Distinct DNA methylation profiles between adenocarcinoma and squamous cell carcinoma of human uterine cervix
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE15113 | GEO | 2009/03/18
SECONDARY ACCESSION(S): PRJNA114863
REPOSITORIES: GEO
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Project description:A microarray-based strategy using digestion with the methylation-sensitive restriction enzyme HpaII, ligation, and PCR, was performed to assess the DNA methylation status of promoter regions in Six human cervical adenocarcinomas (ACA) and four squamous cell carcinomas (SCC). Two array platforms were used; an array of 11,994 ~1.5kb PCR products from 10,445 promoter regions, and an array of 355,264 oligonucleotides for 18,212 HpaII fragments in 12,617 promoter regions. Loci near 21 genes showed significant differences between ACA and SCC. Real time PCR-based validation was performed on 13 loci using other nearby candidate methylation targets in the same promoter. Methylation patterns of eleven of these 13 linked loci concurred with the microarray results. Four loci were further studied using tissues from additional patients. Hyper-methylation of loci in PAK6 and NOGOR most strongly correlated with adenocarcinoma. In this project, we investigated the promoter methylation status in human cervical adenocarcinoma (ACA) and squamous cell carcinoma (SCC) to identify genes with tumor type-specific promoter DNA methylation patterns. Six ACA and four SCC tissue specimens from bulky tumor masses (clinical stage Ib2- IIb) were selected to minimize contamination from normal tissues. Another set of hybridizations was performed using an in situ synthesized high-resolution oligonucleotide microarray (NimbleGen Inc), to increase the reliability of the data. This chip was designed to cover the same promoters as the in-house microarray. Four pools of samples were used to reduce the number of arrays used. These pools consisted of two sets of three ACA samples (total of six ACA samples) and two sets of two SCC samples (total of four SCC samples).
2010-06-21 | E-GEOD-15113 | biostudies-arrayexpress
Project description:Osteosarcoma is the most common primary malignant bone tumor in children. Validated markers for disease prognosis available at diagnosis are lacking. No genome-wide DNA methylation studies linked to clinical outcomes have been reported in osteosarcoma. To address this, we tested the methylome at over 1.1 million loci in 15 osteosarcoma biopsy samples obtained prior to the initiation of therapy and correlated these molecular data with disease outcomes. At the tested loci, samples obtained from patients who experienced disease relapse were generally more methylated than those from patients who did not have recurrence. In samples from patients who went on to have recurrent disease, increased DNA methylation was found at gene bodies, intergenic regions and empirically-annotated candidate enhancers, whereas candidate gene promoters were unusual for a more balanced distribution of increased and decreased DNA methylation. A locus at the TLR4 gene demonstrates one of strongest associations between DNA methylation and five year event-free survival, with empirical annotation of this locus showing promoter characteristics. Our data indicate that DNA methylation information has potential to be predictive of outcome in pediatric osteosarcoma, and that both promoters and non-promoter loci are potentially informative in DNA methylation studies. 15 samples. HpaII libraries were compared to at least 3 MspI libraries from the same sample
2015-01-18 | E-GEOD-59200 | biostudies-arrayexpress
Project description:Estrogen, specifically estradiol, has been shown to mediate DNA methylation changes at mutiple locations downstream of estrogen response elements (EREs). We identified differentially methylated regions associated with long-term estradiol exposure in the hippocampus of C57BL/6J mice. Hippocampus was collected from ovariectomized (OVX) C57BL/6J mice treated with estradiol (n=5) and vehicle controls (n=5). Genomic DNA was extracted and treated with methylation sensitive restriction enzymes HpaII and HinP1I to enrich for the unmethylated fraction. This fraction was interrogated using the Affymetrix GeneChip® Mouse Tiling Promoter 1.0R Array
2013-05-24 | E-GEOD-43460 | biostudies-arrayexpress
Project description:To investigate podocyte gene expression and promoter methylation affected by KLF4, we examine about whole genome expression profiles using Agilent microarray and its correlation with promoter methylation profiles using Illumina DNA methylation array (GSE41689) as shown in supplementary data. Genome wide expression profiling of control (vector-transfected n=1) and KLF4-overexpressing (n=1) human podocytes (parental cell line: Saleem et al. JASN 13:630, 2002) and DNA methylation profile of promoter regions were shown.
