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Induced pluripotency is a promising avenue for disease modeling and therapy, but the molecular principles underlying this process, particularly in human cells, remain poorly understood due to donor-to-donor variability and intercellular heterogeneity. Here, we constructed and characterized a clonal, inducible human reprogramming system that provides a reliable source of cells at any stage of the process. This system enabled integrative transcriptional and epigenomic analysis across the human reprogramming timeline at high resolution. We observed distinct waves of gene network activation, including the ordered re-activation of broad developmental regulators followed by early embryonic patterning genes and culminating in the emergence of a signature reminiscent of pre-implantation stages. Moreover, complementary functional analyses allowed us to identify and validate novel regulators of the reprogramming process. Altogether, this study sheds light on the molecular underpinnings of induced pluripotency in human cells and provides a robust cell platform for further studies. PAPERCLIP.
Assuntos
Reprogramação Celular , Células-Tronco Pluripotentes Induzidas/citologia , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina , Epigênese Genética , Perfilação da Expressão Gênica , Histona Desmetilases/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismoRESUMO
Reprogramming to iPSCs resets the epigenome of somatic cells, including the reversal of X chromosome inactivation. We sought to gain insight into the steps underlying the reprogramming process by examining the means by which reprogramming leads to X chromosome reactivation (XCR). Analyzing single cells in situ, we found that hallmarks of the inactive X (Xi) change sequentially, providing a direct readout of reprogramming progression. Several epigenetic changes on the Xi occur in the inverse order of developmental X inactivation, whereas others are uncoupled from this sequence. Among the latter, DNA methylation has an extraordinary long persistence on the Xi during reprogramming, and, like Xist expression, is erased only after pluripotency genes are activated. Mechanistically, XCR requires both DNA demethylation and Xist silencing, ensuring that only cells undergoing faithful reprogramming initiate XCR. Our study defines the epigenetic state of multiple sequential reprogramming intermediates and establishes a paradigm for studying cell fate transitions during reprogramming.
Assuntos
Reprogramação Celular , Epigênese Genética , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Cromossomo X/metabolismo , Animais , Proteínas Cdh1/metabolismo , Metilação de DNA , Proteínas de Homeodomínio/metabolismo , Camundongos , Proteína Homeobox Nanog , RNA Longo não Codificante/metabolismoRESUMO
An Amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
Intermediary metabolism generates substrates for chromatin modification, enabling the potential coupling of metabolic and epigenetic states. Here we identify a network linking metabolic and epigenetic alterations that is central to oncogenic transformation downstream of the liver kinase B1 (LKB1, also known as STK11) tumour suppressor, an integrator of nutrient availability, metabolism and growth. By developing genetically engineered mouse models and primary pancreatic epithelial cells, and employing transcriptional, proteomics, and metabolic analyses, we find that oncogenic cooperation between LKB1 loss and KRAS activation is fuelled by pronounced mTOR-dependent induction of the serine-glycine-one-carbon pathway coupled to S-adenosylmethionine generation. At the same time, DNA methyltransferases are upregulated, leading to elevation in DNA methylation with particular enrichment at retrotransposon elements associated with their transcriptional silencing. Correspondingly, LKB1 deficiency sensitizes cells and tumours to inhibition of serine biosynthesis and DNA methylation. Thus, we define a hypermetabolic state that incites changes in the epigenetic landscape to support tumorigenic growth of LKB1-mutant cells, while resulting in potential therapeutic vulnerabilities.
