RESUMO
Metazoan chromosomes are sequentially partitioned into topologically associating domains (TADs) and then into smaller sub-domains. One class of sub-domains, insulated neighborhoods, are proposed to spatially sequester and insulate the enclosed genes through self-association and chromatin looping. However, it has not been determined functionally whether promoter-enhancer interactions and gene regulation are broadly restricted to within these loops. Here, we employed published datasets from murine embryonic stem cells (mESCs) to identify insulated neighborhoods that confine promoter-enhancer interactions and demarcate gene regulatory regions. To directly address the functionality of these regions, we depleted estrogen-related receptor ß (Esrrb), which binds the Mediator co-activator complex, to impair enhancers of genes within 222 insulated neighborhoods without causing mESC differentiation. Esrrb depletion reduces Mediator binding, promoter-enhancer looping, and expression of both nascent RNA and mRNA within the insulated neighborhoods without significantly affecting the flanking genes. Our data indicate that insulated neighborhoods represent functional regulons in mammalian genomes.
Assuntos
Cromossomos de Mamíferos , Elementos Facilitadores Genéticos , Elementos Isolantes , Células-Tronco Embrionárias Murinas/fisiologia , Regiões Promotoras Genéticas , Transcrição Gênica , Animais , Sítios de Ligação , Fator de Ligação a CCCTC/genética , Fator de Ligação a CCCTC/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Bases de Dados Genéticas , Regulação para Baixo , Camundongos , Ligação Proteica , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Receptores de Estrogênio/genética , Receptores de Estrogênio/metabolismo , CoesinasRESUMO
Limited knowledge of the mechanisms that govern the self-renewal of human haematopoietic stem cells (HSCs), and why this fails in culture, have impeded the expansion of HSCs for transplantation1. Here we identify MLLT3 (also known as AF9) as a crucial regulator of HSCs that is highly enriched in human fetal, neonatal and adult HSCs, but downregulated in culture. Depletion of MLLT3 prevented the maintenance of transplantable human haematopoietic stem or progenitor cells (HSPCs) in culture, whereas stabilizing MLLT3 expression in culture enabled more than 12-fold expansion of transplantable HSCs that provided balanced multilineage reconstitution in primary and secondary mouse recipients. Similar to endogenous MLLT3, overexpressed MLLT3 localized to active promoters in HSPCs, sustained levels of H3K79me2 and protected the HSC transcriptional program in culture. MLLT3 thus acts as HSC maintenance factor that links histone reader and modifying activities to modulate HSC gene expression, and may provide a promising approach to expand HSCs for transplantation.
Assuntos
Autorrenovação Celular , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Proteínas Nucleares/metabolismo , Animais , Células Cultivadas , Regulação da Expressão Gênica , Transplante de Células-Tronco Hematopoéticas , Humanos , Camundongos , Proteínas Nucleares/genética , Ligação ProteicaRESUMO
Chromobox homolog 3 (Cbx3/heterochromatin protein 1γ [HP1γ]) stimulates cell differentiation, but its mechanism is unknown. We found that Cbx3 binds to gene promoters upon differentiation of murine embryonic stem cells (ESCs) to neural progenitor cells (NPCs) and recruits the Mediator subunit Med26. RNAi knockdown of either Cbx3 or Med26 inhibits neural differentiation while up-regulating genes involved in mesodermal lineage decisions. Thus, Cbx3 and Med26 together ensure the fidelity of lineage specification by enhancing the expression of neural genes and down-regulating genes specific to alternative fates.
