RESUMO
BACKGROUND: The identification of tissue-specific differentially methylated regions (tDMRs) is key to our understanding of mammalian development. Research has indicated that tDMRs are aberrantly methylated in cancer and may affect the oncogenic process. PROCEDURE: We used the MassARRAY EpiTYPER system to determine the quantitative methylation levels of seven neuroblastomas (NBs) and two control adrenal medullas at 12 conserved tDMRs. A second sample set of 19 NBs was also analyzed. Statistical analysis was carried out to determine the relationship of the quantitative methylation levels to other prognostic factors in these sample sets. RESULTS: Screening of 12 tDMRs revealed 2 genomic regions (SLC16A5 and ZNF206) with frequent aberrant methylation patterns in NB. The methylation levels of SLC16A5 and ZNF206 were low compared to the control adrenal medullas. The SLC16A5 methylation level (cut-off point, 13.25%) was associated with age at diagnosis, disease stage, and Shimada classification but not with MYCN amplification. The ZNF206 methylation level (cut-off point, 68.80%) was associated with all of the prognostic factors analyzed. Although the methylation levels at these regions did not reach statistical significance in their association with prognosis in mono-variant analysis, patients with both hypomethylation of SLC16A5 and hypermethylation of ZNF206 had a significantly prolonged event-free survival, when these two variables were analyzed together. CONCLUSIONS: We demonstrated that two tDMRs frequently displayed altered methylation patterns in the NB genome, suggesting their distinct involvement in NB development/differentiation. The combined analysis of these two regions could serve as a diagnostic biomarker for poor clinical outcome.
Assuntos
Metilação de DNA/genética , Neuroblastoma/genética , Neuroblastoma/mortalidade , Fatores de Transcrição/genética , Biomarcadores Tumorais/análise , Biomarcadores Tumorais/genética , Criança , Pré-Escolar , Proteínas de Ligação a DNA , Intervalo Livre de Doença , Feminino , Humanos , Lactente , Estimativa de Kaplan-Meier , Masculino , Reação em Cadeia da PolimeraseRESUMO
BACKGROUND: Changes in DNA methylation in the mammalian genome during development are frequent events and play major roles regulating gene expression and other developmental processes. It is necessary to identify these events so that we may understand how these changes affect normal development and how aberrant changes may impact disease. RESULTS: In this study Methylated DNA ImmunoPrecipitation (MeDIP) was used in conjunction with a NimbleGen promoter plus CpG island (CpGi) array to identify Tissue and Developmental Stage specific Differentially Methylated DNA Regions (T-DMRs and DS-DMRs) on a genome-wide basis. Four tissues (brain, heart, liver, and testis) from C57BL/6J mice were analyzed at three developmental stages (15 day embryo, E15; new born, NB; 12 week adult, AD). Almost 5,000 adult T-DMRs and 10,000 DS-DMRs were identified. Surprisingly, almost all DS-DMRs were tissue specific (i.e. methylated in at least one tissue and unmethylated in one or more tissues). In addition our results indicate that many DS-DMRs are methylated at early development stages (E15 and NB) but are unmethylated in adult. There is a very strong bias for testis specific methylation in non-CpGi promoter regions (94%). Although the majority of T-DMRs and DS-DMRs tended to be in non-CpGi promoter regions, a relatively large number were also located in CpGi in promoter, intragenic and intergenic regions (>15% of the 15,979 CpGi on the array). CONCLUSIONS: Our data suggests the vast majority of unique sequence DNA methylation has tissue specificity, that demethylation has a prominent role in tissue differentiation, and that DNA methylation has regulatory roles in alternative promoter selection and in non-promoter regions. Overall, our studies indicate changes in DNA methylation during development are a dynamic, widespread, and tissue-specific process involving both DNA methylation and demethylation.
