RESUMO
ABSTRACT: Coagulation factor VIII (FVIII) and its carrier protein von Willebrand factor (VWF) are critical to coagulation and platelet aggregation. We leveraged whole-genome sequence data from the Trans-Omics for Precision Medicine (TOPMed) program along with TOPMed-based imputation of genotypes in additional samples to identify genetic associations with circulating FVIII and VWF levels in a single-variant meta-analysis, including up to 45 289 participants. Gene-based aggregate tests were implemented in TOPMed. We identified 3 candidate causal genes and tested their functional effect on FVIII release from human liver endothelial cells (HLECs) and VWF release from human umbilical vein endothelial cells. Mendelian randomization was also performed to provide evidence for causal associations of FVIII and VWF with thrombotic outcomes. We identified associations (P < 5 × 10-9) at 7 new loci for FVIII (ST3GAL4, CLEC4M, B3GNT2, ASGR1, F12, KNG1, and TREM1/NCR2) and 1 for VWF (B3GNT2). VWF, ABO, and STAB2 were associated with FVIII and VWF in gene-based analyses. Multiphenotype analysis of FVIII and VWF identified another 3 new loci, including PDIA3. Silencing of B3GNT2 and the previously reported CD36 gene decreased release of FVIII by HLECs, whereas silencing of B3GNT2, CD36, and PDIA3 decreased release of VWF by HVECs. Mendelian randomization supports causal association of higher FVIII and VWF with increased risk of thrombotic outcomes. Seven new loci were identified for FVIII and 1 for VWF, with evidence supporting causal associations of FVIII and VWF with thrombotic outcomes. B3GNT2, CD36, and PDIA3 modulate the release of FVIII and/or VWF in vitro.
Assuntos
Moléculas de Adesão Celular , Fator VIII , Cininogênios , Lectinas Tipo C , Receptores de Superfície Celular , Fator de von Willebrand , Humanos , Fator de von Willebrand/genética , Fator de von Willebrand/metabolismo , Fator VIII/genética , Fator VIII/metabolismo , Polimorfismo de Nucleotídeo Único , Células Endoteliais da Veia Umbilical Humana/metabolismo , Análise da Randomização Mendeliana , Estudo de Associação Genômica Ampla , Trombose/genética , Trombose/sangue , Estudos de Associação Genética , Masculino , Células Endoteliais/metabolismo , FemininoRESUMO
Mutations in the isocitrate dehydrogenase 1 (IDH1) and IDH2 genes are frequently observed in a wide variety of hematologic malignancies, including myeloid and T-cell leukemias. In this study, we generated Idh2R140Q transgenic mice to examine the role of the Idh2R140Q mutation in leukemia. No leukemia developed in Idh2R140Q transgenic mice, suggesting a need for additional genetic events for leukemia development. Because myeloid cells from NUP98-HOXD13 fusion (NHD13) transgenic mice frequently acquire somatic Idh mutations when they transform to acute myeloid leukemia, we generated Idh2R140Q/NHD13 double transgenic mice. Idh2R140Q/NHD13 transgenic mice developed an immature T-cell leukemia with an immunophenotype similar to double-negative 1 (DN1) or DN2 thymocytes. Idh2R140Q/NHD13 leukemic cells were enriched for an early thymic precursor transcriptional signature, and the gene expression profile for Idh2R140Q/NHD13 DN1/DN2 T-ALL closely matched that of human early/immature T-cell precursor (EITP) acute lymphoblastic leukemia (ALL). Moreover, recurrent mutations found in patients with EITP ALL, including KRAS, PTPN11, JAK3, SH2B3, and EZH2 were also found in Idh2R140Q/NHD13 DN1/DN2 T-ALL. In vitro treatment of Idh2R140Q/NHD13 thymocytes with enasidenib, a selective inhibitor of mutant IDH2, led to a marked decrease in leukemic cell proliferation. These findings demonstrate that Idh2R140Q/NHD13 mice can serve as a useful in vivo model for the study of early/immature thymocyte precursor acute lymphoblastic leukemia development and therapy. SIGNIFICANCE: T-cell leukemia induced in Idh2R140Q/NUP98-HOXD13 mice is immunophenotypically, transcriptionally, and genetically similar to human EITP ALL, providing a model for studying disease development and treatment.