Assuntos
Transformação Celular Neoplásica , Metilação de DNA , Proteínas Serina-Treonina Quinases/deficiência , Proteínas Serina-Treonina Quinases/metabolismo , Serina/metabolismo , Quinases Proteína-Quinases Ativadas por AMP , Proteínas Quinases Ativadas por AMP , Animais , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Cromatina/genética , Cromatina/metabolismo , DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Células Epiteliais/metabolismo , Inativação Gênica , Genes Supressores de Tumor , Glicina/metabolismo , Glicólise , Humanos , Camundongos , Ductos Pancreáticos/citologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Retroelementos/genética , S-Adenosilmetionina/metabolismo , Serina/biossíntese , Serina-Treonina Quinases TOR/metabolismo , Transaminases/metabolismoRESUMO
In mammals, cytosine methylation is predominantly restricted to CpG dinucleotides and stably distributed across the genome, with local, cell-type-specific regulation directed by DNA binding factors. This comparatively static landscape is in marked contrast with the events of fertilization, during which the paternal genome is globally reprogrammed. Paternal genome demethylation includes the majority of CpGs, although methylation remains detectable at several notable features. These dynamics have been extensively characterized in the mouse, with only limited observations available in other mammals, and direct measurements are required to understand the extent to which early embryonic landscapes are conserved. We present genome-scale DNA methylation maps of human preimplantation development and embryonic stem cell derivation, confirming a transient state of global hypomethylation that includes most CpGs, while sites of residual maintenance are primarily restricted to gene bodies. Although most features share similar dynamics to those in mouse, maternally contributed methylation is divergently targeted to species-specific sets of CpG island promoters that extend beyond known imprint control regions. Retrotransposon regulation is also highly diverse, and transitions from maternally to embryonically expressed elements. Together, our data confirm that paternal genome demethylation is a general attribute of early mammalian development that is characterized by distinct modes of epigenetic regulation.
Assuntos
Blastocisto/metabolismo , Metilação de DNA , Animais , Linhagem Celular , Ilhas de CpG/fisiologia , DNA/metabolismo , Células-Tronco Embrionárias , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Despite their central importance in mammalian development, the mechanisms that regulate the DNA methylation machinery and thereby the generation of genomic methylation patterns are still poorly understood. Here, we identify the 5mC-binding protein MeCP2 as a direct and strong interactor of DNA methyltransferase 3 (DNMT3) proteins. We mapped the interaction interface to the transcriptional repression domain of MeCP2 and the ADD domain of DNMT3A and find that binding of MeCP2 strongly inhibits the activity of DNMT3A in vitro. This effect was reinforced by cellular studies where a global reduction of DNA methylation levels was observed after overexpression of MeCP2 in human cells. By engineering conformationally locked DNMT3A variants as novel tools to study the allosteric regulation of this enzyme, we show that MeCP2 stabilizes the closed, autoinhibitory conformation of DNMT3A. Interestingly, the interaction with MeCP2 and its resulting inhibition were relieved by the binding of K4 unmodified histone H3 N-terminal tail to the DNMT3A-ADD domain. Taken together, our data indicate that the localization and activity of DNMT3A are under the combined control of MeCP2 and H3 tail modifications where, depending on the modification status of the H3 tail at the binding sites, MeCP2 can act as either a repressor or activator of DNA methylation.
Assuntos
Cromatina/metabolismo , DNA (Citosina-5-)-Metiltransferases/genética , DNA/química , Epigênese Genética , Histonas/genética , Proteína 2 de Ligação a Metil-CpG/genética , Regulação Alostérica , Animais , Sítios de Ligação , Química Encefálica , Cromatina/química , Clonagem Molecular , DNA/metabolismo , DNA (Citosina-5-)-Metiltransferases/química , DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA , DNA Metiltransferase 3A , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Células HEK293 , Histonas/química , Histonas/metabolismo , Humanos , Proteína 2 de Ligação a Metil-CpG/química , Proteína 2 de Ligação a Metil-CpG/metabolismo , Camundongos , Mutagênese Sítio-Dirigida/métodos , Ligação Proteica , Engenharia de Proteínas/métodos , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Hidradenitis suppurativa (HS) is a chronic inflammatory disease manifesting as painful dermal nodules, abscesses, and tunnels. Activation of the IL-1R/toll-like receptor (TLR) pathway is strongly implicated in the pathogenesis of HS; thus, the role of a key signaling node, IL-1R-associated kinase 4 (IRAK4), was investigated in a noninterventional study (NCT04440410) that enrolled 30 patients with HS. IRAK4 expression was evaluated in blood and lesional, perilesional, and nonlesional skin biopsies. Peripheral blood mononuclear cells (PBMCs) expressed IRAK4, with significantly higher levels in monocytes (P ≤ 0.0001). Ex vivo treatment of PBMCs with KT-474, a targeted degrader of IRAK4, robustly decreased IRAK4 in all immune cell types from healthy volunteers and patients with HS. Ex vivo treatment of TLR-stimulated healthy donor monocytes with KT-474 decreased IRAK4 protein levels and inhibited inflammatory cytokine production. In HS skin samples, IRAK4 protein levels were significantly higher in lesional versus nonlesional tissue (P ≤ 0.0001), and IRAK4-positive immune infiltrate increased with greater disease severity. Multiple inflammatory mediators were upregulated in HS lesional skin, correlating with IRAK4 overexpression. These data confirm the significance of the IL-1R/TLR pathway in the pathogenesis of HS and provide support for ongoing clinical studies evaluating KT-474 in the treatment of HS.