Assuntos
Encéfalo/crescimento & desenvolvimento , Metilação de DNA , Regulação da Expressão Gênica no Desenvolvimento , Estudo de Associação Genômica Ampla , Coração/crescimento & desenvolvimento , Fígado/crescimento & desenvolvimento , Testículo/crescimento & desenvolvimento , Animais , Animais Recém-Nascidos , Encéfalo/embriologia , Encéfalo/metabolismo , Caderinas/genética , Células Cultivadas , Ilhas de CpG , Embrião de Mamíferos , Células-Tronco Embrionárias/metabolismo , Epigênese Genética , Coração/embriologia , Proteínas de Homeodomínio/genética , Fígado/embriologia , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Família Multigênica , Miocárdio/metabolismo , Especificidade de Órgãos , Regiões Promotoras Genéticas , Testículo/embriologia , Testículo/metabolismoRESUMO
Tissue specific differentially methylated regions (TDMRs) were identified and localized in the mouse genome using second generation virtual RLGS (vRLGS). Sequenom MassARRAY quantitative methylation analysis was used to confirm and determine the fine structure of tissue specific differences in DNA methylation. TDMRs have a broad distribution of locations to intragenic and intergenic regions including both CpG islands, and non-CpG islands regions. Somewhat surprising, there is a strong bias for TDMR location in non-promoter intragenic regions. Although some TDMRs are within or close to repeat sequences, overall they are less frequently associated with repetitive elements than expected from a random distribution. Many TDMRs are methylated at early developmental stages, but unmethylated later, suggesting active or passive demethylation, or expansions of populations of cells with unmethylated TDMRs. This is notable during postnatal testis differentiation where many testis specific TDMRs become progressively "demethylated". These results suggest that methylation changes during development are dynamic, involve demethylation and methylation, and may occur at late stages of embryonic development or even postnatally.
Assuntos
Metilação de DNA , Animais , Biologia Computacional/métodos , Ilhas de CpG , Embrião de Mamíferos , Epigênese Genética , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica no Desenvolvimento , Inativação Gênica , Genoma , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Análise de Sequência com Séries de Oligonucleotídeos , Software , Testículo/metabolismo , Distribuição TecidualRESUMO
Both genetic and epigenetic mechanisms contribute to meningioma development by altering gene expression and protein function. To determine the relative contribution of each mechanism to meningioma development, we used an integrative approach measuring copy number and DNA methylation changes genomewide. We found that genetic alterations affected 1.9%, 7.4%, and 13.3% of the 691 loci studied, whereas epigenetic mechanisms affected 5.4%, 9.9%, and 10.3% of these loci in grade I, II, and III meningiomas, respectively. Genetic and epigenetic mechanisms rarely involved the same locus in any given tumor. The predilection for epigenetic rather than genetic silencing was exemplified at the 5' CpG island of WNK2, a serine-threonine kinase gene on chromosome 9q22.31. WNK2 is known to negatively regulate epidermal growth factor receptor signaling via inhibition of MEK1 (mitogen-activated protein kinase kinase 1), and point mutations have been reported in WNK1, WNK2, WNK3, and WNK4. In meningiomas, WNK2 was aberrantly methylated in 83% and 71% of grade II and III meningiomas, respectively, but rarely in a total of 209 tumors from 13 other tumor types. Aberrant methylation of the CpG island was associated with decreased expression in primary tumors. WNK2 could be reactivated with a methylation inhibitor in IOMM-Lee, a meningioma cell line with a densely methylated WNK2 CpG island and lack of WNK2 expression. Expression of exogenous WNK2 inhibited colony formation, implicating it as a potential cell growth suppressor. These findings indicate that epigenetic mechanisms are common across meningiomas of all grades and that for specific genes such as WNK2, epigenetic alteration may be the dominant, grade-specific mechanism of gene inactivation.
