysis of transcription factor binding sites.
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Sequência Conservada , Metilação de DNA/genética , Animais , Sítios de Ligação , Epigenômica , Evolução Molecular , Humanos , Camundongos , Especificidade de Órgãos , Ratos , Fatores de Transcrição/metabolismoRESUMO
Several mechanisms of action have been proposed for DNA methyltransferase and histone deacetylase inhibitors (DNMTi and HDACi), primarily based on candidate-gene approaches. However, less is known about their genome-wide transcriptional and epigenomic consequences. By mapping global transcription start site (TSS) and chromatin dynamics, we observed the cryptic transcription of thousands of treatment-induced non-annotated TSSs (TINATs) following DNMTi and HDACi treatment. The resulting transcripts frequently splice into protein-coding exons and encode truncated or chimeric ORFs translated into products with predicted abnormal or immunogenic functions. TINAT transcription after DNMTi treatment coincided with DNA hypomethylation and gain of classical promoter histone marks, while HDACi specifically induced a subset of TINATs in association with H2AK9ac, H3K14ac, and H3K23ac. Despite this mechanistic difference, both inhibitors convergently induced transcription from identical sites, as we found TINATs to be encoded in solitary long terminal repeats of the ERV9/LTR12 family, which are epigenetically repressed in virtually all normal cells.
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DNA (Citosina-5-)-Metiltransferases/antagonistas & inibidores , Proteínas Quinases Associadas com Morte Celular/genética , Código das Histonas , Inibidores de Histona Desacetilases/farmacologia , Sequências Repetidas Terminais/genética , Sítio de Iniciação de Transcrição/efeitos dos fármacos , Processamento Alternativo/genética , Animais , Benzimidazóis/farmacologia , Linhagem Celular Tumoral , DNA (Citosina-5-)-Metiltransferase 1 , DNA (Citosina-5-)-Metiltransferases/fisiologia , Metilação de DNA , Proteínas Quinases Associadas com Morte Celular/antagonistas & inibidores , Repressão Epigenética , Éxons/genética , Feminino , Perfilação da Expressão Gênica , Inativação Gênica , Humanos , Ácidos Hidroxâmicos/farmacologia , Íntrons/genética , Camundongos , Camundongos Nus , Interferência de RNA , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , VorinostatRESUMO
Empirical models of sequence evolution have spurred progress in the field of evolutionary genetics for decades. We are now realizing the importance and complexity of the eukaryotic epigenome. While epigenome analysis has been applied to genomes from single-cell eukaryotes to human, comparative analyses are still relatively few and computational algorithms to quantify epigenome evolution remain scarce. Accordingly, a quantitative model of epigenome evolution remains to be established. We review here the comparative epigenomics literature and synthesize its overarching themes. We also suggest one mechanism, transcription factor binding site (TFBS) turnover, which relates sequence evolution to epigenetic conservation or divergence. Lastly, we propose a framework for how the field can move forward to build a coherent quantitative model of epigenome evolution.