RESUMO
Toll-like receptor-driven and interleukin-1 (IL-1) receptor-driven inflammation mediated by IL-1 receptor-associated kinase 4 (IRAK4) is involved in the pathophysiology of hidradenitis suppurativa (HS) and atopic dermatitis (AD). KT-474 (SAR444656), an IRAK4 degrader, was studied in a randomized, double-blind, placebo-controlled phase 1 trial where the primary objective was safety and tolerability. Secondary objectives included pharmacokinetics, pharmacodynamics and clinical activity in patients with moderate to severe HS and in patients with moderate to severe AD. KT-474 was administered as a single dose and then daily for 14 d in 105 healthy volunteers (HVs), followed by dosing for 28 d in an open-label cohort of 21 patients. Degradation of IRAK4 was observed in HV blood, with mean reductions after a single dose of ≥93% at 600-1,600 mg and after 14 daily doses of ≥95% at 50-200 mg. In patients, similar IRAK4 degradation was achieved in blood, and IRAK4 was normalized in skin lesions where it was overexpressed relative to HVs. Reduction of disease-relevant inflammatory biomarkers was demonstrated in the blood and skin of patients with HS and patients with AD and was associated with improvement in skin lesions and symptoms. There were no drug-related infections. These results, from what, to our knowledge, is the first published clinical trial using a heterobifunctional degrader, provide initial proof of concept for KT-474 in HS and AD to be further confirmed in larger trials. ClinicalTrials.gov identifier: NCT04772885 .
Assuntos
Dermatite Atópica , Hidradenite Supurativa , Humanos , Hidradenite Supurativa/tratamento farmacológico , Dermatite Atópica/tratamento farmacológico , Quinases Associadas a Receptores de Interleucina-1 , Resultado do Tratamento , Pele/patologia , Método Duplo-Cego , Índice de Gravidade de DoençaRESUMO
Stem and progenitor cells have the capacity to balance self-renewal and differentiation. Hematopoietic myeloid progenitors replenish more than 25 billion terminally differentiated neutrophils every day under homeostatic conditions and can increase this output in response to stress or infection. At what point along the spectrum of maturation do progenitors lose capacity for self-renewal and become irreversibly committed to differentiation? Using a system of conditional myeloid development that can be toggled between self-renewal and differentiation, we interrogate determinants of this "point of no return" in differentiation commitment. Irreversible commitment is due primarily to loss of open regulatory site access and disruption of a positive feedback transcription factor activation loop. Restoration of the transcription factor feedback loop extends the window of cell plasticity and alters the point of no return. These findings demonstrate how the chromatin state enforces and perpetuates cell fate and identify potential avenues for manipulating cell identity.
Assuntos
Medula Óssea/fisiologia , Linhagem da Célula , Cromatina/genética , Hematopoese , Células-Tronco Hematopoéticas/citologia , Células Mieloides/citologia , Fatores de Transcrição/metabolismo , Animais , Diferenciação Celular , Células Cultivadas , Cromatina/metabolismo , Feminino , Perfilação da Expressão Gênica , Camundongos , Fatores de Transcrição/genéticaRESUMO
Zika virus (ZIKV) infection during early pregnancy can cause microcephaly and associated defects at birth, but whether it can induce neurologic sequelae that appear later in life remains unclear. Using a model of the developing brain based on embryonic stem cell-derived brain organoids, we studied the impact of ZIKV infection on the DNA methylation pattern across the entire genome in selected neural cell types. The virus unexpectedly alters the DNA methylome of neural progenitors, astrocytes, and differentiated neurons at genes that have been implicated in the pathogenesis of a number of brain disorders, most prominently mental retardation and schizophrenia. Our results suggest that ZIKV infection during fetal development could lead to a spectrum of delayed-onset neuropsychiatric complications. IMPORTANCE Scientific research on human neural stem cells and cerebral organoids has confirmed the congenital neurotropic and neurodestructive nature of the Zika virus. However, the extent to which prenatal ZIKV infection is associated with more subtle brain alterations, such as epigenetic changes, remains ill defined. Here, we address the question of whether ZIKV infection induces DNA methylation changes with the potential to cause brain disorders later in life.