Assuntos
Metilação de DNA , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Neoplasias Meníngeas/genética , Meningioma/genética , Proteínas Serina-Treonina Quinases/genética , Western Blotting , Ensaio de Unidades Formadoras de Colônias , Hibridização Genômica Comparativa , Ilhas de CpG , Citometria de Fluxo , Imunofluorescência , Genótipo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Luciferases/metabolismo , MAP Quinase Quinase 1/genética , MAP Quinase Quinase 1/metabolismo , Neoplasias Meníngeas/classificação , Neoplasias Meníngeas/patologia , Meningioma/classificação , Meningioma/patologia , Antígenos de Histocompatibilidade Menor , Invasividade Neoplásica , Estadiamento de Neoplasias , Análise de Sequência com Séries de Oligonucleotídeos , Regiões Promotoras Genéticas/genética , Proteínas Serina-Treonina Quinases/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transfecção , Células Tumorais Cultivadas , Proteína Quinase 1 Deficiente de Lisina WNKRESUMO
Tissue-specific differentially methylated regions (tDMRs) have been identified and implicated for their indispensable involvement in mammalian development and tissue differentiation. In this report, a quantitative DNA methylation analysis was performed for 13 human orthologous regions of recently confirmed mouse tDMRs by using Sequenom Mass Array, by which bisulfite-treated fragments are quantitatively detected using time of flight mass spectroscopy analysis. Eight regions were shown as tDMRs in various tissues from three independent individuals. Testis DNA samples from eight individuals were also analyzed for methylation. Interestingly, there is evidence that the DNA methylation level is divergent among individuals. DNA methylation levels of five testis-specific DMRs were significantly inversely correlated with the number of spermatocytes. However, a positive correlation was seen at tDMRs located near the TRIM38 and CASZ1 genes. Our results indicate that tDMRs are conserved between mouse and human and may have an important role in regulating tissue function, differentiation, and aging.
Assuntos
Metilação de DNA , Envelhecimento/genética , Animais , Sequência de Bases , Proteínas de Transporte , Diferenciação Celular/genética , Sequência Conservada , Proteínas de Ligação a DNA/genética , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas/genética , Testículo/metabolismo , Distribuição Tecidual , Fatores de Transcrição/genética , Proteínas com Motivo Tripartido , Ubiquitina-Proteína LigasesRESUMO
BACKGROUND: Restriction landmark genomic scanning (RLGS) is one of the most successfully applied methods for the identification of aberrant CpG island hypermethylation in cancer, as well as the identification of tissue specific methylation of CpG islands. However, a limitation to the utility of this method has been the ability to assign specific genomic sequences to RLGS spots, a process commonly referred to as "RLGS spot cloning." RESULTS: We report the development of a virtual RLGS method (vRLGS) that allows for RLGS spot identification in any sequenced genome and with any enzyme combination. We report significant improvements in predicting DNA fragment migration patterns by incorporating sequence information into the migration models, and demonstrate a median Euclidian distance between actual and predicted spot migration of 0.18 centimeters for the most complex human RLGS pattern. We report the confirmed identification of 795 human and 530 mouse RLGS spots for the most commonly used enzyme combinations. We also developed a method to filter the virtual spots to reduce the number of extra spots seen on a virtual profile for both the mouse and human genomes. We demonstrate use of this filter to simplify spot cloning and to assist in the identification of spots exhibiting tissue-specific methylation. CONCLUSION: The new vRLGS system reported here is highly robust for the identification of novel RLGS spots. The migration models developed are not specific to the genome being studied or the enzyme combination being used, making this tool broadly applicable. The identification of hundreds of mouse and human RLGS spot loci confirms the strong bias of RLGS studies to focus on CpG islands and provides a valuable resource to rapidly study their methylation.
