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1.
Cell ; 167(5): 1354-1368.e14, 2016 11 17.
Artigo em Inglês | MEDLINE | ID: mdl-27863248

RESUMO

Innate immune memory is the phenomenon whereby innate immune cells such as monocytes or macrophages undergo functional reprogramming after exposure to microbial components such as lipopolysaccharide (LPS). We apply an integrated epigenomic approach to characterize the molecular events involved in LPS-induced tolerance in a time-dependent manner. Mechanistically, LPS-treated monocytes fail to accumulate active histone marks at promoter and enhancers of genes in the lipid metabolism and phagocytic pathways. Transcriptional inactivity in response to a second LPS exposure in tolerized macrophages is accompanied by failure to deposit active histone marks at promoters of tolerized genes. In contrast, ß-glucan partially reverses the LPS-induced tolerance in vitro. Importantly, ex vivo ß-glucan treatment of monocytes from volunteers with experimental endotoxemia re-instates their capacity for cytokine production. Tolerance is reversed at the level of distal element histone modification and transcriptional reactivation of otherwise unresponsive genes. VIDEO ABSTRACT.


Assuntos
Tolerância Imunológica , Lipopolissacarídeos/imunologia , Macrófagos/imunologia , Monócitos/imunologia , Sepse/imunologia , Transcrição Gênica , beta-Glucanas/imunologia , Diferenciação Celular , Metilação de DNA , Epigenômica , Redes Reguladoras de Genes , Código das Histonas , Humanos , Imunidade Inata , Memória Imunológica , Macrófagos/citologia , Monócitos/citologia , Sepse/genética
2.
Cell ; 167(5): 1398-1414.e24, 2016 11 17.
Artigo em Inglês | MEDLINE | ID: mdl-27863251

RESUMO

Characterizing the multifaceted contribution of genetic and epigenetic factors to disease phenotypes is a major challenge in human genetics and medicine. We carried out high-resolution genetic, epigenetic, and transcriptomic profiling in three major human immune cell types (CD14+ monocytes, CD16+ neutrophils, and naive CD4+ T cells) from up to 197 individuals. We assess, quantitatively, the relative contribution of cis-genetic and epigenetic factors to transcription and evaluate their impact as potential sources of confounding in epigenome-wide association studies. Further, we characterize highly coordinated genetic effects on gene expression, methylation, and histone variation through quantitative trait locus (QTL) mapping and allele-specific (AS) analyses. Finally, we demonstrate colocalization of molecular trait QTLs at 345 unique immune disease loci. This expansive, high-resolution atlas of multi-omics changes yields insights into cell-type-specific correlation between diverse genomic inputs, more generalizable correlations between these inputs, and defines molecular events that may underpin complex disease risk.


Assuntos
Epigenômica , Doenças do Sistema Imunitário/genética , Monócitos/metabolismo , Neutrófilos/metabolismo , Linfócitos T/metabolismo , Transcrição Gênica , Adulto , Idoso , Processamento Alternativo , Feminino , Predisposição Genética para Doença , Células-Tronco Hematopoéticas/metabolismo , Código das Histonas , Humanos , Masculino , Pessoa de Meia-Idade , Locos de Características Quantitativas , Adulto Jovem
3.
Cell ; 155(1): 121-34, 2013 Sep 26.
Artigo em Inglês | MEDLINE | ID: mdl-24074865

RESUMO

The de novo DNA methyltransferase 3-like (Dnmt3L) is a catalytically inactive DNA methyltransferase that cooperates with Dnmt3a and Dnmt3b to methylate DNA. Dnmt3L is highly expressed in mouse embryonic stem cells (ESCs), but its function in these cells is unknown. Through genome-wide analysis of Dnmt3L knockdown in ESCs, we found that Dnmt3L is a positive regulator of methylation at the gene bodies of housekeeping genes and, more surprisingly, is also a negative regulator of methylation at promoters of bivalent genes. Dnmt3L is required for the differentiation of ESCs into primordial germ cells (PGCs) through the activation of the homeotic gene Rhox5. We demonstrate that Dnmt3L interacts with the Polycomb PRC2 complex in competition with the DNA methyltransferases Dnmt3a and Dnmt3b to maintain low methylation levels at the H3K27me3 regions. Thus, in ESCs, Dnmt3L counteracts the activity of de novo DNA methylases to maintain hypomethylation at promoters of bivalent developmental genes.