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A dramatic difference in global DNA methylation between male and female cells characterizes mouse embryonic stem cells (ESCs), unlike somatic cells. We analyzed DNA methylation changes during reprogramming of male and female somatic cells and in resulting induced pluripotent stem cells (iPSCs). At an intermediate reprogramming stage, somatic and pluripotency enhancers are targeted for partial methylation and demethylation. Demethylation within pluripotency enhancers often occurs at ESC binding sites of pluripotency transcription factors. Late in reprogramming, global hypomethylation is induced in a female-specific manner. Genome-wide hypomethylation in female cells affects many genomic landmarks, including enhancers and imprint control regions, and accompanies the reactivation of the inactive X chromosome. The loss of one of the two X chromosomes in propagating female iPSCs is associated with genome-wide methylation gain. Collectively, our findings highlight the dynamic regulation of DNA methylation at enhancers during reprogramming and reveal that X chromosome dosage dictates global DNA methylation levels in iPSCs.
Assuntos
Reprogramação Celular/genética , Cromossomos de Mamíferos/genética , Metilação de DNA/genética , Células-Tronco Pluripotentes Induzidas/metabolismo , Cromossomo X/genética , Animais , Sítios de Ligação , Ilhas de CpG/genética , Células-Tronco Embrionárias/metabolismo , Elementos Facilitadores Genéticos/genética , Feminino , Genoma , Impressão Genômica , Células-Tronco Pluripotentes Induzidas/citologia , Masculino , Camundongos , Fatores de Transcrição/metabolismoRESUMO
DNA methylation plays an essential role in mammalian genomes and expression of the responsible enzymes is tightly controlled. Deregulation of the de novo DNA methyltransferase DNMT3B is frequently observed across cancer types, yet little is known about its ectopic genomic targets. Here, we used an inducible transgenic mouse model to delineate rules for abnormal DNMT3B targeting, as well as the constraints of its activity across different cell types. Our results explain the preferential susceptibility of certain CpG islands to aberrant methylation and point to transcriptional state and the associated chromatin landscape as the strongest predictors. Although DNA methylation and H3K27me3 are usually non-overlapping at CpG islands, H3K27me3 can transiently co-occur with DNMT3B-induced DNA methylation. Our genome-wide data combined with ultra-deep locus-specific bisulfite sequencing suggest a distributive activity of ectopically expressed Dnmt3b that leads to discordant CpG island hypermethylation and provides new insights for interpreting the cancer methylome.
Assuntos
Ilhas de CpG , DNA (Citosina-5-)-Metiltransferases/biossíntese , Metilação de DNA , Expressão Gênica , Proteínas Recombinantes/biossíntese , Animais , DNA (Citosina-5-)-Metiltransferases/genética , Células-Tronco Embrionárias/fisiologia , Regulação da Expressão Gênica , Humanos , Camundongos Transgênicos , Neoplasias/patologia , Proteínas Recombinantes/genética , DNA Metiltransferase 3BRESUMO
Transcription factors (TFs) direct developmental transitions by binding to target DNA sequences, influencing gene expression and establishing complex gene-regultory networks. To systematically determine the molecular components that enable or constrain TF activity, we investigated the genomic occupancy of FOXA2, GATA4 and OCT4 in several cell types. Despite their classification as pioneer factors, all three TFs exhibit cell-type-specific binding, even when supraphysiologically and ectopically expressed. However, FOXA2 and GATA4 can be distinguished by low enrichment at loci that are highly occupied by these factors in alternative cell types. We find that expression of additional cofactors increases enrichment at a subset of these sites. Finally, FOXA2 occupancy and changes to DNA accessibility can occur in G1-arrested cells, but subsequent loss of DNA methylation requires DNA replication.