Assuntos
Proteínas de Homeodomínio/metabolismo , Isocitrato Desidrogenase/genética , Isocitrato Desidrogenase/metabolismo , Mutação , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Proteínas de Fusão Oncogênica/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/etiologia , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Timócitos/metabolismo , Animais , Biomarcadores Tumorais , Diferenciação Celular/genética , Linhagem Celular Tumoral , Biologia Computacional/métodos , Metilação de DNA , Modelos Animais de Doenças , Suscetibilidade a Doenças , Perfilação da Expressão Gênica , Xenoenxertos , Humanos , Imunofenotipagem , Camundongos , Camundongos Transgênicos , Leucemia-Linfoma Linfoblástico de Células T Precursoras/mortalidade , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologia , Timócitos/patologia , TranscriptomaRESUMO
Platelet aggregation at the site of atherosclerotic vascular injury is the underlying pathophysiology of myocardial infarction and stroke. To build upon prior GWAS, here we report on 16 loci identified through a whole genome sequencing (WGS) approach in 3,855 NHLBI Trans-Omics for Precision Medicine (TOPMed) participants deeply phenotyped for platelet aggregation. We identify the RGS18 locus, which encodes a myeloerythroid lineage-specific regulator of G-protein signaling that co-localizes with expression quantitative trait loci (eQTL) signatures for RGS18 expression in platelets. Gene-based approaches implicate the SVEP1 gene, a known contributor of coronary artery disease risk. Sentinel variants at RGS18 and PEAR1 are associated with thrombosis risk and increased gastrointestinal bleeding risk, respectively. Our WGS findings add to previously identified GWAS loci, provide insights regarding the mechanism(s) by which genetics may influence cardiovascular disease risk, and underscore the importance of rare variant and regulatory approaches to identifying loci contributing to complex phenotypes.
Assuntos
Plaquetas/metabolismo , Mapeamento Cromossômico , Sequenciamento Completo do Genoma , Sequência de Bases , Proteínas de Ligação ao GTP , Estudo de Associação Genômica Ampla , Células HEK293 , Humanos , Células K562 , Fenótipo , Agregação Plaquetária , Testes de Função Plaquetária , Polimorfismo de Nucleotídeo Único , Locos de Características Quantitativas , Proteínas RGS/genética , Proteínas RGS/metabolismo , Receptores de Superfície Celular/genética , Trombose/genéticaRESUMO
BACKGROUND: Use of targeted exome-arrays with common, rare variants and functionally enriched variation has led to discovery of new genes contributing to population variation in risk factors. Plasminogen activator-inhibitor 1 (PAI-1), tissue plasminogen activator (tPA), and the plasma product D-dimer are important components of the fibrinolytic system. There have been few large-scale genome-wide or exome-wide studies of PAI-1, tPA, and D-dimer. OBJECTIVES: We sought to discover new genetic loci contributing to variation in these traits using an exome-array approach. METHODS: Cohort-level analyses and fixed effects meta-analyses of PAI-1 (n = 15 603), tPA (n = 6876,) and D-dimer (n = 19 306) from 12 cohorts of European ancestry with diverse study design were conducted, including single-variant analyses and gene-based burden testing. RESULTS: Five variants located in NME7, FGL1, and the fibrinogen locus, all associated with D-dimer levels, achieved genome-wide significance (P < 5 × 10-8 ). Replication was sought for these 5 variants, as well as 45 well-imputed variants with P < 1 × 10-4 in the discovery using an independent cohort. Replication was observed for three out of the five significant associations, including a novel and uncommon (0.013 allele frequency) coding variant p.Trp256Leu in FGL1 (fibrinogen-like-1) with increased plasma D-dimer levels. Additionally, a candidate-gene approach revealed a suggestive association for a coding variant (rs143202684-C) in SERPINB2, and suggestive associations with consistent effect in the replication analysis include an intronic variant (rs11057830-A) in SCARB1 associated with increased D-dimer levels. CONCLUSION: This work provides new evidence for a role of FGL1 in hemostasis.