2014-11-01 | E-GEOD-54395 | biostudies-arrayexpress
Project description:Background: Mechanisms underlying the development of virus-induced asthma exacerbations remain unclear. Objective: To investigate if epigenetic mechanisms could be involved in virus-induced asthma exacerbations, we undertook DNA methylation profiling in asthmatic and healthy nasal epithelial cells (NECs) during Human Rhinovirus (HRV) infection in vitro. Methods: Global and loci-specific methylation profiles were determined via Alu element and Infinium Human Methylation 450K microarray respectively. Principal components analysis identified the genomic loci influenced the most by disease-status and infection. Real-time PCR and pyrosequencing was used to confirm gene expression and DNA methylation respectively. Results: Global methylation was increased in Asthmatics during infection. Disease status and virus infection influenced the methylation of 389 loci. Healthy and asthmatic NECs were characterized predominately by methylation profiles and patterns in loci that were not influenced by virus infection. However, both groups also exhibited distinct DNA methylation profiles in response to infection. Despite these differences, we found that the methylation of small nucleolar RNA, H/ACA box 12 (SNORA12) was common during infection. Further analysis indicated a relationship existed between SNORA12 DNA methylation and gene expression in response to infection. Conclusions: Findings from our study indicate that in addition to the well described phenotypic and genomic differences, the airway epithelium of asthmatics is characterized by a distinct methylome and that epigenetic mechanism may contribute to the development of virus-induced asthma exacerbations. Bisulphite converted DNA from matched mock and human rhinovirus-16 infected nasal epithelial cells from Healthy (n=3) and Asthmatics (n=6) adults were hybridized to the Illumina DNA methylation 450K Bead Array.
2014-07-28 | E-GEOD-52074 | biostudies-arrayexpress
Project description:CG Methylation of Col, Van and reciprocal hybrids using HpaII and MspI digestion followed by bioprime random labeling, and hybridization to AtTILE1 forward array. Study on constitutive and ploymorphic CG methylation between arabidopsis thaliana accessions Col-0 and Van-0. Study on the inheritance of CG methylation in reciprocal hybrids. Keywords: genomic hybridization
2008-04-11 | GSE8890 | GEO
Project description:This data was generated by ENCODE. If you have questions about the data, contact the submitting laboratory directly (Florencia Pauli mailto:fpauli@hudsonalpha.org). If you have questions about the Genome Browser track associated with this data, contact ENCODE (mailto:genome@soe.ucsc.edu). This track shows average methylation status in CpG islands. In general, methylation of CpG sites within a promoter causes silencing of the gene associated with that promoter For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf CpG regions were assayed via Methyl-seq, a method developed in the Myers laboratory to measure the methylation status at CpGs throughout the genome. It combines DNA digestion by a methyl-sensitive enzyme HpaII and its methyl-insensitive isoschizomer MspI with the Illumina DNA sequencing platform. The method was first applied in a collaboration with the laboratory of Dr. Julie Baker at Stanford University to study methylation and gene expression changes that occur in human embryonic stem cells before and after differentiation to definitive endoderm. A paper describing the results as well as the method has been submitted for publication [1]. This study profiled genomic DNA and mRNA samples derived from two human embryonic stem cell lines: H9 and BG02. These cells were differentiated into definitive endoderm, embryoid bodies, embryoid body-derived cells, and AFP+ (alpha-fetoprotein positive) hepatocytes. These in vitro samples were profiled with Methyl-seq and compared them with normal tissue samples from 11-week and 24-week fetal liver and adult liver. Methyl-seq assays more than 250,000 methyl-sensitive restriction enzyme cleavage sites, representing more than 90,000 genomic regions. These regions include 35,528 annotated CpG islands, while the remaining 55,084 non-CpG island regions are distributed across the genome in promoters, genes, and intergenic regions. Sequence tags present in MspI libraries but not in HpaII libraries are derived from methylated regions. Conversely, sequence tags that occur in HpaII libraries come from at least partially unmethylated regions. In vitro differentiation: Definitive endoderm precursor cells were generated from H9 hES cells by treating them with activin A. Embryoid bodies (EBs) were generated by growing