Assuntos
DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA , Células-Tronco Embrionárias/metabolismo , Regiões Promotoras Genéticas , Animais , Diferenciação Celular , DNA Metiltransferase 3A , Proteína Potenciadora do Homólogo 2 de Zeste , Células Germinativas/metabolismo , Histonas/metabolismo , Proteínas de Homeodomínio/genética , Camundongos , Complexo Repressor Polycomb 2/metabolismo , Proteínas do Grupo Polycomb/metabolismo , Fatores de Transcrição/genética , DNA Metiltransferase 3B
4.
Cell ; 142(6): 967-80, 2010 Sep 17.
Artigo em Inglês | MEDLINE | ID: mdl-20850016

RESUMO

Trimethyl-lysine (me3) modifications on histones are the most stable epigenetic marks and they control chromatin-mediated regulation of gene expression. Here, we determine proteins that bind these marks by high-accuracy, quantitative mass spectrometry. These chromatin "readers" are assigned to complexes by interaction proteomics of full-length BAC-GFP-tagged proteins. ChIP-Seq profiling identifies their genomic binding sites, revealing functional properties. Among the main findings, the human SAGA complex binds to H3K4me3 via a double Tudor-domain in the C terminus of Sgf29, and the PWWP domain is identified as a putative H3K36me3 binding motif. The ORC complex, including LRWD1, binds to the three most prominent transcriptional repressive lysine methylation sites. Our data reveal a highly adapted interplay between chromatin marks and their associated protein complexes. Reading specific trimethyl-lysine sites by specialized complexes appears to be a widespread mechanism to mediate gene expression.


Assuntos
Cromatina/metabolismo , Epigênese Genética , Código das Histonas , Regulação da Expressão Gênica , Estudo de Associação Genômica Ampla , Células HeLa , Histona Acetiltransferases/metabolismo , Humanos , Lisina/metabolismo , Espectrometria de Massas , Metilação , Proteômica/métodos
5.
Proc Natl Acad Sci U S A ; 112(42): 13115-20, 2015 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-26438844

RESUMO

Genes with similar transcriptional activation kinetics can display very different temporal mRNA profiles because of differences in transcription time, degradation rate, and RNA-processing kinetics. Recent studies have shown that a splicing-associated RNA production delay can be significant. To investigate this issue more generally, it is useful to develop methods applicable to genome-wide datasets. We introduce a joint model of transcriptional activation and mRNA accumulation that can be used for inference of transcription rate, RNA production delay, and degradation rate given data from high-throughput sequencing time course experiments. We combine a mechanistic differential equation model with a nonparametric statistical modeling approach allowing us to capture a broad range of activation kinetics, and we use Bayesian parameter estimation to quantify the uncertainty in estimates of the kinetic parameters. We apply the model to data from estrogen receptor α activation in the MCF-7 breast cancer cell line. We use RNA polymerase II ChIP-Seq time course data to characterize transcriptional activation and mRNA-Seq time course data to quantify mature transcripts. We find that 11% of genes with a good signal in the data display a delay of more than 20 min between completing transcription and mature mRNA production. The genes displaying these long delays are significantly more likely to be short. We also find a statistical association between high delay and late intron retention in pre-mRNA data, indicating significant splicing-associated production delays in many genes.