Assuntos
Diferenciação Celular , Linhagem da Célula , Proteínas Cromossômicas não Histona/metabolismo , Células-Tronco Embrionárias/citologia , Regulação da Expressão Gênica , Complexo Mediador/metabolismo , Células-Tronco Neurais/citologia , Animais , Células Cultivadas , Proteínas Cromossômicas não Histona/antagonistas & inibidores , Proteínas Cromossômicas não Histona/genética , Quinase 8 Dependente de Ciclina/genética , Quinase 8 Dependente de Ciclina/metabolismo , Células-Tronco Embrionárias/metabolismo , Complexo Mediador/genética , Mesoderma/citologia , Mesoderma/metabolismo , Camundongos , Células-Tronco Neurais/metabolismo , Regiões Promotoras Genéticas , RNA Interferente Pequeno/genéticaRESUMO
Characterizing the phenotypic diversity and metabolic capabilities of industrially relevant manufacturing cell lines is critical to bioprocess optimization and cell line development. Metabolic capabilities of production hosts limit nutrient and resource channeling into desired cellular processes and can have a profound impact on productivity. These limitations cannot be directly inferred from measured data such as spent media concentrations or transcriptomics. Here, we present an integrated multi-omic analysis pipeline combining exo-metabolomics, transcriptomics, and genome-scale metabolic network analysis and apply it to three antibody-producing Chinese Hamster Ovary cell lines to identify reprogramming features associated with high-producing clones and metabolic bottlenecks limiting product formation in an industrial bioprocess. Analysis of individual datatypes revealed a decreased nitrogenous byproduct secretion in high-producing clones and the topological changes in peripheral metabolic pathway expression associated with phase shifts. An integrated omics analysis in the context of the genome-scale metabolic model elucidated the differences in central metabolism and identified amino acid utilization bottlenecks limiting cell growth and antibody production that were not evident from exo-metabolomics or transcriptomics alone. Thus, we demonstrate the utility of a multi-omics characterization in providing an in-depth understanding of cellular metabolism, which is critical to efforts in cell engineering and bioprocess optimization.
RESUMO
The role of cytosine methylation in the structure and function of enhancers is not well understood. In this study, we investigate the role of DNA methylation at enhancers by comparing the epigenomes of the HCT116 cell line and its highly demethylated derivative, DKO1. Unlike promoters, a portion of regular and super- or stretch enhancers show active H3K27ac marks co-existing with extensive DNA methylation, demonstrating the unexpected presence of bivalent chromatin in both cultured and uncultured cells. Furthermore, our findings also show that bivalent regions have fewer nucleosome-depleted regions and transcription factor-binding sites than monovalent regions. Reduction of DNA methylation genetically or pharmacologically leads to a decrease of the H3K27ac mark. Thus, DNA methylation plays an unexpected dual role at enhancer regions, being anti-correlated focally at transcription factor-binding sites but positively correlated globally with the active H3K27ac mark to ensure structural enhancer integrity.
Assuntos
Metilação de DNA , Elementos Facilitadores Genéticos , Epigênese Genética , Histonas/metabolismo , Processamento de Proteína Pós-Traducional , Acetilação , Sítios de Ligação , Montagem e Desmontagem da Cromatina , Citosina , Células HCT116 , Histonas/genética , Humanos , Fatores de TempoRESUMO
BACKGROUND: Genetic diversity and the heterogeneous nature of cardiac fibroblasts (CFbs) have hindered characterization of the molecular mechanisms that regulate cardiac fibrosis. The Hybrid Mouse Diversity Panel offers a valuable tool to examine genetically diverse cardiac fibroblasts and their role in fibrosis. METHODS: Three strains of mice (C57BL/6J, C3H/HeJ, and KK/HlJ) were selected from the Hybrid Mouse Diversity Panel and treated with either isoproterenol (ISO) or saline by an intraperitoneally implanted osmotic pump. After 21 days, cardiac function and levels of fibrosis were measured by echocardiography and trichrome staining, respectively. Activation and proliferation of CFbs were measured by in vitro and in vivo assays under normal and injury conditions. RNA sequencing was done on isolated CFbs from each strain. Results were analyzed by Ingenuity Pathway Analysis and validated by reverse transcription-qPCR, immunohistochemistry, and ELISA. RESULTS: ISO treatment in C57BL/6J, C3H/HeJ, and KK/HlJ mice resulted in minimal, moderate, and extensive levels of fibrosis, respectively (n=7-8 hearts per condition). Isolated CFbs treated with ISO exhibited strain-specific increases in the levels of activation but showed comparable levels of proliferation. Similar results were found in vivo, with fibroblast activation, and not proliferation, correlating with the differential levels of cardiac fibrosis after ISO treatment. RNA sequencing revealed that CFbs from each strain exhibit unique gene expression changes in response to ISO. We identified Ltbp2 as a commonly upregulated gene after ISO treatment. Expression of LTBP2 was elevated and specifically localized in the fibrotic regions of the myocardium after injury in mice and in human heart failure patients. CONCLUSIONS: This study highlights the importance of genetic variation in cardiac fibrosis by using multiple inbred mouse strains to characterize CFbs and their response to ISO treatment. Our data suggest that, although fibroblast activation is a response that parallels the extent of scar formation, proliferation may not necessarily correlate with levels of fibrosis. In addition, by comparing CFbs from multiple strains, we identified pathways as potential therapeutic targets and LTBP2 as a marker for fibrosis, with relevance to patients with underlying myocardial fibrosis.