Assuntos
Ilhas de CpG/genética , Enzimas de Restrição do DNA/metabolismo , Genoma/genética , Genômica/métodos , Mapeamento por Restrição/métodos , Animais , Biologia Computacional , Metilação de DNA , Eletroforese em Gel Bidimensional , Genoma Humano/genética , Humanos , Mucosa Intestinal/metabolismo , Fígado/metabolismo , Camundongos , Especificidade de Órgãos/genéticaRESUMO
Understanding the role of 'epigenetic' changes such as DNA methylation and chromatin remodeling has now become critical in understanding many biological processes. In order to delineate the global methylation pattern in a given genomic DNA, computer software has been developed to create a virtual image of restriction landmark genomic scanning (Vi-RLGS). When using a methylation- sensitive enzyme such as NotI as the restriction landmark, the comparison between real and in silico RLGS profiles of the genome provides a methylation map of genomic NotI sites. A methylation map of the Arabidopsis genome was created that could be confirmed by a methylation-sensitive PCR assay. The method has also been applied to the mouse genome. Although a complete methylation map has not been completed, a region of methylation difference between two tissues has been tested and confirmed by bisulfite sequencing. Vi-RLGS in conjunction with real RLGS will make it possible to develop a more complete map of genomic sites that are methylated or demethylated as a consequence of normal or abnormal development.
Assuntos
Arabidopsis/genética , Genoma , Camundongos/genética , Mapeamento por Restrição/métodos , Software , Animais , Metilação de DNA , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Genoma de Planta , Genômica/métodos , Camundongos Endogâmicos C57BL , Especificidade de Órgãos , Interface Usuário-ComputadorRESUMO
The related members of the interleukin-6 (IL-6) family of cytokines, leukemia inhibitory factor (LIF), oncostatin M (OSM) and IL-6 are inflammatory mediators that control differentiated cell functions as well as proliferation. The cellular responsiveness to these cytokines is largely determined by the expression of the appropriate receptor proteins. The receptor expression profile for each cell type is established during differentiation and is often altered during oncogenic transformation. Since inhibition of histone deacetylases (HDAC) has the potential to re-activate epigenetically silenced genes, we asked whether inhibition of HDAC enhances the expression of IL-6 cytokine receptors and, thus, increase desirable cytokine responses. We demonstrate that treatment with FR901228 (FR), an HDAC inhibitor, increases the responsiveness to LIF in different cell types, including normal fibroblasts, epithelial cells, macrophages and splenocytes, as well as various tumor cell lines. Depending on the cell type, FR treatment also enhances the responsiveness to OSM and IL-6. These effects involve a transcriptional induction of the cytokine receptor subunits LIFRalpha, OSMRbeta, gp130, or the transcription factor STAT3. FR-specific induction of LIFRalpha occurs independently of de novo protein synthesis and cell proliferation and is mediated in part by the CBP/p300 coactivator. Chromatin immunoprecipitation experiments indicate that the expression of LIFRalpha and gp130 genes correlates with the level of acetylated histone 3 associated with the receptor promoter regions. The FR-stimulated expression of IL-6-type cytokine receptors in certain tumor cells also provided improved conditions for suppression of cell growth by taking advantage of the growth inhibitory effect of these cytokines.
Assuntos
Antibacterianos/farmacologia , Antibióticos Antineoplásicos/farmacologia , Depsipeptídeos , Inibidores Enzimáticos/farmacologia , Inibidores de Histona Desacetilases , Histonas/metabolismo , Interleucina-6/farmacologia , Peptídeos Cíclicos , Receptores de Interleucina-6/metabolismo , Acetilação/efeitos dos fármacos , Antígenos CD/genética , Antígenos CD/metabolismo , Sequência de Bases , Receptor gp130 de Citocina , Proteínas de Ligação a DNA/metabolismo , Sinergismo Farmacológico , Humanos , Subunidade alfa de Receptor de Fator Inibidor de Leucemia , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Receptores de Citocinas/genética , Receptores de Citocinas/metabolismo , Receptores de OSM-LIF , Fator de Transcrição STAT3 , Transdução de Sinais/efeitos dos fármacos , Transativadores/metabolismo , Células Tumorais CultivadasRESUMO
BACKGROUND: The comprehensive methylation analysis of tumor-specific differently methylated regions in malignant melanomas and brain tumors has led to the identification of non-promoter hypermethylation of zygote arrest 1 (ZAR1). To search the non-promoter ZAR1 hypermethylation in neuroblastomas, we analyzed the levels of the methylation and transcript expression of ZAR1. METHODS: The MassARRAY® EpiTYPER (Sequenom Inc., San Diego, CA, USA) system was optimized to determine the quantitative methylation levels of ZAR1 for 12 neuroblastoma cell lines, 23 neuroblastoma samples and four adrenal samples. ZAR1 expression levels were evaluated through a quantitative, real-time reverse transcription-polymerase chain reaction. The quantitative methylation levels of ZAR1 were subjected to correlation studies with the established markers of progressive disease and outcome. RESULTS: Strikingly, the hypermethylation of ZAR1 regions and ZAR1 expression levels was observed in the neuroblastoma cell lines and neuroblastoma samples, compared to the adrenal samples. Somatic changes in ZAR1 methylation and ZAR1 expression were found in all three neuroblastoma patients. In the ZAR1 regions, poor-outcome tumors that were MYCN-amplified and/or Stage 3 or 4 and/or the age at diagnosis was≥18months, and/or showed an unfavorable histology were frequently hypermethylated. CONCLUSION: Our results indicate that the hypermethylation of ZAR1 regions is extremely frequent in neuroblastomas and correlates with established markers of progressive disease and outcome.