Assuntos
Genoma Humano , Modelos Genéticos , RNA/biossíntese , Transcrição Gênica , Receptor alfa de Estrogênio/metabolismo , Humanos , Cinética , Células MCF-7 , RNA/genética , Transdução de Sinais
6.
J Biol Chem ; 291(14): 7313-24, 2016 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-26841866

RESUMO

Recent work from others and us revealed interactions between the Sin3/HDAC complex, the H3K4me3 demethylase KDM5A, GATAD1, and EMSY. Here, we characterize the EMSY/KDM5A/SIN3B complex in detail by quantitative interaction proteomics and ChIP-sequencing. We identify a novel substoichiometric interactor of the complex, transcription factor ZNF131, which recruits EMSY to a large number of active, H3K4me3 marked promoters. Interestingly, using an EMSY knock-out line and subsequent rescue experiments, we show that EMSY is in most cases positively correlated with transcriptional activity of its target genes and stimulates cell proliferation. Finally, by immunohistochemical staining of primary breast tissue microarrays we find that EMSY/KDM5A/SIN3B complex subunits are frequently overexpressed in primary breast cancer cases in a correlative manner. Taken together, these data open venues for exploring the possibility that sporadic breast cancer patients with EMSY amplification might benefit from epigenetic combination therapy targeting both the KDM5A demethylase and histone deacetylases.


Assuntos
Neoplasias da Mama/metabolismo , Proteínas de Ligação a DNA/metabolismo , Histonas/metabolismo , Complexos Multiproteicos/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Repressoras/metabolismo , Fatores de Transcrição/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Proteínas de Ligação a DNA/genética , Feminino , Técnicas de Inativação de Genes , Células HeLa , Histonas/genética , Humanos , Complexos Multiproteicos/genética , Proteínas de Neoplasias/genética , Proteínas Nucleares/genética , Proteínas Repressoras/genética , Proteína 2 de Ligação ao Retinoblastoma/genética , Proteína 2 de Ligação ao Retinoblastoma/metabolismo , Fatores de Transcrição/genética
7.
Genome Res ; 22(6): 1128-38, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22466170

RESUMO

Cross-talk between DNA methylation and histone modifications drives the establishment of composite epigenetic signatures and is traditionally studied using correlative rather than direct approaches. Here, we present sequential ChIP-bisulfite-sequencing (ChIP-BS-seq) as an approach to quantitatively assess DNA methylation patterns associated with chromatin modifications or chromatin-associated factors directly. A chromatin-immunoprecipitation (ChIP)-capturing step is used to obtain a restricted representation of the genome occupied by the epigenetic feature of interest, for which a single-base resolution DNA methylation map is then generated. When applied to H3 lysine 27 trimethylation (H3K27me3), we found that H3K27me3 and DNA methylation are compatible throughout most of the genome, except for CpG islands, where these two marks are mutually exclusive. Further ChIP-BS-seq-based analysis in Dnmt triple-knockout (TKO) embryonic stem cells revealed that total loss of CpG methylation is associated with alteration of H3K27me3 levels throughout the genome: H3K27me3 in localized peaks is decreased while broad local enrichments (BLOCs) of H3K27me3 are formed. At an even broader scale, these BLOCs correspond to regions of high DNA methylation in wild-type ES cells, suggesting that DNA methylation prevents H3K27me3 deposition locally and at a megabase scale. Our strategy provides a unique way of investigating global interdependencies between DNA methylation and other chromatin features.


Assuntos
Imunoprecipitação da Cromatina/métodos , Cromatina/genética , Ilhas de CpG , Metilação de DNA , Histonas/metabolismo , Análise de Sequência de DNA/métodos , Animais , Linhagem Celular , Linhagem Celular Tumoral , Cromatina/efeitos dos fármacos , Neoplasias do Colo/genética , Células-Tronco Embrionárias/fisiologia , Epigênese Genética , Técnicas de Inativação de Genes , Genômica/métodos , Humanos , Lisina/metabolismo , Camundongos , Sulfitos/farmacologia
8.
PLoS Comput Biol ; 10(5): e1003598, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24830797