Assuntos
Cardiomiopatias/genética , Cardiomiopatias/patologia , Proliferação de Células , Fibroblastos/patologia , Variação Genética , Proteínas de Ligação a TGF-beta Latente/genética , Animais , Cardiomiopatias/induzido quimicamente , Cardiomiopatias/metabolismo , Células Cultivadas , Modelos Animais de Doenças , Feminino , Fibroblastos/metabolismo , Fibrose , Predisposição Genética para Doença , Isoproterenol , Proteínas de Ligação a TGF-beta Latente/metabolismo , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Fenótipo , Especificidade da Espécie , TranscriptomaRESUMO
Fibroblast Growth Factor (FGF) signaling promotes self-renewal in progenitor cells by encouraging proliferation and inhibiting cellular senescence. Yet, these beneficial effects can be hijacked by disease-causing mutations in FGF receptor (FGFR) during embryogenesis. By studying dominant FGFR2 mutations that are germline in bent bone dysplasia syndrome (BBDS), we reveal a mechanistic connection between FGFR2, ribosome biogenesis, and cellular stress that links cell fate determination to disease pathology. We previously showed that FGFR2 mutations in BBDS, which amplify nucleolar targeting of FGFR2, activate ribosomal DNA (rDNA) transcription and delay differentiation in osteoprogenitor cells and patient-derived bone. Here we find that the BBDS mutations augment the ability of FGFR2 to recruit histone-remodeling factors that epigenetically activate transcriptionally silent rDNA. Nucleolar morphology is controlled by chromatin structure, and the high levels of euchromatic rDNA induced by the BBDS mutations direct nucleolar disorganization, alter ribosome biogenesis, and activate the Rpl11-Mdm2-p53 nucleolar stress response pathway. Inhibition of p53 in cells expressing the FGFR2 mutations in BBDS rescues delayed osteoblast differentiation, suggesting that p53 activation is an essential pathogenic factor in, and potential therapeutic target for, BBDS. This work establishes rDNA as developmentally regulated loci that receive direct input from FGF signaling to balance self-renewal and cell fate determination.
Assuntos
Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/genética , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/metabolismo , Doenças do Desenvolvimento Ósseo/genética , Doenças do Desenvolvimento Ósseo/metabolismo , Diferenciação Celular/fisiologia , Proliferação de Células/genética , Proliferação de Células/fisiologia , DNA Ribossômico/metabolismo , Humanos , Mutação , Osteoblastos/metabolismo , Osteogênese/genética , Fragmentos de Peptídeos/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Ribossomos/metabolismo , Transdução de Sinais , Proteína Supressora de Tumor p53/metabolismoRESUMO
The holistic role of DNA methylation in the organization of the cancer epigenome is not well understood. Here we perform a comprehensive, high-resolution analysis of chromatin structure to compare the landscapes of HCT116 colon cancer cells and a DNA methylation-deficient derivative. The NOMe-seq accessibility assay unexpectedly revealed symmetrical and transcription-independent nucleosomal phasing across active, poised, and inactive genomic elements. DNA methylation abolished this phasing primarily at enhancers and CpG island (CGI) promoters, with little effect on insulators and non-CGI promoters. Abolishment of DNA methylation led to the context-specific reestablishment of the poised and active states of normal colon cells, which were marked in methylation-deficient cells by distinct H3K27 modifications and the presence of either well-phased nucleosomes or nucleosome-depleted regions, respectively. At higher-order genomic scales, we found that long, H3K9me3-marked domains had lower accessibility, consistent with a more compact chromatin structure. Taken together, our results demonstrate the nuanced and context-dependent role of DNA methylation in the functional, multiscale organization of cancer epigenomes.
