Systematic evaluation of genome-wide methylated DNA enrichment using a CpG island array
Ontology highlight
ABSTRACT: Methylated DNA enrichment is a key step in a microarray based genome-wide methylation profiling study, and even for future high-throughput sequencing based methylome analysis. In order to evaluate the sensitivity and accuracy of methylated DNA enrichment, we investigated and optimized a number of important parameters to improve the performance of several enrichment assays, including differential methylation hybridization (DMH), microarray-based methylation assessment of single samples (MMASS), and methylated DNA immunoprecipitation (MeDIP). With advantages and disadvantages unique to each approach, we found that assays based on methylation-sensitive enzyme digestion and those based on immunoprecipitation detected different methylated DNA fragments, indicating that they are complementary in their relative ability to detect methylation differences. Our study provides the first comprehensive evaluation for widely used methodologies for methylated DNA enrichment, and could be helpful for developing a cost effective approach for DNA methylation profiling.
Project description:Cellular differentiation involves widespread epigenetic reprogramming, including modulation of DNA methylation patterns. Using Differential Methylation Hybridization (DMH) in combination with a custom DMH array containing more than 53,000 features covering more than 16,000 murine genes, we carried out a genome-wide screen for cell- and tissue-specific differentially methylated regions (tDMRs) in undifferentiated embryonic stem cells (ESCs), in in-vitro induced neural stem cells (NSCs) and 8 differentiated embryonic and adult tissues. Unsupervised clustering of the generated data showed distinct cell- and tissue-specific DNA methylation profiles, revealing 202 significant tDMRs (p<0.005) between ESCs and NSCs and a further 380 tDMRs (p<0.05) between NSCs/ESCs and embryonic brain tissue. We validated these tDMRs using direct bisulfite sequencing (DBS) and methylated DNA immunoprecipitation on chip (MeDIP-chip). Gene ontology (GO) analysis of the genes associated with these tDMRs showed significant (absolute Z score >1.96) enrichment for genes involved in neural differentiation, including e.g. Jag1 and Tcf4. Our results provide robust evidence for the relevance of DNA methylation in early neural development and identify novel marker candidates for neural cell differentiation.
Project description:To globally define methylation-M-bM-^@M-^YproneM-bM-^@M-^Y and -M-bM-^@M-^YprotectedM-bM-^@M-^Y CpG islands in cancer, we analyzed the methylation status of 23,000 CpG islands of the human genome in 19 colorectal carcinoma samples as well as normal colon using our previously described methyl-CpG immunoprecipitation (MCIp) technique (Gebhard et al. 2006; Schilling and Rehli 2007). This method enriches for highly CpG methylated DNA that can be directly applied to fluorescent labeling and oligonucleotide microarray hybridization without an additional amplification step. CpG-methylated genomic DNA was enriched using methyl-CpG immunoprecipitation (MCIp). On each microarray, the enriched material from colorectal carcinoma samples was compared to the enriched material from normal colon to identify aberrantly methylated regions.
Project description:To globally define methylation-’prone’ and -’protected’ CpG islands in cancer, we analyzed the methylation status of 23,000 CpG islands of the human genome in 19 colorectal carcinoma samples as well as normal colon using our previously described methyl-CpG immunoprecipitation (MCIp) technique (Gebhard et al. 2006; Schilling and Rehli 2007). This method enriches for highly CpG methylated DNA that can be directly applied to fluorescent labeling and oligonucleotide microarray hybridization without an additional amplification step.
Project description:Cisplatin and carboplatin are the primary first-line therapies for the treatment of ovarian cancer. However, resistance to these platinum-based drugs occurs in the large majority of initially responsive tumors, subsequently resulting in a poor long-term prognosis. To model the onset of drug resistance, and investigate the DNA methylation alterations associated with cisplatin resistance, we treated clonally derived, drug-sensitive A2780 epithelial ovarian cancer cells with increasing concentrations of cisplatin. After several cycles of drug selection, the isogenic drug-sensitive and -resistant pairs were subjected to global CGI methylation microarray analyses. We treated clonally derived, drug-sensitive A2780 epithelial ovarian cancer cells with increasing concentrations of cisplatin. After several cycles of drug selection, the isogenic drug-sensitive and -resistant pairs were subjected to global CGI methylation analyses by differential methylation hybridization (DMH) using a customed 44K promoter CGI microarray.
Project description:Microarray analysis of DNA methylation has a great promise in biomedical research. However, its accuracy in quantification of DNA methylation, especially at intermediate levels, is still unreliable due to the lack of an appropriate parameter that takes account of unique distribution nature of methylation levels and highly variable mean methylation levels depending upon individual samples. Here, for methylated DNA immunoprecipitation-microarray analysis, we developed a parameter, "Me value" as a parameter that linearly correlates with methylation levels in CpG islands (CGIs) in various samples with various mean methylation levels. The Me value had a linear correlation with the fraction of methylated DNA molecules obtained by bisulfite sequencing (R=0.86). Analysis of cells treated with 5-aza-2'-deoxycitidine showed that distribution of the Me values shifted toward lower methylation levels. For analysis of completely unmethylated or methylated CGIs, common threshold values stood valid with accuracy of 80 % for cell lines with various methylation levels, which was shown by methylation-specific PCR of more than one hundred loci. Combined with expression microarray analyses, it was shown that methylation of promoter CGIs was significantly associated with low gene expression while that of far downstream was not. It was also shown that various regions against TSSs are demethylated evenly by a demethylating agent, and that the degree of demethylation of any region was not associated with the degree of re-expression.