RESUMO

Gene transcription mediated by RNA polymerase II (pol-II) is a key step in gene expression. The dynamics of pol-II moving along the transcribed region influence the rate and timing of gene expression. In this work, we present a probabilistic model of transcription dynamics which is fitted to pol-II occupancy time course data measured using ChIP-Seq. The model can be used to estimate transcription speed and to infer the temporal pol-II activity profile at the gene promoter. Model parameters are estimated using either maximum likelihood estimation or via Bayesian inference using Markov chain Monte Carlo sampling. The Bayesian approach provides confidence intervals for parameter estimates and allows the use of priors that capture domain knowledge, e.g. the expected range of transcription speeds, based on previous experiments. The model describes the movement of pol-II down the gene body and can be used to identify the time of induction for transcriptionally engaged genes. By clustering the inferred promoter activity time profiles, we are able to determine which genes respond quickly to stimuli and group genes that share activity profiles and may therefore be co-regulated. We apply our methodology to biological data obtained using ChIP-seq to measure pol-II occupancy genome-wide when MCF-7 human breast cancer cells are treated with estradiol (E2). The transcription speeds we obtain agree with those obtained previously for smaller numbers of genes with the advantage that our approach can be applied genome-wide. We validate the biological significance of the pol-II promoter activity clusters by investigating cluster-specific transcription factor binding patterns and determining canonical pathway enrichment. We find that rapidly induced genes are enriched for both estrogen receptor alpha (ERα) and FOXA1 binding in their proximal promoter regions.


Assuntos
Imunoprecipitação da Cromatina/métodos , RNA Polimerases Dirigidas por DNA/genética , Modelos Genéticos , Modelos Estatísticos , Regiões Promotoras Genéticas/genética , Transcrição Gênica/genética , Ativação Transcricional/genética , Animais , Simulação por Computador , Humanos , Ligação Proteica
9.
PLoS Comput Biol ; 9(6): e1003100, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23818839

RESUMO

Identification of responsive genes to an extra-cellular cue enables characterization of pathophysiologically crucial biological processes. Deep sequencing technologies provide a powerful means to identify responsive genes, which creates a need for computational methods able to analyze dynamic and multi-level deep sequencing data. To answer this need we introduce here a data-driven algorithm, SPINLONG, which is designed to search for genes that match the user-defined hypotheses or models. SPINLONG is applicable to various experimental setups measuring several molecular markers in parallel. To demonstrate the SPINLONG approach, we analyzed ChIP-seq data reporting PolII, estrogen receptor α (ERα), H3K4me3 and H2A.Z occupancy at five time points in the MCF-7 breast cancer cell line after estradiol stimulus. We obtained 777 ERa early responsive genes and compared the biological functions of the genes having ERα binding within 20 kb of the transcription start site (TSS) to genes without such binding site. Our results show that the non-genomic action of ERα via the MAPK pathway, instead of direct ERa binding, may be responsible for early cell responses to ERα activation. Our results also indicate that the ERα responsive genes triggered by the genomic pathway are transcribed faster than those without ERα binding sites. The survival analysis of the 777 ERα responsive genes with 150 primary breast cancer tumors and in two independent validation cohorts indicated the ATAD3B gene, which does not have ERα binding site within 20 kb of its TSS, to be significantly associated with poor patient survival.


Assuntos
Adenosina Trifosfatases/genética , Neoplasias da Mama/genética , Receptor alfa de Estrogênio/genética , Proteínas de Membrana/genética , Proteínas Mitocondriais/genética , ATPases Associadas a Diversas Atividades Celulares , Neoplasias da Mama/patologia , Feminino , Humanos , Células MCF-7 , Análise de Sobrevida , Transcrição Gênica
10.
Mol Syst Biol ; 7: 562, 2011 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-22186736

RESUMO

The discovery of the Ten-Eleven-Translocation (TET) oxygenases that catalyze the hydroxylation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) has triggered an avalanche of studies aiming to resolve the role of 5hmC in gene regulation if any. Hitherto, TET1 is reported to bind to CpG-island (CGI) and bivalent promoters in mouse embryonic stem cells, whereas binding at DNAseI hypersensitive sites (HS) had escaped previous analysis. Significant enrichment/accumulation of 5hmC but not 5mC can indeed be detected at bivalent promoters and at DNaseI-HS. Surprisingly, however, 5hmC is not detected or present at very low levels at CGI promoters notwithstanding the presence of TET1. Our meta-analysis of DNA methylation profiling points to potential issues with regard to the various methodologies that are part of the toolbox used to detect 5mC and 5hmC. Discrepancies between published studies and technical limitations prevent an unambiguous assignment of 5hmC as a 'true' epigenetic mark, that is, read and interpreted by other factors and/or as a transiently accumulating intermediary product of the conversion of 5mC to unmodified cytosines.