Assuntos
Cromatina/genética , Neoplasias do Colo/genética , Metilação de DNA/genética , Linhagem Celular Tumoral , Ilhas de CpG/genética , DNA (Citosina-5-)-Metiltransferase 1 , DNA (Citosina-5-)-Metiltransferases/biossíntese , DNA (Citosina-5-)-Metiltransferases/genética , Epigênese Genética , Células HCT116 , Histonas/genética , Humanos , Nucleossomos/genética , Regiões Promotoras Genéticas/genética , DNA Metiltransferase 3BRESUMO
Extracellular cues play critical roles in the establishment of the epigenome during development and may also contribute to epigenetic perturbations found in disease states. The direct role of the local tissue environment on the post-development human epigenome, however, remains unclear due to limitations in studies of human subjects. Here, we use an isogenic human ileal neobladder surgical model and compare global DNA methylation levels of intestinal epithelial cells pre- and post-neobladder construction using the Infinium HumanMethylation450 BeadChip. Our study is the first to quantify the effect of environmental cues on the human epigenome and show that the local tissue environment directly modulates DNA methylation patterns in normal differentiated cells in vivo. In the neobladder, the intestinal epithelial cells lose their tissue-specific epigenetic landscape in a time-dependent manner following the tissue's exposure to a bladder environment. We find that de novo methylation of many intestine-specific enhancers occurs at the rate of 0.41% per month (P < 0.01, Pearson = 0.71), while demethylation of primarily non-intestine-specific transcribed regions occurs at the rate of -0.37% per month (P < 0.01, Pearson = -0.57). The dynamic resetting of the DNA methylome in the neobladder not only implicates local environmental cues in the shaping and maintenance of the epigenome but also illustrates an unexpected cross-talk between the epigenome and the cellular environment.
Assuntos
Diferenciação Celular/genética , Metilação de DNA/genética , Epigênese Genética , Intestinos/crescimento & desenvolvimento , Idoso , Ilhas de CpG , Genoma Humano , Humanos , Intestinos/cirurgia , Intestinos/transplante , Pessoa de Meia-Idade , Transplante de TecidosRESUMO
DNA methylation and nucleosome positioning work together to generate chromatin structures that regulate gene expression. Nucleosomes are typically mapped using nuclease digestion requiring significant amounts of material and varying enzyme concentrations. We have developed a method (NOMe-seq) that uses a GpC methyltransferase (M.CviPI) and next generation sequencing to generate a high resolution footprint of nucleosome positioning genome-wide using less than 1 million cells while retaining endogenous DNA methylation information from the same DNA strand. Using a novel bioinformatics pipeline, we show a striking anti-correlation between nucleosome occupancy and DNA methylation at CTCF regions that is not present at promoters. We further show that the extent of nucleosome depletion at promoters is directly correlated to expression level and can accommodate multiple nucleosomes and provide genome-wide evidence that expressed non-CpG island promoters are nucleosome-depleted. Importantly, NOMe-seq obtains DNA methylation and nucleosome positioning information from the same DNA molecule, giving the first genome-wide DNA methylation and nucleosome positioning correlation at the single molecule, and thus, single cell level, that can be used to monitor disease progression and response to therapy.