Assuntos
Proteínas do Ovo/genética , Neuroblastoma/genética , Criança , Pré-Escolar , Metilação de DNA , Progressão da Doença , Feminino , Humanos , Lactente , Masculino , Estadiamento de Neoplasias , Neuroblastoma/patologia , Reação em Cadeia da Polimerase em Tempo Real , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Estatísticas não Paramétricas , Taxa de SobrevidaRESUMO
BACKGROUND: Epigenetic changes such as aberrant DNA methylation and histone modification have been shown to play an important role in the tumorigenesis of malignant melanoma. OBJECTIVE: To identify novel tumor-specific differentially methylated regions (DMRs) in human malignant melanoma. METHODS: The aberrant methylation at 14 candidate human genomic regions identified through a mouse model study with quantitative DNA methylation analysis using the Sequenom MassARRAY system was performed. RESULTS: The CpG island Exon 1 region of the Zygote arrest 1 (ZAR1) gene, which is responsible for oocyte-to-embryo transition, showed frequent aberrant methylation of 28 out of 30 (93%) melanoma surgical specimens, 16 of 17 (94%) melanoma cell lines, 0% of 4 normal human epidermal melanocyte (NHEM) cell lines, 0% of 10 melanocytic nevi and 100% of 51 various cancer cell lines. According to the real-time RT-PCR, the ZAR1 gene was overexpressed in part of the hypermethylated cell lines, while its low expression with bivalent histone methylation status was seen in unmethylated cell lines. CONCLUSION: Our findings suggest that the ZAR1 intra-genic differentially methylated region would be a useful tumor marker for malignant melanoma and may be other type of cancers. The involvement of ZAR1 in the carcinogenesis of melanoma, still remains unclear, although we have examined tumorigenic capacities by exogenous full-length ZAR1 over-expression and siRNA knock-down experiments.
Assuntos
Biomarcadores Tumorais/metabolismo , Metilação de DNA , Proteínas do Ovo/metabolismo , Melanoma/metabolismo , Neoplasias Cutâneas/metabolismo , 9,10-Dimetil-1,2-benzantraceno/efeitos adversos , Animais , Linhagem Celular , Linhagem Celular Tumoral , Diagnóstico Diferencial , Histonas/metabolismo , Humanos , Melanócitos/metabolismo , Melanócitos/patologia , Melanoma/diagnóstico , Melanoma/patologia , Camundongos , Camundongos Endogâmicos C57BL , Nevo/diagnóstico , Nevo/metabolismo , Nevo/patologia , Neoplasias Cutâneas/diagnóstico , Neoplasias Cutâneas/patologiaRESUMO
Alterations in DNA methylation have been implicated in mammalian development. Hence, the identification of tissue-specific differentially methylated regions (TDMs) is indispensable for understanding its role. Using restriction landmark genomic scanning of six mouse tissues, 150 putative TDMs were identified and 14 were further analyzed. The DNA sequences of the 14 mouse TDMs are analyzed in this study. Six of the human homologous regions show TDMs to both mouse and human and genes in five of these regions have conserved tissue-specific expression: preferential expression in testis. A TDM, DDX4, is further analyzed in nine testis tissues. An increase in methylation of the promoter region is significantly associated with a marked reduction of the gene expression and defects in spermatogenesis, suggesting that hypomethylation of the DDX4 promoter region regulates DDX4 gene expression in spermatogenic cells. Our results indicate that some genomic regions with tissue-specific methylation and expression are conserved between mouse and human and suggest that DNA methylation may have an important role in regulating differentiation and tissue-/cell-specific gene expression of some genes.