Assuntos
Citosina/análogos & derivados , Metilação de DNA , Regulação da Expressão Gênica , 5-Metilcitosina/fisiologia , Animais , Linhagem Celular , Cromatina , Análise por Conglomerados , Citosina/fisiologia , Células-Tronco Embrionárias , Epigênese Genética , Humanos , Camundongos , Oxigenases , Transcrição Gênica , Translocação Genética
11.
Sci Rep ; 11(1): 9252, 2021 04 29.
Artigo em Inglês | MEDLINE | ID: mdl-33927210

RESUMO

Celiac disease is an auto-immune disease in which an immune response to dietary gluten leads to inflammation and subsequent atrophy of small intestinal villi, causing severe bowel discomfort and malabsorption of nutrients. The major instigating factor for the immune response in celiac disease is the activation of gluten-specific CD4+ T cells expressing T cell receptors that recognize gluten peptides presented in the context of HLA-DQ2 and DQ8. Here we provide an in-depth characterization of 28 gluten-specific T cell clones. We assess their transcriptional and epigenetic response to T cell receptor stimulation and link this to genetic factors associated with celiac disease. Gluten-specific T cells have a distinct transcriptional profile that mostly resembles that of Th1 cells but also express cytokines characteristic of other types of T-helper cells. This transcriptional response appears not to be regulated by changes in chromatin state, but rather by early upregulation of transcription factors and non-coding RNAs that likely orchestrate the subsequent activation of genes that play a role in immune pathways. Finally, integration of chromatin and transcription factor binding profiles suggest that genes activated by T cell receptor stimulation of gluten­specific T cells may be impacted by genetic variation at several genetic loci associated with celiac disease.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Doença Celíaca/genética , Doença Celíaca/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Biomarcadores/metabolismo , Linfócitos T CD4-Positivos/metabolismo , Doença Celíaca/induzido quimicamente , Doença Celíaca/patologia , Citocinas/imunologia , Citocinas/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Glutens/administração & dosagem , Glutens/imunologia , Humanos , Receptores de Antígenos de Linfócitos T/genética , Transcriptoma
12.
Cell Rep ; 26(4): 1059-1069.e6, 2019 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-30673601

RESUMO

Global investigation of histone marks in acute myeloid leukemia (AML) remains limited. Analyses of 38 AML samples through integrated transcriptional and chromatin mark analysis exposes 2 major subtypes. One subtype is dominated by patients with NPM1 mutations or MLL-fusion genes, shows activation of the regulatory pathways involving HOX-family genes as targets, and displays high self-renewal capacity and stemness. The second subtype is enriched for RUNX1 or spliceosome mutations, suggesting potential interplay between the 2 aberrations, and mainly depends on IRF family regulators. Cellular consequences in prognosis predict a relatively worse outcome for the first subtype. Our integrated profiling establishes a rich resource to probe AML subtypes on the basis of expression and chromatin data.


Assuntos
Cromatina , Subunidade alfa 2 de Fator de Ligação ao Core , Leucemia Mieloide Aguda , Mutação , Proteínas Nucleares , Proteínas de Fusão Oncogênica , Cromatina/genética , Cromatina/metabolismo , Cromatina/patologia , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Humanos , Leucemia Mieloide Aguda/classificação , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Nucleofosmina , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo
13.
Mol Biol Cell ; 14(12): 5051-9, 2003 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-12960425