Assuntos
Mapeamento Cromossômico , Metilação de DNA , DNA/genética , Epigenômica/métodos , Nucleossomos/genética , Alelos , Linhagem Celular , Montagem e Desmontagem da Cromatina , Ilhas de CpG , Pegada de DNA , Deleção de Genes , Regulação da Expressão Gênica , Loci Gênicos , Humanos , Metiltransferases , Nucleossomos/metabolismo , Regiões Promotoras Genéticas , Alinhamento de Sequência , Análise de Sequência de DNARESUMO
Neonatal gene therapy has the potential to ameliorate abnormalities before disease onset. Our gene knockout of arginase I (AI) deficiency is characterized by increasing hyperammonemia, neurological deterioration, and early death. We constructed a helper-dependent adenoviral vector (HDV) carrying AI and examined for correction of this defect. Neonates were administered 5 x 10(9) viral particles/g and analyzed for survival, arginase activity, and ammonia and amino acids levels. The life expectancy of arg(-/-) mice increased to 27 days while controls died at 14 days with hyperammonemia and in extremis. Death correlated with a decrease in viral DNA/RNA per cell as liver mass increased. Arginase assays demonstrated that vector-injected hepatocytes had ~20% activity of heterozygotes at 2 weeks of age. Hepatic arginine and ornithine in treated mice were similar to those of saline-injected heterozygotes at 2 weeks, whereas ammonia was normal. By 26 days, arginase activity in the treated arg(-/-) livers declined to <10%, and arginine and ornithine increased. Ammonia levels began increasing by day 25, suggesting the cause of death to be similar to that of uninjected arg(-/-) mice, albeit at a later time. These studies demonstrate that the AI deficient newborn mouse can be temporarily corrected and rescued using a HDV.
Assuntos
Adenoviridae/genética , Arginase/genética , Terapia Genética/métodos , Vetores Genéticos/genética , Hiperamonemia/terapia , Hiperargininemia/terapia , Aminoácidos/metabolismo , Amônia/metabolismo , Animais , Animais Recém-Nascidos , Arginase/metabolismo , Arginase/fisiologia , Camundongos , Camundongos KnockoutRESUMO
To better understand the impact of chromatin structure on regulation of the prostate cancer transcriptome, we develop high-resolution chromatin interaction maps in normal and prostate cancer cells using in situ Hi-C. By combining the in situ Hi-C data with active and repressive histone marks, CTCF binding sites, nucleosome-depleted regions, and transcriptome profiling, we identify topologically associating domains (TADs) that change in size and epigenetic states between normal and prostate cancer cells. Moreover, we identify normal and prostate cancer-specific enhancer-promoter loops and involved transcription factors. For example, we show that FOXA1 is enriched in prostate cancer-specific enhancer-promoter loop anchors. We also find that the chromatin structure surrounding the androgen receptor (AR) locus is altered in the prostate cancer cells with many cancer-specific enhancer-promoter loops. This creation of 3D epigenomic maps enables a better understanding of prostate cancer biology and mechanisms of gene regulation.
Assuntos
Epigenômica , Neoplasias da Próstata/genética , Transcriptoma/genética , Linhagem Celular Tumoral , Cromatina/metabolismo , Elementos Facilitadores Genéticos , Epigênese Genética , Loci Gênicos , Código das Histonas/genética , Humanos , Masculino , Regiões Promotoras Genéticas , Receptores Androgênicos/genéticaRESUMO
Various methodologies are available to interrogate specific components of epigenetic mechanisms such as DNA methylation or nucleosome occupancy at both the locus-specific and the genome-wide level. It has become increasingly clear, however, that comprehension of the functional interactions between epigenetic mechanisms is critical for understanding how cellular transcription programs are regulated or deregulated during normal and disease development. The Nucleosome Occupancy and Methylome sequencing (NOMe-seq) assay allows us to directly measure the relationship between DNA methylation and nucleosome occupancy by taking advantage of the methyltransferase M.CviPI, which methylates unprotected GpC dinucleotides to create a footprint of chromatin accessibility. This assay generates dual nucleosome occupancy and DNA methylation information at a single-DNA molecule resolution using as little as 200,000 cells and in as short as 15 min reaction time. DNA methylation levels and nucleosome occupancy status of genomic regions of interest can be subsequently interrogated by cloning PCR-amplified bisulfite DNA and sequencing individual clones. Alternatively, NOMe-seq can be combined with next-generation sequencing in order to generate an integrated global map of DNA methylation and nucleosome occupancy, which allows for comprehensive examination as to how these epigenetic components correlate with each other.