Assuntos
Inibidor 1 de Ativador de Plasminogênio , Ativador de Plasminogênio Tecidual , Exoma , Produtos de Degradação da Fibrina e do Fibrinogênio , Fibrinogênio/genética , Fibrinólise , Humanos , Inibidor 1 de Ativador de Plasminogênio/genética , Ativador de Plasminogênio Tecidual/genéticaRESUMO
Type 2 diabetes is a major risk factor for cardiovascular disease. Given the contribution of platelets to atherothrombosis-which in turn is a major contributor to cardiac events, there may be cause to consider platelet function in management of diabetes. Despite the large body of research concerning the role of platelets in cardiovascular complications of type 2 diabetes, evidence from population-based studies of platelet aggregation in diabetes is limited. Mean Platelet Volume (MPV), a cell trait partially associated with markers of platelet activity, is more commonly available. We investigated the association of metabolic syndrome and diabetes with platelet aggregation to three physiological agonists, ADP, collagen, and epinephrine, in the Framingham Heart Study Offspring cohort. We further examined the relationship between MPV measured with Beckman Coulter LH750 instruments and self-reported diabetes as well as MPV and diabetes medication in the UK BioBank cohort, performing the largest such analysis to date. Increased platelet aggregation associated with prevalent diabetes was observed for low concentration epinephrine (0.1 µM) alone and only in analyses of participants stratified either by male sex and/or having metabolic syndrome. Other agonists and concentrations were not significant for prevalent diabetes, or in opposite direction to the main hypothesis (i.e., they showed lower platelet aggregation associated with diabetes). After a median of 18.1 years follow-up, no platelet aggregation trait was associated with increased risk of diabetes (n = 344 cases). As expected, increased MPV was significantly associated with diabetes (ß = 0.0976; P = 8.62 × 10-33). Interestingly, sex-stratified analyses indicated the association of MPV with diabetes is markedly stronger in males (ß = 0.1232; P = 1.00 × 10-31) than females (ß = 0.0514; P = 7.37 × 10-5). Among diabetes medications increased MPV was associated with Insulin (ß = 0.1341; P = 1.38 × 10-11) and decreased MPV with both Metformin (ß = 0.0763; P = 1.99 × 10-6) as well as the sulphonylureas (ß = 0.0559; P = 0.0034). Each drug showed the same direction of effect in both sexes, however, the association with MPV was nearly twice as great or more in women compared to men. In conclusion, platelet function as measured by aggregation to ADP, collagen, or epinephrine does not appear to be consistently associated with diabetes, however, MPV is robustly associated suggesting future work may focus on how MPV segments pre-diabetics and diabetics for risk prediction.
RESUMO
RATIONALE: Mean platelet volume (MPV) and platelet count (PLT) are platelet measures that have been linked to cardiovascular disease (CVD) and mortality risk. Identifying protein biomarkers for these measures may yield insights into CVD mechanisms. OBJECTIVE: We aimed to identify causal protein biomarkers for MPV and PLT among 71 CVD-related plasma proteins measured in FHS (Framingham Heart Study) participants. METHODS AND RESULTS: We conducted integrative analyses of genetic variants associated with PLT/MPV with protein quantitative trait locus variants associated with plasma proteins followed by Mendelian randomization to infer causal relations of proteins for PLT/MPV. We also tested protein-PLT/MPV association in FHS participants. Using induced pluripotent stem cell-derived megakaryocyte clones that produce functional platelets, we conducted RNA-sequencing and analyzed expression differences between low- and high-platelet producing clones. We then performed small interfering RNA gene knockdown experiments targeting genes encoding proteins with putatively causal platelet effects in megakaryocyte clones to examine effects on platelet production. In protein-trait association analyses, ten proteins were associated with MPV and 31 with PLT. Mendelian randomization identified 4 putatively causal proteins for MPV and 4 for PLT. GP-5 (Glycoprotein V), GRN (granulin), and MCAM (melanoma cell adhesion molecule) were associated with PLT, while MPO (myeloperoxidase) showed significant association with MPV in both analyses. RNA-sequencing analysis results were directionally concordant with observed and Mendelian randomization-inferred associations for GP-5, GRN, and MCAM. In siRNA gene knockdown experiments, silencing GP-5, GRN, and MPO decreased PLTs. Genome-wide association study results suggest several of these may be linked to CVD risk. CONCLUSIONS: We identified 4 proteins that are causally linked to PLTs. These proteins may also have roles in the pathogenesis of CVD via a platelet/blood coagulation-based mechanism.
