Methylation arrays delineate the extent of 5mC and 5hmC in human brain and glioma samples.
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
SUBMITTER: Agustin F. Fernandez
PROVIDER: E-MTAB-6003 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:In this array series, we have characterized the extent and the location of 5hmC loss in colon cancer. We performed genome-wide methylation oxBS and BS analyses with the Illumina Infinium HumanMethylation450 BeadChip platform in 11 normal colon and 11 matched tumor samples plus 2 colon cancer cell lines, and we measured the extent of 5mC and 5hmC in normal versus tumoral conditions.
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Project description:To characterize the largely unknown functions of oxidised methylcytosines (oxi-mC; 5hmC/5fC/5caC) in DNA, several sequencing protocols have been recently developed. Quantitative analysis is complicated because DNA methylation modifications need to be deconvoluted from the data which is affected by several experimental parameters, including e.g. imperfect bisulphite conversion, oxidation efficiencies, various chemical labeling and protection steps, and sequencing errors. Here, we present a hierarchical generative model, Lux, for integrative analysis of any combination of BS-seq and âoxi-mCâ-seq (BS-seq/oxBS-seq/TAB-seq/fCAB-seq/CAB-seq/redBS-seq/MAB-seq) data. We show that Lux improves quantification and comparison of methylation levels over existing methods and that Lux can easily process any oxi-mC-seq data sets to quantify all cytosine modifications simultaneously together with their experimental parameters. Application of Lux to targeted data from Tet2 knockdown ESCs and T cells during development demonstrates DNA modification quantification at unprecedented detail, quantifies active demethylation pathway and reveals 5hmC localization in putative regulatory regions. Examine the distribution of C, 5mC, and 5hmC in ES and Tet2 knock-down cells within selected loci. Half of the samples are measured using the traditional bisulphite-seq protocol but the other half is measured using the oxidative bisulphite-seq protocol (oxBS-seq).
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Project description:To characterize the largely unknown functions of oxidised methylcytosines (oxi-mC; 5hmC/5fC/5caC) in DNA, several sequencing protocols have been recently developed. Quantitative analysis is complicated because DNA methylation modifications need to be deconvoluted from the data which is affected by several experimental parameters, including e.g. imperfect bisulphite conversion, oxidation efficiencies, various chemical labeling and protection steps, and sequencing errors. Here, we present a hierarchical generative model, Lux, for integrative analysis of any combination of BS-seq and âoxi-mCâ-seq (BS-seq/oxBS-seq/TAB-seq/fCAB-seq/CAB-seq/redBS-seq/MAB-seq) data. We show that Lux improves quantification and comparison of methylation levels over existing methods and that Lux can easily process any oxi-mC-seq data sets to quantify all cytosine modifications simultaneously together with their experimental parameters. Application of Lux to targeted data from Tet2 knockdown ESCs and T cells during development demonstrates DNA modification quantification at unprecedented detail, quantifies active demethylation pathway and reveals 5hmC localization in putative regulatory regions. Examine the distribution of C, 5mC, and 5hmC in DP, CD4 SP, and naïve CD4 T cells within selected loci. Half of the samples are measured using the traditional bisulphite-seq protocol but the other half is measured using the oxidative bisulphite-seq protocol (oxBS-seq).
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Project description:The Infinium 450K Methylation array is an established tool for measuring methylation. However, the bisulfite (BS) reaction commonly used with the 450K array cannot distinguish between 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC). The oxidative-bisulfite assay disambiguates 5mC and 5hmC. We describe the use of oxBS in conjunction with the 450K array (oxBS-array) to analyse 5hmC/5mC in cerebellum DNA. The methylation level derived by the BS reaction is the combined level of 5mC and 5hmC at a given base, while the oxBS reaction gives the level of 5mC alone. The level of 5hmC is derived by subtracting the oxBS level from the BS level. Here we present an analysis method that distinguishes genuine positive levels of 5hmC at levels as low as 3 %. We preformed four replicates of the same sample of cerebellum and found a high level of reproducibility (average r for BS = 98.3, and average r for oxBS = 96.8). In total, 114,734 probes showed a significant positive measurement for 5hmC. The range at which we were able to distinguish 5hmC occupancy was between 3 % and 42 %. In order to investigate the effects of multiple replicates on 5hmC detection we also simulated fewer replicates and found that decreasing the number of replicates to two reduced the number of positive probes identified by > 50%. We validated our results using qPCR in conjunction with glucosylation of 5hmC sites followed by MspI digestion and we found good concordance with the array estimates (r = 0.94). This experiment provides a map of 5hmC in the cerebellum and a robust dataset for use as a standard in future 5hmC analyses. We also provide a novel method for validating the presence of 5hmC at low levels, and highlight some of the pitfalls associated with measuring 5hmC and 5mC.
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Project description:DNA hydroxymethylation is frequently lost in glioblastoma. We hypothesized that reduced 5hmC levels might be related to the impaired expression of TET proteins in brain tumors. In this study we performed a genome-wide methylation analysis of LN229 cells stably transfected with scramble or TET3 overexpressing vectors. TET3 overexpression partially restored the genome-wide patterns of 5hmC characteristic of control brain samples in glioblastoma cell lines.
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Project description:5-hydroxymethylcytosine (5hmC), converted from 5-methylcytosine (5mC) by Tet enzymes, has recently drawn attention as the ‘sixth base’ of DNA since it is considered to be an intermediate of the demethylation pathway. Nonetheless, it remains to be addressed how 5hmC is linked to the development of human imprinting disorders. In this regard, conventional bisulfite (BS) treatment is unable to differentiate 5hmC from 5mC. It is thus hypothesized that BS conversion-derived ‘hypermethylation’ at imprinting control regions (ICRs), which may cause human imprinting disorders, would in fact be attributable to excessively increased levels of 5hmC as well as 5mC. To test this hypothesis, we applied the newly developed oxidative BS (oxBS) treatment to detect 5hmC in blood samples from Kagami-Ogata syndrome (KOS14) patients caused by perturbed expression of clustered imprinted genes on 14q32.2 resulting from the hypermethylation of ICRs at this locus, IG-DMR and MEG3-DMR. oxBS with pyrosequencing and cloning-based sequencing revealed that there were few amounts of 5hmC at the hypermethylated IG-DMR in blood samples from KOS14 patients. oxBS with genome-wide methylation array analysis demonstrated that global levels of 5hmC were very low with similar distribution patterns in blood samples from KOS14 patients and normal controls. We also confirmed the presence of a large amount of 5hmC in the brain sample from a normal control. 5hmC is not a major component in abnormally hypermethylated ICRs or at a global level, at least in blood from KOS14 patients. As the brain sample contained a large amount of 5hmC, the neural tissues of KOS14 patients are promising candidates for analysis in elucidating the role of 5hmC in the neurodevelopmental context.
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