Assuntos
Metilação de DNA , Nucleossomos/metabolismo , Análise de Sequência de DNA/métodos , Ilhas de CpG , Epigênese Genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Metiltransferases/metabolismo , Regiões Promotoras GenéticasRESUMO
As part of PsychENCODE, we developed a three-dimensional (3D) epigenomic map of primary cultured neuronal cells derived from olfactory neuroepithelium (CNON). We mapped topologically associating domains and high-resolution chromatin interactions using Hi-C and identified regulatory elements using chromatin immunoprecipitation and nucleosome positioning assays. Using epigenomic datasets from biopsies of 63 living individuals, we found that epigenetic marks at distal regulatory elements are more variable than marks at proximal regulatory elements. By integrating genotype and metadata, we identified enhancers that have different levels corresponding to differences in genetic variation, gender, smoking, and schizophrenia. Motif searches revealed that many CNON enhancers are bound by neuronal-related transcription factors. Last, we combined 3D epigenomic maps and gene expression profiles to predict enhancer-target gene interactions on a genome-wide scale. This study not only provides a framework for understanding individual epigenetic variation using a primary cell model system but also contributes valuable data resources for epigenomic studies of neuronal epithelium.
Assuntos
Epigênese Genética , Epigenômica , Regulação da Expressão Gênica , Neurônios Receptores Olfatórios/metabolismo , Sítios de Ligação , Imunoprecipitação da Cromatina , Mapeamento Cromossômico , Biologia Computacional/métodos , Elementos Facilitadores Genéticos , Epigenômica/métodos , Perfilação da Expressão Gênica , Variação Genética , Heterocromatina/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Motivos de Nucleotídeos , Ligação Proteica , Sequências Reguladoras de Ácido Nucleico , Fatores de Transcrição/metabolismo , Transcriptoma , Fluxo de TrabalhoRESUMO
Prostate cancer (PCa) is the leading cancer among men in the United States, with genetic factors contributing to â¼42% of the susceptibility to PCa. We analyzed a PCa risk region located at 7p15.2 to gain insight into the mechanisms by which this noncoding region may affect gene regulation and contribute to PCa risk. We performed Hi-C analysis and demonstrated that this region has long-range interactions with the HOXA locus, located â¼873 kb away. Using the CRISPR/Cas9 system, we deleted a 4-kb region encompassing several PCa risk-associated SNPs and performed RNA-seq to investigate transcriptomic changes in prostate cells lacking the regulatory element. Our results suggest that the risk element affects the expression of HOXA13 and HOTTIP, but not other genes in the HOXA locus, via a repressive loop. Forced expression of HOXA13 was performed to gain further insight into the mechanisms by which this risk element affects PCa risk.
Assuntos
Proteínas de Homeodomínio/metabolismo , Sistemas CRISPR-Cas/genética , Linhagem Celular Tumoral , Proteínas Correpressoras , Proteínas de Ligação a DNA , Loci Gênicos , Proteínas de Homeodomínio/genética , Humanos , Masculino , Proteínas de Neoplasias/genética , Polimorfismo de Nucleotídeo Único , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , RNA/química , RNA/isolamento & purificação , RNA/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Risco , Análise de Sequência de RNA , TranscriptomaRESUMO
Changes in ribosome biogenesis are tightly linked to cell growth, proliferation, and differentiation. The rate of ribosome biogenesis is established by RNA Pol I-mediated transcription of ribosomal RNA (rRNA). Thus, rRNA gene transcription is a key determinant of cell behavior. Here, we show that ribosome biogenesis is dynamically regulated during osteoblast differentiation. Upon osteoinduction, osteoprogenitor cells transiently silence a subset of rRNA genes through a reversible mechanism that is initiated through biphasic nucleolar depletion of UBF1 and then RNA Pol I. Nucleolar depletion of UBF1 is coincident with an increase in the number of silent but transcriptionally permissible rRNA genes. This increase in the number of silent rRNA genes reduces levels of ribosome biogenesis and subsequently, protein synthesis. Together these findings demonstrate that fluctuations in rRNA gene transcription are determined by nucleolar occupancy of UBF1 and closely coordinated with the early events necessary for acquisition of the osteoblast cell fate.