Assuntos
Doenças Cardiovasculares/genética , Granulinas , Volume Plaquetário Médio , Peroxidase , Contagem de Plaquetas , Glicoproteínas da Membrana de Plaquetas , Biomarcadores/sangue , Proteínas Sanguíneas/análise , Antígeno CD146/genética , Antígeno CD146/metabolismo , Doenças Cardiovasculares/sangue , Doenças Cardiovasculares/etiologia , Doenças Cardiovasculares/mortalidade , Diferenciação Celular , Feminino , Inativação Gênica , Estudo de Associação Genômica Ampla , Granulinas/genética , Granulinas/metabolismo , Humanos , Estudos Longitudinais , Masculino , Células Progenitoras de Megacariócitos , Megacariócitos/citologia , Análise da Randomização Mendeliana , Pessoa de Meia-Idade , Peroxidase/genética , Peroxidase/metabolismo , Fenótipo , Glicoproteínas da Membrana de Plaquetas/genética , Glicoproteínas da Membrana de Plaquetas/metabolismo , Células-Tronco Pluripotentes , RNA Interferente Pequeno , Risco , Análise de Sequência de RNARESUMO
Dual antiplatelet therapy reduces ischemic events in cardiovascular disease, but it increases bleeding risk. Thrombin receptors PAR1 and PAR4 are drug targets, but the role of thrombin in platelet aggregation remains largely unexplored in large populations. We performed a genome-wide association study (GWAS) of platelet aggregation in response to full-length thrombin, followed by clinical association analyses, Mendelian randomization, and functional characterization including iPSC-derived megakaryocyte and platelet experiments. We identified a single sentinel variant in the GRK5 locus (rs10886430-G, p = 3.0 × 10-42) associated with increased thrombin-induced platelet aggregation (ß = 0.70, SE = 0.05). We show that disruption of platelet GRK5 expression by rs10886430-G is associated with enhanced platelet reactivity. The proposed mechanism of a GATA1-driven megakaryocyte enhancer is confirmed in allele-specific experiments. Utilizing further data, we demonstrate that the allelic effect is highly platelet- and thrombin-specific and not likely due to effects on thrombin levels. The variant is associated with increased risk of cardiovascular disease outcomes in UK BioBank, most strongly with pulmonary embolism. The variant associates with increased risk of stroke in the MEGASTROKE, UK BioBank, and FinnGen studies. Mendelian randomization analyses in independent samples support a causal role for rs10886430-G in increasing risk for stroke, pulmonary embolism, and venous thromboembolism through its effect on thrombin-induced platelet reactivity. We demonstrate that G protein-coupled receptor kinase 5 (GRK5) promotes platelet activation specifically via PAR4 receptor signaling. GRK5 inhibitors in development for the treatment of heart failure and cancer could have platelet off-target deleterious effects. Common variants in GRK5 may modify clinical outcomes with PAR4 inhibitors, and upregulation of GRK5 activity or signaling in platelets may have therapeutic benefits.
Assuntos
Plaquetas/fisiologia , Doenças Cardiovasculares/genética , Receptores de Trombina/genética , Transdução de Sinais/genética , Trombina/genética , Alelos , Embolia/genética , Feminino , Estudo de Associação Genômica Ampla/métodos , Insuficiência Cardíaca/genética , Humanos , Pulmão/fisiologia , Masculino , Pessoa de Meia-Idade , Neoplasias/genética , Ativação Plaquetária/genética , Agregação Plaquetária/genética , Receptor PAR-1/genética , Acidente Vascular Cerebral/genéticaRESUMO
Mutations in DNA methyltransferase 3A (DNMT3A) are common in acute myeloid leukemia and portend a poor prognosis; thus, new therapeutic strategies are needed. The likely mechanism by which DNMT3A loss contributes to leukemogenesis is altered DNA methylation and the attendant gene expression changes; however, our current understanding is incomplete. We observed that murine hematopoietic stem cells (HSCs) in which Dnmt3a had been conditionally deleted markedly overexpress the histone 3 lysine 79 (H3K79) methyltransferase, Dot1l. We demonstrate that Dnmt3a(-/-) HSCs have increased H3K79 methylation relative to wild-type (WT) HSCs, with the greatest increases noted at DNA methylation canyons, which are regions highly enriched for genes dysregulated in leukemia and prone to DNA methylation loss with Dnmt3a deletion. These findings led us to explore DOT1L as a therapeutic target for the treatment of DNMT3A-mutant AML. We show that pharmacologic inhibition of DOT1L resulted in decreased expression of oncogenic canyon-associated genes and led to dose- and time-dependent inhibition of proliferation, induction of apoptosis, cell-cycle arrest, and terminal differentiation in DNMT3A-mutant cell lines in vitro. We show in vivo efficacy of the DOT1L inhibitor EPZ5676 in a nude rat xenograft model of DNMT3A-mutant AML. DOT1L inhibition was also effective against primary patient DNMT3A-mutant AML samples, reducing colony-forming capacity (CFC) and inducing terminal differentiation in vitro. These studies suggest that DOT1L may play a critical role in DNMT3A-mutant leukemia. With pharmacologic inhibitors of DOT1L already in clinical trials, DOT1L could be an immediately actionable therapeutic target for the treatment of this poor prognosis disease.