RESUMO

Estrogens control cell growth and viability in target cells via an interplay of genomic and extragenomic pathways not yet elucidated. Here, we show evidence that cell proliferation and survival are differentially regulated by estrogen in rat pituitary tumor PR1 cells. Pico- to femtomolar concentrations of 17beta-estradiol (E2) are sufficient to foster PR1 cell proliferation, whereas nanomolar concentrations of the same are needed to prevent cell death that occurs at a high rate in these cells in the absence of hormone. Activation of endogenous (PRL) or transfected estrogen-responsive genes occurs at the same, higher concentrations of E2 required to promote cell survival, whereas stimulation of cyclin D3 expression and DNA synthesis occur at lower E2 concentrations. Similarly, the pure antiestrogen ICI 182,780 inhibits estrogen response element-dependent trans-activation and cell death more effectively than cyclin-cdk activity, G1-S transition, or DNA synthesis rate. In antiestrogen-treated and/or estrogen-deprived cells, death is due predominantly to apoptosis. Estrogen-induced cell survival, but not E2-dependent cell cycle progression, can be prevented by an inhibitor of c-Src kinase or by blockade of the mitogen-activated protein kinase kinase/extracellular signal-regulated kinase signaling pathway. These data indicate the coexistence of two distinguishable estrogen signaling pathways in PR1 cells, characterized by different functions and sensitivity to hormones and antihormones.


Assuntos
Estradiol/análogos & derivados , Estrogênios/metabolismo , Hipófise/metabolismo , Animais , Apoptose/fisiologia , Proteína Tirosina Quinase CSK , Divisão Celular/fisiologia , Sobrevivência Celular/fisiologia , Ciclina D3 , Ciclinas/metabolismo , Replicação do DNA/fisiologia , Inibidores Enzimáticos/farmacologia , Estradiol/farmacologia , Antagonistas de Estrogênios/farmacologia , Feminino , Fulvestranto , Regulação da Expressão Gênica/fisiologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas Tirosina Quinases/metabolismo , Ratos , Transdução de Sinais/fisiologia , Quinases da Família src
14.
PeerJ ; 5: e3742, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28970965

RESUMO

We have developed a machine learning approach to predict stimulation-dependent enhancer-promoter interactions using evidence from changes in genomic protein occupancy over time. The occupancy of estrogen receptor alpha (ERα), RNA polymerase (Pol II) and histone marks H2AZ and H3K4me3 were measured over time using ChIP-Seq experiments in MCF7 cells stimulated with estrogen. A Bayesian classifier was developed which uses the correlation of temporal binding patterns at enhancers and promoters and genomic proximity as features to predict interactions. This method was trained using experimentally determined interactions from the same system and was shown to achieve much higher precision than predictions based on the genomic proximity of nearest ERα binding. We use the method to identify a genome-wide confident set of ERα target genes and their regulatory enhancers genome-wide. Validation with publicly available GRO-Seq data demonstrates that our predicted targets are much more likely to show early nascent transcription than predictions based on genomic ERα binding proximity alone.

15.
Sci Rep ; 7(1): 11725, 2017 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-28916762

RESUMO

Interactions between DNA and proteins are mainly studied through chemical procedures involving bi-functional reagents, mostly formaldehyde. Chromatin immunoprecipitation is used to identify the binding between transcription factors (TFs) and chromatin, and to evaluate the occurrence and impact of histone/DNA modifications. The current bottleneck in probing DNA-protein interactions using these approaches is caused by the fact that chemical crosslinkers do not discriminate direct and indirect bindings or short-lived chromatin occupancy. Here, we describe a novel application of UV laser-induced (L-) crosslinking and demonstrate that a combination of chemical and L-crosslinking is able to distinguish between direct and indirect DNA-protein interactions in a small number of living cells. The spatial and temporal dynamics of TF bindings to chromatin and their role in gene expression regulation may thus be assessed. The combination of chemical and L-crosslinking offers an exciting and unprecedented tool for biomedical applications.