Assuntos
DNA/metabolismo , Epigênese Genética/fisiologia , Redes Reguladoras de Genes/fisiologia , Fatores de Transcrição/metabolismo , Células A549 , Sítios de Ligação/genética , Linhagem da Célula/efeitos dos fármacos , Linhagem da Célula/genética , Células Cultivadas , Biologia Computacional , DNA/genética , Epistasia Genética/fisiologia , Fator de Transcrição GATA4/metabolismo , Regulação da Expressão Gênica , Genes de Troca , Células HEK293 , Células Hep G2 , Fator 3-beta Nuclear de Hepatócito/metabolismo , Humanos , Fator 3 de Transcrição de Octâmero/metabolismo , Ligação ProteicaRESUMO
The generation of genomic binding or accessibility data from massively parallel sequencing technologies such as ChIP-seq and DNase-seq continues to accelerate. Yet state-of-the-art computational approaches for the identification of DNA binding motifs often yield motifs of weak predictive power. Here we present a novel computational algorithm called MotifSpec, designed to find predictive motifs, in contrast to over-represented sequence elements. The key distinguishing feature of this algorithm is that it uses a dynamic search space and a learned threshold to find discriminative motifs in combination with the modeling of motifs using a full PWM (position weight matrix) rather than k-mer words or regular expressions. We demonstrate that our approach finds motifs corresponding to known binding specificities in several mammalian ChIP-seq datasets, and that our PWMs classify the ChIP-seq signals with accuracy comparable to, or marginally better than motifs from the best existing algorithms. In other datasets, our algorithm identifies novel motifs where other methods fail. Finally, we apply this algorithm to detect motifs from expression datasets in C. elegans using a dynamic expression similarity metric rather than fixed expression clusters, and find novel predictive motifs.
Assuntos
Biologia Computacional/métodos , Elementos Reguladores de Transcrição , Sequências Reguladoras de Ácido Nucleico , Software , Algoritmos , Animais , Sítios de Ligação , Caenorhabditis elegans , Imunoprecipitação da Cromatina , Conjuntos de Dados como Assunto , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Camundongos , Motivos de Nucleotídeos , Matrizes de Pontuação de Posição Específica , Fatores de Transcrição/metabolismo , LevedurasRESUMO
Mouse embryonic stem cells (mESCs) cultured under serum/LIF conditions exhibit heterogeneous expression of pluripotency-associated factors that can be overcome by two inhibitors (2i) of the MEK and GSK3 pathways. Several studies have shown that the "ground state" induced by 2i is characterized by global hypomethylation and specific transcriptional profiles, but little is known about the contributing effectors. Here we show that 2i conditions rapidly alter the global binding landscape of OCT4, SOX2, and NANOG. The dynamic binding influences enhancer activity and shows enrichment for regulators linked to Wnt and Erk signaling. Epigenomic characterization provided limited insights to the immediate transcriptional dynamics, suggesting that these are likely more secondary effects. Likewise, loss of the PRC2 component EED to prevent H3K27me3 deposition had minimal effect on the transcriptome, implying that it is largely dispensable for continued repression of bivalent genes and de novo silencing in 2i.
Assuntos
Epigênese Genética , Células-Tronco Embrionárias Murinas/fisiologia , Animais , Células Cultivadas , Inibidores Enzimáticos/farmacologia , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Quinase 3 da Glicogênio Sintase/genética , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Histona Desmetilases com o Domínio Jumonji/genética , MAP Quinase Quinase 1/antagonistas & inibidores , MAP Quinase Quinase 1/genética , Camundongos , Proteína Homeobox Nanog , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Complexo Repressor Polycomb 2/genética , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , TranscriptomaRESUMO
DNA methylation is a key epigenetic modification involved in regulating gene expression and maintaining genomic integrity. Here we inactivated all three catalytically active DNA methyltransferases (DNMTs) in human embryonic stem cells (ESCs) using CRISPR/Cas9 genome editing to further investigate the roles and genomic targets of these enzymes. Disruption of DNMT3A or DNMT3B individually as well as of both enzymes in tandem results in viable, pluripotent cell lines with distinct effects on the DNA methylation landscape, as assessed by whole-genome bisulfite sequencing. Surprisingly, in contrast to findings in mouse, deletion of DNMT1 resulted in rapid cell death in human ESCs. To overcome this immediate lethality, we generated a doxycycline-responsive tTA-DNMT1* rescue line and readily obtained homozygous DNMT1-mutant lines. However, doxycycline-mediated repression of exogenous DNMT1* initiates rapid, global loss of DNA methylation, followed by extensive cell death. Our data provide a comprehensive characterization of DNMT-mutant ESCs, including single-base genome-wide maps of the targets of these enzymes.
