DNA demethylation by 5-aza-2'-deoxycytidine is imprinted targeted to euchromatin and has limited transcriptional consequences
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
SUBMITTER: Maria-Paz Ramos
PROVIDER: E-GEOD-62590 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
Similar Datasets
Project description:DNA methylation can be abnormally regulated in human disease and associated with effects on gene transcription that appear to be causally related to pathogenesis. The potential to use pharmacological agents that reverse this dysregulation is therefore an attractive possibility. To test how 5-aza-2M-bM-^@M-^Y-deoxycytidine (5-aza-CdR) influences the genome therapeutically, we exposed non-malignant cells in culture to the agent and used genome-wide assays to assess the cellular response. We found that cells allowed to recover from 5-aza-CdR treatment only partially recover DNA methylation levels, retaining an epigenetic M-bM-^@M-^\imprintM-bM-^@M-^] of drug exposure. We show very limited transcriptional responses to demethylation of not only protein-coding genes but also loci encoding non-coding RNAs, with a limited proportion of the induced genes acquiring new promoter activation within gene bodies. The data revealed an uncoupling of DNA methylation effects at promoters, with demethylation mostly unaccompanied by transcriptional changes. The limited panel of genes induced by 5-aza-CdR resembles those activated in other human cell types exposed to the drug, and represents loci targeted for Polycomb-mediated silencing in stem cells, suggesting a model for the therapeutic effects of the drug. Our results do not support the hypothesis of DNA methylation having a predominant role to regulate transcriptional noise in the genome, and indicate that DNA methylation acts only as part of a larger complex system of transcriptional regulation. The targeting of 5-aza-CdR effects with its clastogenic consequences to euchromatin raises concerns that the use of 5-aza-CdR has innate tumorigenic consequences, requiring its cautious use in diseases involving epigenetic dysregulation. Cytosine methylation profile of 4 different samples of HEK 293T treated with 5-aza-CdR
2015-02-13 | E-GEOD-62592 | biostudies-arrayexpress
Project description:Obesity-associated asthma is recognized as a distinct entity with non-atopic T-helper 1 polarized systemic inflammation. DNA methylation is linked with T helper cell maturation and is associated with inflammatory patterns in asthma and obesity. However, it is unknown whether pathologic dysregulation of DNA methylation patterns occurs in obesity-associated asthma. Using HELP-tagging, we studied epigenome wide DNA methylation in peripheral blood mononuclear cells in 8 urban minority obese asthmatic pre-adolescent children and compared it to methylation in groups of 8 children with asthma alone, obesity alone and healthy controls. Ingenuity Pathway Analysis was used to identify biological pathways that were differentially targeted by methylation dysregulation. We found that obese asthmatics had distinct epigenome wide methylation patterns associated with decreased promoter methylation of a subset of genes, including RANTES, IL-12R and TBX21 and increased promoter methylation of CD23, a low affinity receptor for IgE and of TGFM-NM-2, inhibitor of Th cell activation. T cell signaling and macrophage activation were the two primary pathways that were selectively hypomethylated in obese asthmatics. These methylation patterns suggest that methylation is associated with non-atopic inflammation observed in obese asthmatic children compared to children with asthma alone and obesity alone. Our findings suggest a role of DNA methylation in the observed inflammatory patterns in pediatric obesity-associated asthma in minorities. 32 HpaII test
2014-04-14 | E-GEOD-43705 | biostudies-arrayexpress
Project description:Background: 5-hydroxymethylcytosine (5-hmC) is a recently discovered epigenetic modification that is altered in cancers. Genome wide assays for 5-hmC determination are needed as many of the techniques commonly used to assay 5-methylcytosine (5-mC), including conventional methyl-sensitive restriction digest and bisulfite sequencing, are incapable of distinguishing between 5-mC and 5-hmC. Results: Glycosylation of 5-hmC residues by beta-Glucosyl Transferase (beta-GT) can make CCGG residues insensitive to digestion by MspI. We used this premise to modify the HELP-tagging assay to identify both 5-mC and 5-hmC loci in the genome. Comparison of sequencing libraries after HpaII, MspI and MspI+ beta-GT conversion resulted in locus specific 5-mC and 5-hmC determination. A custom bioinformatics pipeline was created to identify 5-hmC sites that were validated at global level by LS-MS and the locus specific level by qRT-PCR of 5-hmC pulldown DNA. Hydroxymethylation at both promoter and intragenic locations correlated positively with gene expression. Analysis of pancreatic cancer samples revealed striking redistribution of 5-hmC sites in cancer cells and demonstrated enrichment of this modification at many oncogenic promoters such as GATA6. Conclusions: The HELP-GT assay allows a high resolution, simultaneous determination of 5-hmC and 5-mC loci from small amounts of DNA with the utilisation of modest sequencing resources. Redistribution of 5-hmC seen in cancer highlights the importance of examining this modification in conjugation with conventional methylome analysis. We did methylation and hydroxymethylation tests for one control and two pancreatic cancer cases
2013-07-30 | E-GEOD-42723 | biostudies-arrayexpress