Assuntos
Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Lasers , Raios Ultravioleta , Linhagem Celular Tumoral , Cromatina/metabolismo , Reagentes de Ligações Cruzadas , Regulação da Expressão Gênica , Humanos , Fatores de Tempo , Fatores de Transcrição/metabolismo
16.
Cell Rep ; 17(3): 783-798, 2016 10 11.
Artigo em Inglês | MEDLINE | ID: mdl-27732854

RESUMO

NuRD (nucleosome remodeling and histone deacetylase) is a versatile multi-protein complex with roles in transcription regulation and the DNA damage response. Here, we show that ZMYND8 bridges NuRD to a number of putative DNA-binding zinc finger proteins. The MYND domain of ZMYND8 directly interacts with PPPLΦ motifs in the NuRD subunit GATAD2A. Both GATAD2A and GATAD2B exclusively form homodimers and define mutually exclusive NuRD subcomplexes. ZMYND8 and NuRD share a large number of genome-wide binding sites, mostly active promoters and enhancers. Depletion of ZMYND8 does not affect NuRD occupancy genome-wide and only slightly affects expression of NuRD/ZMYND8 target genes. In contrast, the MYND domain in ZMYND8 facilitates the rapid, poly(ADP-ribose)-dependent recruitment of GATAD2A/NuRD to sites of DNA damage to promote repair by homologous recombination. Thus, these results show that a specific substoichiometric interaction with a NuRD subunit paralogue provides unique functionality to distinct NuRD subcomplexes.


Assuntos
Dano ao DNA , Fatores de Transcrição GATA/metabolismo , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/metabolismo , Poli Adenosina Difosfato Ribose/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Sequência de Aminoácidos , Dano ao DNA/genética , Reparo do DNA/genética , Elementos Facilitadores Genéticos/genética , Genoma Humano , Células HEK293 , Células HeLa , Humanos , Regiões Promotoras Genéticas , Ligação Proteica , Domínios Proteicos , Domínios e Motivos de Interação entre Proteínas , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Proteínas Repressoras , Proteínas Supressoras de Tumor/química
17.
Cell Rep ; 17(8): 2087-2100, 2016 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-27851970

RESUMO

The t(8;21) acute myeloid leukemia (AML)-associated oncoprotein AML1-ETO disrupts normal hematopoietic differentiation. Here, we have investigated its effects on the transcriptome and epigenome in t(8,21) patient cells. AML1-ETO binding was found at promoter regions of active genes with high levels of histone acetylation but also at distal elements characterized by low acetylation levels and binding of the hematopoietic transcription factors LYL1 and LMO2. In contrast, ERG, FLI1, TAL1, and RUNX1 bind at all AML1-ETO-occupied regulatory regions, including those of the AML1-ETO gene itself, suggesting their involvement in regulating AML1-ETO expression levels. While expression of AML1-ETO in myeloid differentiated induced pluripotent stem cells (iPSCs) induces leukemic characteristics, overexpression increases cell death. We find that expression of wild-type transcription factors RUNX1 and ERG in AML is required to prevent this oncogene overexpression. Together our results show that the interplay of the epigenome and transcription factors prevents apoptosis in t(8;21) AML cells.


Assuntos
Apoptose/genética , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Hematopoese/genética , Leucemia Mieloide Aguda/genética , Proteínas de Fusão Oncogênica/metabolismo , Proteína 1 Parceira de Translocação de RUNX1/metabolismo , Translocação Genética , Acetilação , Sequência de Bases , Linhagem Celular Tumoral , Linhagem da Célula/genética , Sobrevivência Celular/genética , Cromossomos Humanos Par 21/genética , Cromossomos Humanos Par 8/genética , Técnicas de Silenciamento de Genes , Genoma Humano , Histona Desacetilases/metabolismo , Humanos , Leucemia Mieloide Aguda/patologia , Oncogenes , Regiões Promotoras Genéticas , Ligação Proteica/genética , Regulador Transcricional ERG/metabolismo
18.
Cell Rep ; 17(8): 2137-2150, 2016 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-27851974

RESUMO

Epigenome-wide association studies (EWAS) provide an alternative approach for studying human disease through consideration of non-genetic variants such as altered DNA methylation. To advance the complex interpretation of EWAS, we developed eFORGE (http://eforge.cs.ucl.ac.uk/), a new standalone and web-based tool for the analysis and interpretation of EWAS data. eFORGE determines the cell type-specific regulatory component of a set of EWAS-identified differentially methylated positions. This is achieved by detecting enrichment of overlap with DNase I hypersensitive sites across 454 samples (tissues, primary cell types, and cell lines) from the ENCODE, Roadmap Epigenomics, and BLUEPRINT projects. Application of eFORGE to 20 publicly available EWAS datasets identified disease-relevant cell types for several common diseases, a stem cell-like signature in cancer, and demonstrated the ability to detect cell-composition effects for EWAS performed on heterogeneous tissues. Our approach bridges the gap between large-scale epigenomics data and EWAS-derived target selection to yield insight into disease etiology.