Project description:Following erasure in the early animal embryo, a new bimodal DNA methylation pattern is regenerated at the time of implantation in each individual through a wave of generalized de novo modification with CpG islands specifically left unmethylated. In this paper, we ask whether CpG islands are M-bM-^@M-^\protectedM-bM-^@M-^] from de novo methylation or, alternatively, acquire their unmodified state through M-bM-^@M-^\demethylation.M-bM-^@M-^] Using a genetic approach, we demonstrate that ES cells indeed have the ability to specifically demethylate CpG islands through pathways involving hydroxymethylation (Tet1), deamination (Aid), glycosylation (Mbd4) and excision repair (Gadd45a) genes. Surprisingly, this demethylation system is not actually necessary for generating the overall bimodal methylation pattern in ES cells. This general activity does, however, appear to be involved both in somatic cell reprogramming, as well as the normal methylation reprogramming that takes place in the early embryo. CpG-methylated genomic DNA was enriched using a methyl-DNA immunoprecipitation (mDIP) assay. DNA from the input and bound (enriched) DNA for each sample were labeled and hybridized on the array to define the methylation state of each region.
Project description:In order to identify methylation changes in prostate cancer, we performed a genome-wide analysis of DNA methylation using Agilent human CpG island arrays. We then chose specific genes to validate methylation both in the same cases as were hybridized to the array (using quantitative EpiTYPER analysis) and in an independent series of prostate cancer samples (using MethyLight quantitative methylation specific PCR). We specifically chose low grade (Gleason score 6 cases) and high grade (Gleason score 8 cases) to discover methylated genes/loci that may be involved in the progression to a higher grade of prostate cancer. We collected 20 specimens consisting of 10 Gleason 6 and 10 Gleason 8 prostate cancers, and compared these to a reference lymphocyte pool (6 age matched, healthy men) to determine cancer associated methylation changes as well as disease progression associated methylation changes. We performed the differential methylation hybridization procedure as described by Yan et al. (Methods, 2002) on each case to enrich for methylated DNA. Each specimen in the reference pool underwent the same enrichment with amplicons being pooled at the end of the procedure. Each prostate cancer case was subsequently co-hybridized to the microarray with the reference pool.
Project description:We assayed 114 human transcription factors (TFs) for DNA binding using methylation-sensitive selective microfluidics-based ligand enrichment followed by sequencing (meSMiLE-seq). Across 23 experiments, we identified DNA binding sites for 48 TFs. Among these, 13 showed aversion towards methylated DNA, while 11 exhibited either a preference for methylated DNA or an alternative binding site including the modification. The DNA binding for the remaining TFs was unaffected by DNA methylation under the conditions of the assay.
Project description:Protein methylation plays important roles in DNA damage signaling. To date, there is still a lack of global profiling of whole-cell methylation changes during the DNA damage response and repair. In this study, using HILIC affinity enrichment combined with MS analysis, we conducted a quantitative analysis of the methylated proteins in HEK293T cells in response to IR-induced DNA damage. In total, 235 distinct methylation sites responding to IR treatment were identified, and 38% of them were previously unknown. Multiple RNA-binding proteins were differentially methylated upon DNA damage stress. Furthermore, we identified 14 novel methylations in DNA damage response-related proteins. Moreover, we validated the function of PARP1 K23 methylation in repairing IR-induced DNA lesions.
Project description:Epigenetic changes largely contribute to the regulation of gene expression in cancer cells. DNA methylation is part of the epigenetic gene regulation complex which is relevant for the pathogenesis of cancer. We performed a genome-wide search for methylated CpG islands in tumors and corresponding non-malignant lung tissue samples of 101 stage I-III non-small cell lung cancer (NSCLC) patients by combining methylated DNA immunoprecipitation and microarray analysis using NimbleGenM-BM-4s 385K Human CpG Island plus Promoter arrays. By testing for differences in methylation between tumors and corresponding non-malignant lung tissues, we identified 298 tumor-specifically methylated genes. From many of these genes epigenetic regulation was unknown so far. Gene Ontology analysis revealed an over-representation of genes involved in regulation of gene expression and cell adhesion. Expression of 182 of 298 genes was found to be upregulated after 5-aza-2M-BM-4-deoxycytidine (Aza-dC) and/or trichostatin A (TSA) treatment of 3 NSCLC cell lines by Affymetrix microarray analysis. In addition, methylation of selected genes in primary NSCLCs and corresponding non-malignant lung tissue samples were analyzed by methylation-sensitive high resolution melting analysis (MS-HRM). Our results obtained by MS-HRM analysis confirmed our data obtained by MeDIP-chip analysis. Moreover, by comparing methylation results from MeDIP-chip analysis with clinico-pathological parameters of the patients we observed methylation of HOXA2 as potential parameter for shorter disease-free survival of NSCLC patients. In conclusion, using a genome-wide approach we identified a large number of tumor-specifically methylated genes in NSCLC patients. Our results stress the importance of DNA methylation for the pathogenesis of NSCLCs. Overall, samples of 3 untreated, with Aza-dC treated and with Aza-dC/TSA treated NSCLC cell lines were hybridized to Affymetrix HG-U133_plus_2.0 microarrays (18 in total).