Assuntos
Metilação de DNA , Regulação da Expressão Gênica , Especificidade de Órgãos , Idoso , Idoso de 80 Anos ou mais , Animais , Sequência de Bases , Ilhas de CpG/genética , RNA Helicases DEAD-box/genética , Genoma Humano , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Espermatogênese , Testículo/metabolismoRESUMO
Restriction landmark genomic scanning (RLGS), a method for the two-dimensional display of end-labeled DNA restriction fragments, was utilized to identify genomic regions of CpG island methylation associated with human colon cancer. An average of 1.5% of the RLGS loci/spots are lost or significantly reduced in sporadic primary colon tumors relative to normal colon mucosa from the same patient. This may represent tumor specific methylation of about 400 CpG islands in sporadic colon cancer. A number of RLGS loci exhibiting frequent loss associated with colon cancer were cloned. DNA sequence analysis indicated that the RLGS loci identified genomic regions characteristic of CpG islands. A number of methods including bisulfite genomic sequencing as well as quantitative MassARRAY methylation analysis (www.sequenom.com) confirmed tumor specific methylation at several of these loci. DNA database searches indicated that candidate genes associated with these loci include transcription factors and genes involved in signal transduction (52%), and genes of unknown function (37%). Expression analysis using quantitative real time RT-PCR indicates that methylation of some CpG islands located in non-promoter regions were associated with upregulation of gene expression in colorectal cancer. These results indicate that alterations in methylation status within CpG islands in colon tumors may have complex consequences on gene expression and tumorigenesis, sometimes resulting in up regulation or ectopic gene expression that may involve novel regulatory mechanisms.
Assuntos
Regiões 3' não Traduzidas/genética , Neoplasias do Colo/genética , Ilhas de CpG/genética , Metilação de DNA , Regulação Neoplásica da Expressão Gênica/genética , Genes Neoplásicos/genética , Genoma Humano/genética , Neoplasias do Colo/metabolismo , Perfilação da Expressão Gênica , Humanos , Mucosa Intestinal/metabolismo , Locos de Características Quantitativas/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Regulação para Cima/genéticaRESUMO
Early studies proposed that DNA methylation could have a role in regulating gene expression during development [Riggs, A.D. (1975) Cytogenet. Cell Genet. 14, 9-25]. However, some studies of DNA methylation in known tissue-specific genes during development do not support a major role for DNA methylation. In the results presented here, tissue-specific differentially methylated regions (TDMs) were first identified, and then expression of genes associated with these regions correlated with methylation status. Restriction landmark genomic scanning (RLGS) was used in conjunction with virtual RLGS to identify 150 TDMs [Matsuyama, T., Kimura, M.T., Koike, K., Abe, T., Nakao, T., Asami, T., Ebisuzaki, T., Held, W.A., Yoshida, S. & Nagase, H. (2003) Nucleic Acids Res. 31, 4490-4496]. Analysis of 14 TDMs by methylation-specific PCR and by bisulfite genomic sequencing confirms that the regions identified by RLGS are differentially methylated in a tissue-specific manner. The results indicate that 5% or more of the CpG islands are TDMs, disputing the general notion that all CpG islands are unmethylated. Some of the TDMs are within 5' promoter CpG islands of genes, which exhibit a tissue-specific expression pattern that is consistent with methylation status and a role in tissue differentiation.