undifferentitated H9 and BG02 hESCs in suspension. EB-derived cells were obtained by plating clumps of the cells from the EBs. AFP+ fetal hepatocytes were derived from EBs by plating EB cells with FgF, followed by fluorescence activated cell sorting (FACS) to isolate cells expressing the green fluorescent protein (GFP) reporter gene driven from the AFP promoter. Isolation of genomic DNA: Genomic DNA is isolated from biological replicates of each cell line by using the QIAGEN DNeasy Blood & Tissue Kit according to the instructions provided by the manufacturer. DNA concentrations and a level of quality of each preparation is determined by UV absorbance. HpaII and MspI digestions: Cleavage of DNA by restriction endonuclease HpaII is prevented by the presence of a 5-methyl group at the internal C residue of its recognition sequence CCGG. MspI, an isoschizomer of HpaII, cleaves DNA irrespective of the presence of a methyl group at this position. For the MspI library, 5 µg genomic DNA was digested in a 100 µl reaction with 1X NEB Buffer2 and 20 units MspI restriction enzyme and incubated for 18 hr at 37°C. For the HpaII library, 5 µg genomic DNA was digested in a 100 µl reaction with 1X NEB Buffer1 and 20 units HpaII restriction enzyme and incubated for 18 hr at 37°C. Note that in subsequent versions of the Methyl-seq protocol, which will be described later, much lower amounts of genomic DNA were used (1 µg and potentially lower). DNA library construction and sequencing: High-throughput sequencing libraries were generated from DNA fragments of the HpaII or MspI digested genomic DNA according to the protocol posted at the website: http://myers.hudsonalpha.org/content/protocols.html. This approach was recently modified by removing the first PCR amplification step, just prior to the gel electrophoresis size-selection step, which was found to reduce a fragment-size bias in the sequencing libraries. These libraries were sequenced with an Illumina Genome Analyzer (GA2) according to the manufacturer's recommendations. Data analysis: For this analyis, reads that align to human genome sequence version hg19 and contain the 5'-CGG-3' HpaII-cut signature on their 5' end were used. These aligned sequence reads were mapped to CCGG sites predicted in silico on hg19. Sites with four or more Msp1 tags occurring in either the forward or reverse direction were retained for analysis. These "assayable" sites were then grouped with neighboring sites that are within 35-75 bp of each other. Thus, a "region" can be comprised of between 2 and 18 digestion sites that are each within 35-75 bp of another site. Methylated and non-methylated calls were made by using HpaII tag data from all assayable cut sites. For each site across each region, the larger of either the forward read count or reverse read count was used. Regions that have an average of 0 or 1 read per cut site are called methylated, and regions with more than one sequence read per site are called unmethylated.
2012-10-03 | E-GEOD-41304 | biostudies-arrayexpress
Project description:Epidemiological studies indicate that adverse intrauterine and postnatal environment has a long-lasting role in chronic kidney disease (CKD) development. Epigenetic information can represent a plausible carrier for mediating this "programming" effect. Here we demonstrate that genome-wide cytosine methylation patterns of healthy and CKD tubule samples obtained from patients show significant differences. We rarely observed differentially methylated regions (DMR) on promoters. Histone modification-based kidney specific genome-wide gene regulatory region annotation maps (promoters, enhancers, transcribed and repressed regions) were generated. DMRs mostly overlapped with putative enhancer regions and were enriched in consensus binding sequences for important renal transcription factors, indicating their importance in gene expression regulation. A core set of genes, including transforming growth factors and collagens, showed cytosine methylation changes correlating with downstream transcript levels. Our report raises the possibility that epigenetic dysregulation plays a role in CKD development via influencing core profibrotic pathways. HG18_HELP array We used custom-commercial array to detail the differences of methylation regions of human tubule epithelial cells between chronic kidney disease and normal. We sought to decrease the cell type heterogeneity of kidney tissues to increase the resolution of methylation profiles. To that end, microdissected human kidney tissue from both chronic kidney disease patient and normal are used for the HELP-assay (HpaII tiny fragment Enrichment by Ligation-mediated PCR) and hybridization on Roche NimbleGen microarrays.