Assuntos
DNA (Citosina-5-)-Metiltransferases/genética , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/enzimologia , Metiltransferases/genética , Metiltransferases/metabolismo , Terapia de Alvo Molecular , Mutação/genética , Adenosina/análogos & derivados , Adenosina/farmacologia , Adenosina/uso terapêutico , Animais , Apoptose , Pontos de Checagem do Ciclo Celular , Diferenciação Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , DNA Metiltransferase 3A , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/metabolismo , Histona-Lisina N-Metiltransferase , Histonas/metabolismo , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Lisina/metabolismo , Metilação , Camundongos Endogâmicos C57BL , Compostos de Fenilureia/farmacologia , Compostos de Fenilureia/uso terapêutico , Ratos , Fatores de Tempo , Ensaio Tumoral de Célula-Tronco , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Hematopoietic stem cells (HSCs) possess unique gene expression programs that enforce their identity and regulate lineage commitment. Long non-coding RNAs (lncRNAs) have emerged as important regulators of gene expression and cell fate decisions, although their functions in HSCs are unclear. Here we profiled the transcriptome of purified HSCs by deep sequencing and identified 323 unannotated lncRNAs. Comparing their expression in differentiated lineages revealed 159 lncRNAs enriched in HSCs, some of which are likely HSC specific (LncHSCs). These lncRNA genes share epigenetic features with protein-coding genes, including regulated expression via DNA methylation, and knocking down two LncHSCs revealed distinct effects on HSC self-renewal and lineage commitment. We mapped the genomic binding sites of one of these candidates and found enrichment for key hematopoietic transcription factor binding sites, especially E2A. Together, these results demonstrate that lncRNAs play important roles in regulating HSCs, providing an additional layer to the genetic circuitry controlling HSC function.
Assuntos
Células da Medula Óssea/fisiologia , Células-Tronco Hematopoéticas/fisiologia , RNA Longo não Codificante/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Sítios de Ligação/genética , Diferenciação Celular/genética , Linhagem da Célula/genética , Autorrenovação Celular/genética , Células Cultivadas , DNA (Citosina-5-)-Metiltransferases/genética , Metilação de DNA/genética , DNA Metiltransferase 3A , Epigênese Genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento/genética , Sequenciamento de Nucleotídeos em Larga Escala , Camundongos , Camundongos Endogâmicos , Camundongos Knockout , RNA Longo não Codificante/genética , RNA Interferente Pequeno/genéticaRESUMO
This report describes an improved protocol to generate stranded, barcoded RNA-seq libraries to capture the whole transcriptome. By optimizing the use of duplex specific nuclease (DSN) to remove ribosomal RNA reads from stranded barcoded libraries, we demonstrate improved efficiency of multiplexed next generation sequencing (NGS). This approach detects expression profiles of all RNA types, including miRNA (microRNA), piRNA (Piwi-interacting RNA), snoRNA (small nucleolar RNA), lincRNA (long non-coding RNA), mtRNA (mitochondrial RNA) and mRNA (messenger RNA) without the use of gel electrophoresis. The improved protocol generates high quality data that can be used to identify differential expression in known and novel coding and non-coding transcripts, splice variants, mitochondrial genes and SNPs (single nucleotide polymorphisms).
Assuntos
Perfilação da Expressão Gênica/métodos , RNA Mensageiro/genética , Análise de Sequência de RNA , Linhagem Celular Tumoral , Biblioteca Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Anotação de Sequência Molecular , Polimorfismo de Nucleotídeo Único , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Mensageiro/isolamento & purificação , RNA Mensageiro/metabolismo , RNA Ribossômico/química , RNA Ribossômico/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ribonucleases/químicaRESUMO
BACKGROUND: Advances in whole genome profiling have revolutionized the cancer research field, but at the same time have raised new bioinformatics challenges. For next generation sequencing (NGS), these include data storage, computational costs, sequence processing and alignment, delineating appropriate statistical measures, and data visualization. Currently there is a lack of workflows for efficient analysis of large, MethylCap-seq datasets containing multiple sample groups. METHODS: The NGS application MethylCap-seq involves the in vitro capture of methylated DNA and subsequent analysis of enriched fragments by massively parallel sequencing. The workflow we describe performs MethylCap-seq experimental Quality Control (QC), sequence file processing and alignment, differential methylation analysis of multiple biological groups, hierarchical clustering, assessment of genome-wide methylation patterns, and preparation of files for data visualization. RESULTS: Here, we present a scalable, flexible workflow for MethylCap-seq QC, secondary data analysis, tertiary analysis of multiple experimental groups, and data visualization. We demonstrate the experimental QC procedure with results from a large ovarian cancer study dataset and propose parameters which can identify problematic experiments. Promoter methylation profiling and hierarchical clustering analyses are demonstrated for four groups of acute myeloid leukemia (AML) patients. We propose a Global Methylation Indicator (GMI) function to assess genome-wide changes in methylation patterns between experimental groups. We also show how the workflow facilitates data visualization in a web browser with the application Anno-J. CONCLUSIONS: This workflow and its suite of features will assist biologists in conducting methylation profiling projects and facilitate meaningful biological interpretation.