Project description:Background: To perform epigenome-wide association studies in human disease, assays need to be comprehensive and quantitative while remaining cost-effective. We explored how the strengths of prior tag-based cytosine methylation assays based on massively-parallel sequencing can be maximised analytically. Results: We find that the use of the EcoP15I restriction enzyme to generate long tags and the normalisation of methylation-sensitive by methylation-insensitive restriction enzyme representations greatly improve assay performance. When exploring sources of bias, we find that the length of the restriction fragment has moderate effects on EcoP15I digestion, while base composition exerts minimal effects. We detail the analytical workflow that maximises the quantitative capabilities of this modified assay. Also revealed are polymorphic sequences in the genome that could confound microarray, bisulphite sequencing or mass spectrometry-based assays, and a position effect causing hypomethylation of transposable elements near gene promoters. Conclusions: The new combined assay, referred to as HELP-tagging, interrogates over 1.8 million loci in the human genome quantitatively with a single lane of Illumina sequencing. When the goal is to study not only CG-dense sequence but also the CG-depleted majority of the genome, this assay system should be suitable. Three MspI reference, one HpaII test
2010-04-12 | E-GEOD-19937 | biostudies-arrayexpress
Project description:DNA methylation can be abnormally regulated in human disease and associated with effects on gene transcription that appear to be causally related to pathogenesis. The potential to use pharmacological agents that reverse this dysregulation is therefore an attractive possibility. To test how 5-aza-2M-bM-^@M-^Y-deoxycytidine (5-aza-CdR) influences the genome therapeutically, we exposed non-malignant cells in culture to the agent and used genome-wide assays to assess the cellular response. We found that cells allowed to recover from 5-aza-CdR treatment only partially recover DNA methylation levels, retaining an epigenetic M-bM-^@M-^\imprintM-bM-^@M-^] of drug exposure. We show very limited transcriptional responses to demethylation of not only protein-coding genes but also loci encoding non-coding RNAs, with a limited proportion of the induced genes acquiring new promoter activation within gene bodies. The data revealed an uncoupling of DNA methylation effects at promoters, with demethylation mostly unaccompanied by transcriptional changes. The limited panel of genes induced by 5-aza-CdR resembles those activated in other human cell types exposed to the drug, and represents loci targeted for Polycomb-mediated silencing in stem cells, suggesting a model for the therapeutic effects of the drug. Our results do not support the hypothesis of DNA methylation having a predominant role to regulate transcriptional noise in the genome, and indicate that DNA methylation acts only as part of a larger complex system of transcriptional regulation. The targeting of 5-aza-CdR effects with its clastogenic consequences to euchromatin raises concerns that the use of 5-aza-CdR has innate tumorigenic consequences, requiring its cautious use in diseases involving epigenetic dysregulation. Examination of RNAPII Ser5(P) localization by ChIP-seq in HEK 293T cell after treatment with 5-aza-CdR.
2015-02-13 | E-GEOD-62695 | biostudies-arrayexpress
Project description:DNA methylation can be abnormally regulated in human disease and associated with effects on gene transcription that appear to be causally related to pathogenesis. The potential to use pharmacological agents that reverse this dysregulation is therefore an attractive possibility. To test how 5-aza-2M-bM-^@M-^Y-deoxycytidine (5-aza-CdR) influences the genome therapeutically, we exposed non-malignant cells in culture to the agent and used genome-wide assays to assess the cellular response. We found that cells allowed to recover from 5-aza-CdR treatment only partially recover DNA methylation levels, retaining an epigenetic M-bM-^@M-^\imprintM-bM-^@M-^] of drug exposure. We show very limited transcriptional responses to demethylation of not only protein-coding genes but also loci encoding non-coding RNAs, with a limited proportion of the induced genes acquiring new promoter activation within gene bodies. The data revealed an uncoupling of DNA methylation effects at promoters, with demethylation mostly unaccompanied by transcriptional changes. The limited panel of genes induced by 5-aza-CdR resembles those activated in other human cell types exposed to the drug, and represents loci targeted for Polycomb-mediated silencing in stem cells, suggesting a model for the therapeutic effects of the drug. Our results do not support the hypothesis of DNA methylation having a predominant role to regulate transcriptional noise in the genome, and indicate that DNA methylation acts only as part of a larger complex system of transcriptional regulation. The targeting of 5-aza-CdR effects with its clastogenic consequences to euchromatin raises concerns that the use of 5-aza-CdR has innate tumorigenic consequences, requiring its cautious use in diseases involving epigenetic dysregulation. mRNA profile of 5 different samples of HEK 293T cells treated with 5-aza-CdR
2015-02-13 | E-GEOD-62591 | biostudies-arrayexpress
Project description:Epigenetic pathways that regulate DNA methylation and chromatin modifications are frequently found to be dysregulated in human cancers. The TET methylcytosine dioxygenase 1 (TET1) enzyme is an important regulator of hydroxymethylcytosine (5hmC) in embryonic stem cells, neural progenitors,adult cells and reprogrammed cells. Decreased expression of TET proteins and loss of 5hmC has been reported in many tumors, suggesting a critical role for the maintenance of this epigenetic modification in normal cellular function. However, loss of TET1 function in the etiology of cancer has not been directly investigated. Here, we show that deletion of the Tet1 gene promotes the development of B cell lymphoma. Tet1 is required for maintaining normal levels of 5hmC, preventing aberrant DNA hypermethylation and for the regulation of transcriptional programs involved in B-cell lineage specification, chromosome maintenance, and DNA repair. Progenitor B cells in the absence of Tet1 accumulate DNA damage and whole-exome sequencing of Tet1-deficient tumors revealed a high correlation of mutations with those most frequently found in Non-Hodgkin B cell lymphoma (B-NHL) patients. In addition, we show that the TET1 gene is deleted, hypermethylated and transcriptionally silenced in B-NHL patients. These findings provide the first in vivo evidence of TET1 function as a tumor suppressor of hematopoietic malignancy. We did hydroxymethylation tests for two wild type mice and two Tet1 knockout mice.