Assuntos
Epigenômica , Transdução de Sinais , Software , Estatística como Assunto , Metilação de DNA/genética , Estudo de Associação Genômica Ampla , Humanos , Cariotipagem , Esclerose Múltipla/genética , Especificidade de Órgãos/genética , Transdução de Sinais/genética , Células-Tronco/metabolismo
19.
PLoS One ; 9(6): e99603, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24927503

RESUMO

MBD2 is a subunit of the NuRD complex that is postulated to mediate gene repression via recruitment of the complex to methylated DNA. In this study we adopted an MBD2 tagging-approach to study its genome wide binding characteristics. We show that in vivo MBD2 is mainly recruited to CpG island promoters that are highly methylated. Interestingly, MBD2 binds around 1 kb downstream of the transcription start site of a subset of ∼ 400 CpG island promoters that are characterized by the presence of active histone marks, RNA polymerase II (Pol2) and low to medium gene expression levels and H3K36me3 deposition. These tagged-MBD2 binding sites in MCF-7 show increased methylation in a cohort of primary breast cancers but not in normal breast samples, suggesting a putative role for MBD2 in breast cancer.


Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Metilação de DNA , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , RNA Polimerase II/metabolismo , Sítios de Ligação , Ilhas de CpG , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Células MCF-7 , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Análise de Sequência de RNA
20.
Cancer Res ; 74(8): 2328-39, 2014 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-24566867

RESUMO

HDAC inhibitors (HDACi) are widely used in the clinic to sensitize tumorigenic cells for treatment with other anticancer compounds. The major drawback of HDACi is the broad inhibition of the plethora of HDAC-containing complexes. In acute promyelocytic leukemia (APL), repression by the PML-RARα oncofusion protein is mediated by an HDAC-containing complex that can be dissociated by pharmacologic doses of all trans retinoic acid (ATRA) inducing differentiation and cell death at the expense of side effects and recurrence. We hypothesized that the context-specific close physical proximity of a retinoid and HDACi-binding protein in the repressive PML-RARα-HDAC complex may permit selective targeting by a hybrid molecule of ATRA with a 2-aminoanilide tail of the HDAC inhibitor MS-275, yielding MC2392. We show that MC2392 elicits weak ATRA and essentially no HDACi activity in vitro or in vivo. Genome-wide epigenetic analyses revealed that in NB4 cells expressing PML-RARα, MC2392 induces changes in H3 acetylation at a small subset of PML-RARα-binding sites. RNA-seq reveals that MC2392 alters expression of a number of stress-responsive and apoptotic genes. Concordantly, MC2392 induced rapid and massive, caspase-8-dependent cell death accompanied by RIP1 induction and ROS production. Solid and leukemic tumors are not affected by MC2392, but expression of PML-RARα conveys efficient MC2392-induced cell death. Our data suggest a model in which MC2392 binds to the RARα moiety and selectively inhibits the HDACs resident in the repressive complex responsible for the transcriptional impairment in APLs. Our findings provide proof-of-principle of the concept of a context-dependent targeted therapy.


Assuntos
Inibidores de Histona Desacetilases/farmacologia , Leucemia Mieloide Aguda/tratamento farmacológico , Retinoides/farmacologia , Acetilação/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Apoptose/genética , Caspase 8/metabolismo , Morte Celular , Diferenciação Celular/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Proteínas de Fusão Oncogênica/antagonistas & inibidores , Proteínas de Fusão Oncogênica/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacos , Transfecção , Tretinoína/farmacologia , Células U937
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