Assuntos
Metilação de DNA , DNA/análise , Análise de Sequência de DNA/métodos , Análise por Conglomerados , Ilhas de CpG , DNA/normas , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Controle de Qualidade , Análise de Sequência de DNA/normasRESUMO
The transcriptional response driven by Hypoxia-inducible factor (HIF) is central to the adaptation to oxygen restriction. Despite recent characterization of genome-wide HIF DNA binding locations and hypoxia-regulated transcripts in different cell types, the molecular bases of HIF target selection remain unresolved. Herein, we combined multi-level experimental data and computational predictions to identify sequence motifs that may contribute to HIF target selectivity. We obtained a core set of bona fide HIF binding regions by integrating multiple HIF1 DNA binding and hypoxia expression profiling datasets. This core set exhibits evolutionarily conserved binding regions and is enriched in functional responses to hypoxia. Computational prediction of enriched transcription factor binding sites identified sequence motifs corresponding to several stress-responsive transcription factors, such as activator protein 1 (AP1), cAMP response element-binding (CREB), or CCAAT-enhancer binding protein (CEBP). Experimental validations on HIF-regulated promoters suggest a functional role of the identified motifs in modulating HIF-mediated transcription. Accordingly, transcriptional targets of these factors are over-represented in a sorted list of hypoxia-regulated genes. Altogether, our results implicate cooperativity among stress-responsive transcription factors in fine-tuning the HIF transcriptional response.
Assuntos
Fator 1 Induzível por Hipóxia/metabolismo , Estresse Fisiológico , Fatores de Transcrição/metabolismo , Animais , Sequência de Bases , Sítios de Ligação , Imunoprecipitação da Cromatina , DNA/química , Perfilação da Expressão Gênica , Células HeLa , HumanosRESUMO
BACKGROUND: DNA methylation is an important epigenetic mark and dysregulation of DNA methylation is associated with many diseases including cancer. Advances in next-generation sequencing now allow unbiased methylome profiling of entire patient cohorts, greatly facilitating biomarker discovery and presenting new opportunities to understand the biological mechanisms by which changes in methylation contribute to disease. Enrichment-based sequencing assays such as MethylCap-seq are a cost effective solution for genome-wide determination of methylation status, but the technical reliability of methylation reconstruction from raw sequencing data has not been well characterized. METHODS: We analyze three MethylCap-seq data sets and perform two different analyses to assess data quality. First, we investigate how data quality is affected by excluding samples that do not meet quality control cutoff requirements. Second, we consider the effect of additional reads on enrichment score, saturation, and coverage. Lastly, we verify a method for the determination of the global amount of methylation from MethylCap-seq data by comparing to a spiked-in control DNA of known methylation status. RESULTS: We show that rejection of samples based on our quality control parameters leads to a significant improvement of methylation calling. Additional reads beyond ~13 million unique aligned reads improved coverage, modestly improved saturation, and did not impact enrichment score. Lastly, we find that a global methylation indicator calculated from MethylCap-seq data correlates well with the global methylation level of a sample as obtained from a spike-in DNA of known methylation level. CONCLUSIONS: We show that with appropriate quality control MethylCap-seq is a reliable tool, suitable for cohorts of hundreds of patients, that provides reproducible methylation information on a feature by feature basis as well as information about the global level of methylation.
Assuntos
Metilação de DNA , Sequenciamento de Nucleotídeos em Larga Escala , Ilhas de CpG , DNA/metabolismo , Neoplasias do Endométrio/genética , Neoplasias do Endométrio/metabolismo , Feminino , Sequenciamento de Nucleotídeos em Larga Escala/normas , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Regiões Promotoras Genéticas , Controle de QualidadeRESUMO
While tumor suppressor genes frequently undergo epigenetic silencing in cancer, how the instructions directing this transcriptional repression are transmitted in cancer cells remain largely unclear. Expression of cyclin-dependent kinase inhibitor 1C (CDKN1C), an imprinted gene on chromosomal band 11 p15.5, is reduced or lost in the majority of breast cancers. Here, we report that CDKN1C is suppressed by estrogen through epigenetic mechanisms involving the chromatin-interacting noncoding RNA KCNQ1OT1 and CCCTC-binding factor (CTCF). Activation of estrogen signaling reduced CDKN1C expression 3-fold (P < 0.001) and established repressive histone modifications at the 5' regulatory region of the locus. These events were concomitant with induction of KCNQ1OT1 expression as well as increased recruitment of CTCF to both the distal KCNQ1OT1 promoter-associated imprinting control region (ICR) and the CDKN1C locus. Transient depletion of CTCF by small interfering RNA increased CDKN1C expression and significantly reduced the estrogen-mediated repression of CDKN1C. Further studies in breast cancer cell lines indicated that the epigenetic silencing of CDKN1C occurs in part as the result of genetic loss of the inactive methylated 11p15.5 ICR allele (R(2) = 0.612, P < 0.001). We also found a novel cis-encoded antisense transcript, CDKN1C-AS, which is induced by estrogen signaling following pharmacologic inhibition of DNA methyltransferase and histone deacetylase activity. Forced expression of CDKN1C-AS was capable of repressing endogenous CDKN1C in vivo. Our findings suggest that in addition to promoter hypermethylation, epigenetic repression of tumor suppressor genes by CTCF and noncoding RNA transcripts could be more common and important than previously understood.