2015-04-12 | E-GEOD-65895 | biostudies-arrayexpress
Project description:The C57BL/6J mouse model develops obesity and pre-diabetes when fed a high-fat diet. In this experiment, DNA methylation was assessed globally at specific CpG sites in liver tissue from mice receiving high-fat diet (45E% from fat) for 13 weeks (Control) or high-fat diet supplemented with 20% (w/w) of freeze-dried lingonberries (n=4). Our findings show that lingonberries prevent development of high-fat induced obesity, hepatic steatosis and low-grade inflammation, and the DNA was hypermethylated in mice receiving lingonberries compared to control. Genome wide hepatic DNA methylation comparison between mice fed high-fat diet with or without a lingonberry supplement (n=4/group).
2015-09-20 | E-GEOD-67277 | biostudies-arrayexpress
Project description:Multiple endocrine neoplasia type 1 (MEN1) syndrome is the result of mutations in the MEN1 gene and results in tumor formation via mechanisms that are not well understood. Using a novel genome-wide methylation analysis, we studied tissues from patients with MEN1-parathyroid tumors, tissues from Men1 knockout (KO) mouse models, and mouse Men1 null mouse embryonic fibroblast (MEF) cell lines. Tissues from KO mice were used to confirm and assess the findings from the MEN1 clinical samples and further explore the molecular mechanisms of global epigenetic changes following the inactivation of menin. We demonstrated that the inactivation of menin results in enhanced activity of DNA (cytosine-5)-methyltransferase 1 (DNMT1) by retinoblastoma-binding protein 5 (Rbbp5) activation in MEN1 tumor tissues. The increased activity of DNMT1 mediated global DNA hypermethylation, which in turn resulted in aberrant activation of the Wnt/β-catenin signaling pathway through inactivation of Sox regulatory genes. Our study provides important insights into the possible regulatory role of menin in DNA methylation and its impact on the pathogenesis of MEN1 tumor development. Global DNA methylation in tissues from patients with MEN1-parathyroid tumors. Thirty-eight human parathyroid specimens were used: 13 sporadic (non-MEN1) parathyroid adenomas, 12 MEN1-parathyroid tumors, 4 parathyroid carcinomas, and 9 normal parathyroids.
2015-12-22 | E-GEOD-64412 | biostudies-arrayexpress
Project description:Dengue viruses (DENV) are generally maintained in a cycle which requires horizontal transmission via their arthropod vector, Ae. Aegypti, to the vertebrate host. One important consequence of this process is the interference of these arboviruses with both invertebrate and vertebrate immune systems. While infection of vertebrates causes disease, the presence of DENV gives rise to life-long, persistent infection in mosquitoes. The results of a comparative transcriptome analysis between DENV-infected and uninfected salivary glands revealed activation of both the immune deficiency (IMD) and the Toll pathways, as well as involvement of the putative antibacterial cecropin-like peptide (AAEL000598), in controlling DENV infection in Ae. aegypti. The mature form of this peptide was found to be active against DENV and Chikungunya viruses, whereas its precursor also had a strong anti-Leishmania effect. This study is the first to establish a comparative transcriptome analysis of DENV-infected and uninfected salivary glands and demonstrates that certain DENV-induced peptides, that are part of the IMD pathway, possess broad-spectrum anti-pathogenic activity and may have therapeutic potential in human. Infectious blood meals were offered to 3-day-old, adult, female Ae. aegypti Liverpool mosquitoes using a silicone membrane feeder system (Alto et al., 2005). Human blood was combined with DENV-2 16681 to provide a blood meal titer of 5.106 plaque forming units (PFU)/ml. At different time-points after the blood meal, salivary glands were dissected in acid guanidium thiocyanate-phenol-chloroform (RNAble; Eurobio, France) or phosphate-buffered saline (PBS), and the samples were frozen at -70°C until use.
2011-03-21 | E-GEOD-21528 | biostudies-arrayexpress