Assuntos
Diferenciação Celular , Osteoblastos/citologia , Ribossomos/metabolismo , Células 3T3 , Animais , Camundongos , Biossíntese de Proteínas , RNA Ribossômico/genética , Transcrição GênicaRESUMO
Chromatin is structurally involved in the transcriptional regulation of all genes. While the nucleosome positioning at RNA polymerase II (pol II) promoters has been extensively studied, less is known about the chromatin structure at pol III promoters in human cells. We use a high-resolution analysis to show substantial differences in chromatin structure of pol II and pol III promoters, and between subtypes of pol III genes. Notably, the nucleosome depleted region at the transcription start site of pol III genes extends past the termination sequences, resulting in nucleosome free gene bodies. The +1 nucleosome is located further downstream than at pol II genes and furthermore displays weak positioning. The variable position of the +1 location is seen not only within individual cell populations and between cell types, but also between different pol III promoter subtypes, suggesting that the +1 nucleosome may be involved in the transcriptional regulation of pol III genes. We find that expression and DNA methylation patterns correlate with distinct accessibility patterns, where DNA methylation associates with the silencing and inaccessibility at promoters. Taken together, this study provides the first high-resolution map of nucleosome positioning and occupancy at human pol III promoters at specific loci and genome wide.
Assuntos
Montagem e Desmontagem da Cromatina , Nucleossomos/química , Nucleossomos/genética , Regiões Promotoras Genéticas , RNA Polimerase III/genética , Sítio de Iniciação de Transcrição , Transcrição Gênica , Metilação de DNA , Humanos , Células K562 , RNA Polimerase II/genéticaRESUMO
DNA methylation in promoters is well known to silence genes and is the presumed therapeutic target of methylation inhibitors. Gene body methylation is positively correlated with expression, yet its function is unknown. We show that 5-aza-2'-deoxycytidine treatment not only reactivates genes but decreases the overexpression of genes, many of which are involved in metabolic processes regulated by c-MYC. Downregulation is caused by DNA demethylation of the gene bodies and restoration of high levels of expression requires remethylation by DNMT3B. Gene body methylation may, therefore, be an unexpected therapeutic target for DNA methylation inhibitors, resulting in the normalization of gene overexpression induced during carcinogenesis. Our results provide direct evidence for a causal relationship between gene body methylation and transcription.
Assuntos
Metilação de DNA , Expressão Gênica , Divisão Celular , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , Humanos , Regiões Promotoras GenéticasRESUMO
Patterns of DNA methylation are established during embryonic development and faithfully copied through somatic cell divisions. Based on current understanding of DNA methylation and other interrelated epigenetic modifications, a comprehensive view of the 'epigenetic landscape' and cancer epigenome is evolving. The cancer methylome is highly disrupted, making DNA methylation an excellent target for anticancer therapies. During the last few decades, an increasing number of drugs targeting DNA methylation have been developed to increase efficacy and stability and to decrease toxicity. The earliest and the most successful epigenetic drug to date, 5-Azacytidine, is currently recommended as the first-line treatment of high-risk myelodysplastic syndromes (MDS). Encouraging results from clinical trials have prompted further efforts to elucidate epigenetic alterations in cancer, and to subsequently develop new epigenetic therapies. This review delineates the latest cancer epigenetic models, the recent discovery of hypomethylation agents as well as their application in the clinic.