2013-08-09 | E-GEOD-49557 | biostudies-arrayexpress
Project description:Genome wide DNA methylation profiling of normal and squamous carcinoma of the cervix. The Illumina Infinium 27k Human DNA methylation Beadchip v1.2 was used to obtain DNA methylation profiles across approximately 27,000 CpGs in frozen cervical samples. Samples included 4 normal ectocervices (Ecto 1 to 4) and 5 squamous cell carcinoma of the cervix (SCC 1 to 5). In order to identify epigenetic biomarkers for early cervix cancer diagnosis, we performed a methylation screening using Illumina Infinium 27k Human DNA methylation Beadchip v1.2 of stem cell marker promoters during cervical carcinogenesis and demonstrated a strong hypermethylation of Undifferentiated cell Transcription Factor 1 (UTF1) promoter in squamous cell carcinoma (SCC) in comparison with normal ectocervix. Direct bisulfite pyrosequencing of DNA isolated from liquid-based cytological samples showed that UTF1 promoter methylation increases with lesion severity and is associated with higher expression. By RT-PCR, Western Blot and immunofluorescence, we demonstrated that UTF1 mRNA and protein are expressed in epithelial cancer cell lines, even in the absence of its two main described regulators Oct4A and Sox2. Methyl-Specific PCR experiments revealed that the inhibition of DNA methyltransferase by 5-aza-2’-deoxycytidine is associated with decreased UTF1 gene methylation and expression. These findings provide evidence that UTF1 promoter methylation profile might be a useful biomarker for cervix cancer diagnosis and raise the questions of its role during epithelial carcinogenesis and of the mechanisms involved in the regulation of its expression. Bisulfite converted DNA from the 9 samples were hybridised to the Illumina Infinium 27k Human Methylation Beadchip v1.2
2012-03-21 | E-GEOD-36637 | biostudies-arrayexpress
Project description:We have developed a method for mapping unmethylated sites in human genome based on the resistant of TspR1 digested ends to exoIII nuclease degradation. Digestion with TspR1 and methylation-sensitive restriction endonuclease, HpaII, followed by exoIII and single strand DNA nuclease allows the removal of DNA fragments containing unmethylated HpaII sites. We then use array CGH to map the sequences depleted by this procedures in human genomes derived from five human tissues, a primary breast tumor and two breast tumor cell lines. Analysis of methylation patterns of the normal tissue genomes indicates that the hypomethylated sites are enriched in the 5’ end of widely expressed genes including promoter, first exon and first intron. In contrast, genomes of the MCF-7 and MDA-MB-231 cell lines show extensive hypomethylation in the intragenic and intergenic regions whereas primary tumor exhibits intermediate pattern between normal tissue and cell lines. A striking characteristic of tumor genomes is the presence of megabase-sized hypomethylated zones. These hypomethylated zones are associated with large genes, fragile sites, evolutionary breakpoints, chromosomal rearrangement breakpoints, tumor supperessor genes, and with regions containing tissue-specific gene clusters or with gene poor region containing novel tissue-specific genes. Bisulfite sequencing analysis shows a novel mosaic methylation pattern with alternative methylated and unmethylated zones was found in human histone gene clusters in chromosome 6. Correlation with microarray analysis show that genes with hypomethylated sequence 2kb up- or down-stream of transcription start site are highly expressed whereas genes with extensive intragenic and 3’ UTR hypomethylation are silenced. The method described herein can be used for large scale screening of changes in methylation pattern in the genome of interest. Keywords: Genome-Wide Mapping of Hypomethylated Sites in Human Genomes
2007-09-29 | GSE9015 | GEO