Assuntos
DNA (Citosina-5-)-Metiltransferases/genética , Metilação de DNA , Células-Tronco Embrionárias/enzimologia , Animais , Apoptose , Sequência de Bases , Domínio Catalítico , Diferenciação Celular , Proliferação de Células , Sobrevivência Celular , Células Cultivadas , Técnicas de Cocultura , Ilhas de CpG , DNA (Citosina-5-)-Metiltransferase 1 , DNA Metiltransferase 3A , Células-Tronco Embrionárias/fisiologia , Epigênese Genética , Expressão Gênica , Técnicas de Inativação de Genes , Humanos , Camundongos , DNA Metiltransferase 3BRESUMO
Polycomb Repressive Complex 2 (PRC2) function and DNA methylation (DNAme) are typically correlated with gene repression. Here, we show that PRC2 is required to maintain expression of maternal microRNAs (miRNAs) and long non-coding RNAs (lncRNAs) from the Gtl2-Rian-Mirg locus, which is essential for full pluripotency of iPSCs. In the absence of PRC2, the entire locus becomes transcriptionally repressed due to gain of DNAme at the intergenic differentially methylated regions (IG-DMRs). Furthermore, we demonstrate that the IG-DMR serves as an enhancer of the maternal Gtl2-Rian-Mirg locus. Further analysis reveals that PRC2 interacts physically with Dnmt3 methyltransferases and reduces recruitment to and subsequent DNAme at the IG-DMR, thereby allowing for proper expression of the maternal Gtl2-Rian-Mirg locus. Our observations are consistent with a mechanism through which PRC2 counteracts the action of Dnmt3 methyltransferases at an imprinted locus required for full pluripotency.
Assuntos
Metilação de DNA , Células-Tronco Embrionárias/metabolismo , Impressão Genômica , Complexo Repressor Polycomb 2/metabolismo , RNA Longo não Codificante/genética , Animais , Linhagem Celular , DNA (Citosina-5-)-Metiltransferases/metabolismo , DNA Metiltransferase 3A , Camundongos , MicroRNAs/genética , Proteínas Nucleares/genética , Complexo Repressor Polycomb 2/genética , Ligação ProteicaRESUMO
Mutations in the metabolic enzymes isocitrate dehydrogenase-1 (IDH1) and IDH2 that produce the oncometabolite D-2-hydroxyglutarate (2-HG) occur frequently in human acute myeloid leukemia (AML). 2-HG modulates numerous biological pathways implicated in malignant transformation, but the contribution of mutant IDH proteins to maintenance and progression of AML in vivo is currently unknown. To answer this crucial question we have generated transgenic mice that express IDH2(R140Q) in an on/off- and tissue-specific manner using a tetracycline-inducible system. We found that IDH2(R140Q) can cooperate with overexpression of HoxA9 and Meis1a and with mutations in FMS-like tyrosine kinase 3 (FLT3) to drive acute leukemia in vivo. Critically, we show that genetic deinduction of mutant IDH2 in leukemic cells in vivo has profound effects on their growth and/or maintenance. Our data demonstrate the proto-oncogenic role of mutant IDH2 and support its relevance as a therapeutic target for the treatment of human AML.
Assuntos
Carcinogênese/patologia , Isocitrato Desidrogenase/genética , Leucemia Mieloide Aguda/patologia , Mutação/genética , Oncogenes , Animais , Medula Óssea/patologia , Carcinogênese/genética , Diferenciação Celular , Proliferação de Células , Transformação Celular Neoplásica/patologia , Modelos Animais de Doenças , Células Eritroides/metabolismo , Células Eritroides/patologia , Hematopoese , Células-Tronco Hematopoéticas/patologia , Proteínas de Homeodomínio/metabolismo , Isocitrato Desidrogenase/metabolismo , Leucemia Mieloide Aguda/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteína Meis1 , Proteínas de Neoplasias/metabolismo , Baço/patologia , Transcrição Gênica , Tirosina Quinase 3 Semelhante a fms/metabolismoRESUMO
Over the past years we have witnessed an explosion in the generation of freely available genome-wide data sets, including maps of various histone modifications, transcription factor binding, DNase hypersensitivity, and DNA methylation, which provide valuable resources for data validation, exploration, and hypothesis generation. The goal of this review is to provide the reader with information on where to find many of the data sets and how to utilize the various (epi)genome browsers for display and initial analysis. We provide selected examples to highlight key features and demonstrate the application of these browsers to stem cell biology.