Assuntos
Neoplasias da Mama/genética , Inibidor de Quinase Dependente de Ciclina p57/genética , Metilação de DNA , Epigênese Genética/genética , Estrogênios/farmacologia , Inativação Gênica/efeitos dos fármacos , Impressão Genômica , Fator de Ligação a CCCTC , Imunoprecipitação da Cromatina , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Canais de Potássio de Abertura Dependente da Tensão da Membrana/genética , Regiões Promotoras Genéticas/genética , RNA Mensageiro/genética , RNA não Traduzido/genética , Proteínas Repressoras/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais CultivadasRESUMO
The current concept of epigenetic repression is based on one repressor unit corresponding to one silent gene. This notion, however, cannot adequately explain concurrent silencing of multiple loci observed in large chromosome regions. The long-range epigenetic silencing (LRES) can be a frequent occurrence throughout the human genome. To comprehensively characterize the influence of estrogen signaling on LRES, we analyzed transcriptome, methylome, and estrogen receptor alpha (ESR1)-binding datasets from normal breast epithelia and breast cancer cells. This "omics" approach uncovered 11 large repressive zones (range, 0.35 approximately 5.98 megabases), including a 14-gene cluster located on 16p11.2. In normal cells, estrogen signaling induced transient formation of multiple DNA loops in the 16p11.2 region by bringing 14 distant loci to focal ESR1-docking sites for coordinate repression. However, the plasticity of this free DNA movement was reduced in breast cancer cells. Together with the acquisition of DNA methylation and repressive chromatin modifications at the 16p11.2 loci, an inflexible DNA scaffold may be a novel determinant used by breast cancer cells to reinforce estrogen-mediated repression.
Assuntos
Neoplasias da Mama/metabolismo , Cromossomos Humanos Par 16 , Epigênese Genética/fisiologia , Estrogênios/fisiologia , Inativação Gênica , Animais , Sítios de Ligação , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Células Cultivadas , Metilação de DNA , Receptor alfa de Estrogênio/metabolismo , Estrogênios/metabolismo , Feminino , Humanos , Glândulas Mamárias Humanas/citologia , Glândulas Mamárias Humanas/metabolismo , Família MultigênicaRESUMO
Early exposure to xenoestrogens may predispose to breast cancer risk later in adult life. It is likely that long-lived, self-regenerating epithelial progenitor cells are more susceptible to these exposure injuries over time and transmit the injured memory through epigenetic mechanisms to their differentiated progeny. Here, we used progenitor-containing mammospheres as an in vitro exposure model to study this epigenetic effect. Expression profiling identified that, relative to control cells, 9.1% of microRNAs (82 of 898 loci) were altered in epithelial progeny derived from mammospheres exposed to a synthetic estrogen, diethylstilbestrol. Repressive chromatin marks, trimethyl Lys27 of histone H3 (H3K27me3) and dimethyl Lys9 of histone H3 (H3K9me2), were found at a down-regulated locus, miR-9-3, in epithelial cells preexposed to diethylstilbestrol. This was accompanied by recruitment of DNA methyltransferase 1 that caused an aberrant increase in DNA methylation of its promoter CpG island in mammosphere-derived epithelial cells on diethylstilbestrol preexposure. Functional analyses suggest that miR-9-3 plays a role in the p53-related apoptotic pathway. Epigenetic silencing of this gene, therefore, reduces this cellular function and promotes the proliferation of breast cancer cells. Promoter hypermethylation of this microRNA may be a hallmark for early breast cancer development, and restoration of its expression by epigenetic and microRNA-based therapies is another viable option for future treatment of this disease.