Assuntos
Metilação de DNA , Expressão Gênica , Animais , Ilhas de CpG , Genoma , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Regiões Promotoras Genéticas , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The related members of the interleukin 6 (IL-6) family of cytokines, IL-6, leukemia inhibitory factor (LIF), and oncostatin M, act as major inflammatory mediators and induce the hepatic acute phase reaction. Normal parenchymal liver cells express the receptors for these cytokines, and these receptors activate, to a comparable level, the intracellular signaling through signal transducer and activator of transcription (STAT) proteins and extracellular-regulated kinase (ERK). In contrast, hepatoma cell lines show attenuated responsiveness to some of these cytokines that is correlated with lower expression of the corresponding ligand-binding receptor subunits. This study tests the hypothesis that the reduced expression of LIF receptor (LIFR) observed in hepatoma cells is mediated by altered DNA methylation. H-35 rat hepatoma cells that have a greatly reduced LIF responsiveness were treated with 5-aza-2'-deoxycytidine, an inhibitor of DNA methyltransferase. Surviving and proliferating cells showed reestablished expression of LIFR protein and function. Restriction landmark genomic scanning (RLGS) demonstrated genome-wide drug-induced alterations in DNA methylation status, with striking similarities in the demethylation pattern among independently derived clonal lines. Upon extended growth in the absence of 5-aza-2'-deoxycytidine, the cells exhibit partial reversion to pretreatment patterns. Demethylation and remethylation of the CpG island within the LIFR promoter that is active in normal liver cells correlate with increased and decreased usage of this promoter in H-35 cells. In conclusion, these results indicate that transformed liver cells frequently undergo epigenetic alterations that suppress LIFR gene expression and modify the responsiveness to this IL-6 type cytokine.
Assuntos
Azacitidina/análogos & derivados , Carcinoma Hepatocelular/metabolismo , Metilação de DNA , Interleucina-6/farmacologia , Neoplasias Hepáticas/metabolismo , Receptores de Citocinas/genética , Animais , Azacitidina/farmacologia , Proteínas de Ligação a DNA/metabolismo , Decitabina , Fator Inibidor de Leucemia , Subunidade alfa de Receptor de Fator Inibidor de Leucemia , Regiões Promotoras Genéticas , RNA Mensageiro/análise , Ratos , Receptores de Citocinas/análise , Receptores de OSM-LIF , Fator de Transcrição STAT3 , Transdução de Sinais , Transativadores/metabolismoRESUMO
We report the construction of the mouse full-length cDNA encyclopedia,the most extensive view of a complex transcriptome,on the basis of preparing and sequencing 246 libraries. Before cloning,cDNAs were enriched in full-length by Cap-Trapper,and in most cases,aggressively subtracted/normalized. We have produced 1,442,236 successful 3'-end sequences clustered into 171,144 groups, from which 60,770 clones were fully sequenced cDNAs annotated in the FANTOM-2 annotation. We have also produced 547,149 5' end reads,which clustered into 124,258 groups. Altogether, these cDNAs were further grouped in 70,000 transcriptional units (TU),which represent the best coverage of a transcriptome so far. By monitoring the extent of normalization/subtraction, we define the tentative equivalent coverage (TEC),which was estimated to be equivalent to >12,000,000 ESTs derived from standard libraries. High coverage explains discrepancies between the very large numbers of clusters (and TUs) of this project,which also include non-protein-coding RNAs,and the lower gene number estimation of genome annotations. Altogether,5'-end clusters identify regions that are potential promoters for 8637 known genes and 5'-end clusters suggest the presence of almost 63,000 transcriptional starting points. An estimate of the frequency of polyadenylation signals suggests that at least half of the singletons in the EST set represent real mRNAs. Clones accounting for about half of the predicted TUs await further sequencing. The continued high-discovery rate suggests that the task of transcriptome discovery is not yet complete.