Assuntos
Dietilestilbestrol/farmacologia , Epigênese Genética/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , MicroRNAs/genética , Azacitidina/análogos & derivados , Azacitidina/farmacologia , Mama/citologia , Mama/metabolismo , Linhagem Celular Tumoral , Células Cultivadas , Ilhas de CpG/genética , Metilação de DNA , Decitabina , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Imunofluorescência , Expressão Gênica/efeitos dos fármacos , Perfilação da Expressão Gênica , Redes Reguladoras de Genes , Humanos , Ácidos Hidroxâmicos/farmacologia , Modelos Biológicos , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , TransfecçãoRESUMO
Several studies have reported that a high expression ratio of HOXB13 to IL17BR predicts tumor recurrence in node-negative, estrogen receptor (ER) alpha-positive breast cancer patients treated with tamoxifen. The molecular mechanisms underlying this dysregulation of gene expression remain to be explored. Our epigenetic analysis has found that increased promoter methylation of one of these genes, HOXB13, correlate with the decreased expression of its transcript in breast cancer cell lines (P < 0.005). Transcriptional silencing of this gene can be reversed by a demethylation treatment. HOXB13 is suppressed by the activation of estrogen signaling in ERalpha-positive breast cancer cells. However, treatment with 4-hydroxytamoxifen (4-OHT), an antiestrogen, abrogates the ERalpha-mediated suppression in cancer cells. The notion that this transcriptional induction of HOXB13 occurs in vitro with simultaneous exposure to both estrogen and 4-OHT may provide a biological explanation for its aberrant expression in many node-negative patients undergoing tamoxifen therapy. Interestingly, promoter hypermethylation of HOXB13 is more frequently observed in ERalpha-positive patients with increased lymph node metastasis (P = 0.031) and large tumor sizes (>5 cm) (P = 0.008). In addition, this aberrant epigenetic event is associated with shorter disease-free survival (P = 0.029) in cancer patients. These results suggest that hypermethylation of HOXB13 is a late event of breast tumorigenesis and a poor prognostic indicator of node-positive cancer patients.
Assuntos
Neoplasias da Mama/genética , Metilação de DNA , Estradiol/farmacologia , Regulação Neoplásica da Expressão Gênica , Proteínas de Homeodomínio/genética , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Ilhas de CpG , Receptor alfa de Estrogênio/biossíntese , Estrogênios/metabolismo , Feminino , Inativação Gênica , Proteínas de Homeodomínio/biossíntese , Humanos , Regiões Promotoras Genéticas , Transdução de Sinais , Tamoxifeno/análogos & derivados , Tamoxifeno/farmacologiaRESUMO
Estrogen imprinting is used to describe a phenomenon in which early developmental exposure to endocrine disruptors increases breast cancer risk later in adult life. We propose that long-lived, self-regenerating stem and progenitor cells are more susceptible to the exposure injury than terminally differentiated epithelial cells in the breast duct. Mammospheres, containing enriched breast progenitors, were used as an exposure system to simulate this imprinting phenomenon in vitro. Using MeDIP-chip, a methylation microarray screening method, we found that 0.5% (120 loci) of human CpG islands were hypermethylated in epithelial cells derived from estrogen-exposed progenitors compared with the non-estrogen-exposed control cells. This epigenetic event may lead to progressive silencing of tumor suppressor genes, including RUNX3, in these epithelial cells, which also occurred in primary breast tumors. Furthermore, normal tissue in close proximity to the tumor site also displayed RUNX3 hypermethylation, suggesting that this aberrant event occurs in early breast carcinogenesis. The high prevalence of estrogen-induced epigenetic changes in primary tumors and the surrounding histologically normal tissues provides the first empirical link between estrogen injury of breast stem/progenitor cells and carcinogenesis. This finding also offers a mechanistic explanation as to why a tumor suppressor gene, such as RUNX3, can be heritably silenced by epigenetic mechanisms in breast cancer.
Assuntos
Neoplasias da Mama/induzido quimicamente , Neoplasias da Mama/patologia , Mama/efeitos dos fármacos , Estradiol/farmacologia , Células-Tronco/efeitos dos fármacos , Adolescente , Adulto , Mama/citologia , Mama/patologia , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Processos de Crescimento Celular/efeitos dos fármacos , Subunidade alfa 3 de Fator de Ligação ao Core/biossíntese , Subunidade alfa 3 de Fator de Ligação ao Core/genética , Metilação de DNA/efeitos dos fármacos , Células Epiteliais/patologia , Receptor alfa de Estrogênio/metabolismo , Feminino , Inativação Gênica , Humanos , Pessoa de Meia-Idade , Transdução de Sinais/efeitos dos fármacos , Células-Tronco/citologia , Células-Tronco/patologiaRESUMO
Interactions between protein and DNA are essential for cellular function. The incremental process of developing global approaches to study chromatin began with the in vitro characterization of chromatin structural components and modifications of the versatile chromatin immunoprecipitation (ChIP) assay, capable of analyzing protein-DNA interactions in vivo. Among the emerging global approaches are ChIP cloning, ChIP display, differential chromatin scanning, ChIP-chip, DamID chromatin profiling, and chromatin array. These methods have been used to assess transcription-factor binding and (or) histone modification. This review describes these global methods and illustrates their potential in answering biological questions.