Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Application of a low cost array-based technique - TAB-Array - for quantifying and mapping both 5mC and 5hmC at single base resolution.

ABSTRACT: 5-hydroxymethylcytosine (5hmC), an oxidized derivative of 5-methylcytosine (5mC), has been implicated as an important epigenetic regulator of mammalian development. Current procedures use cost-prohibitive DNA sequencing methods to discriminate 5hmC from 5mC, limiting their accessibility to the scientific community. Here we report a method that combines TET-assisted bisulfite conversion with Illumina 450K DNA methylation arrays for a low-cost high-throughput approach that distinguishes 5hmC and 5mC signals. Implementing this approach, termed TAB-array, we assessed DNA methylation dynamics in the differentiation of human pluripotent stem cells into cardiovascular and neural progenitors. With the ability to discriminate 5mC and 5hmC, we found a much larger number of dynamically methylated genomic regions implicated in the development of these lineages than we could detect by 5mC analysis alone. The increased resolution and accuracy afforded by this approach provides a powerful means to investigate the distinct contributions of 5mC and 5hmC in human development and disease. We generated illumina 450k DNA methylation data for a total of 9 sample groups with two biological replicates for each group. Data for 4/9 groups were generated from glucosylated and bisulfite converted DNA, from human induced plurupotent stem cells (hIPSCs), differentiated cardiovascular progenitors (CVPs), differentiated neural progenitors (NPCs), and fibroblasts. Data for the next 4/9 groups were generated from glucosylated, TET-oxidized and bisulfite converted DNA, from and included replicates of hIPSCs, CVPs, NPCs, and fibroblasts. Data for the last group was generated from standard bisulfite converted DNA (not glucosylated) from fibroblasts.

ORGANISM(S): Homo sapiens

SUBMITTER: Kristopher Nazor

PROVIDER: E-GEOD-60225 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

ACCESS DATA

Publications

Application of a low cost array-based technique - TAB-Array - for quantifying and mapping both 5mC and 5hmC at single base resolution in human pluripotent stem cells.

Nazor Kristopher L KL Boland Michael J MJ Bibikova Marina M Klotzle Brandy B Yu Miao M Glenn-Pratola Victoria L VL Schell John P JP Coleman Ronald L RL Cabral-da-Silva Mauricio C MC Schmidt Ulrich U Peterson Suzanne E SE He Chuan C Loring Jeanne F JF Fan Jian-Bing JB

Genomics 20140829 5

5-hydroxymethylcytosine (5hmC), an oxidized derivative of 5-methylcytosine (5mC), has been implicated as an important epigenetic regulator of mammalian development. Current procedures use DNA sequencing methods to discriminate 5hmC from 5mC, limiting their accessibility to the scientific community. Here we report a method that combines TET-assisted bisulfite conversion with Illumina 450K DNA methylation arrays for a low-cost high-throughput approach that distinguishes 5hmC and 5mC signals at bas ...[more]

PMID: 25179373

Similar Datasets

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. We generated illumina 450k DNA methylation data for BS or oxBS converted samples of three KOS14 blood samples, one control pooled blood sample and one control brain sample with two technical replicates for each sample.

Project description:TET proteins oxidize 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). 5fC and 5caC are excised by mammalian DNA glycosylase TDG, implicating 5mC oxidation in DNA demethylation. Here we show that the genomic locations of 5fC can be determined by coupling chemical reduction with biotin tagging. Genome-wide mapping of 5fC in mouse embryonic stem cells (mESCs) reveals that 5fC preferentially occurs at poised enhancers among other gene regulatory elements. Application to Tdg null mESCs further suggests that 5fC production coordinates with p300 in remodeling epigenetic states of enhancers. This process, which is not influenced by 5hmC, appears to be associated with further oxidation of 5hmC and commitment to demethylation through 5fC. Finally, we resolved 5fC at base-resolution by hydroxylamine-based protection from bisulfite-mediated deamination, thereby confirming sites of 5fC accumulation. Our results reveal roles of active 5mC/5hmC oxidation and TDG-mediated demethylation in epigenetic tuning at regulatory elements. We report here a chemical labeling method that effectively differentiates 5fC from 5mC, 5hmC, and 5caC in genomic DNA. First, we quantitatively protect endogenous 5hmC with a regular glucose using b-glucosytransferase-catalyzed 5hmC glucosylation. Then, we selectively reduce 5fC with NaBH4 to 5hmC, and chemically label the resulting 5hmC (from 5fC) with an azide-modified glucose. Biotin can be installed subsequently for specific enrichment of 5fC. Our method thereby provides an effective tool of general utility for the genomic localization of 5fC. Here we provide genome-wide profiles of 5hmC, 5fC, and p300 in Tdg fl/fl and Tdg-/- mESCs as well as a 5fC control (Non-NaBH4) and polyA RNA-Seq expression data. Genome-wide profiles of 5hmC and 5fC in mESCs differentiated to embryoid bodies are also included. We also report the development and application of a single-base resolution method for the detection of 5fC in genomic DNA by hydroylamine mediated protection of 5fC from deamination during bisulfite treatment, or 5fC Chemical Assisted Bisulfite Sequencing (fCAB-Seq). We applied this method in parallel with conventional ChIP-Methyl-Seq to H3K4me1 ChIP enriched DNA from Tdg fl/fl and Tdg-/- mice.

Dataset Information

Application of a low cost array-based technique - TAB-Array - for quantifying and mapping both 5mC and 5hmC at single base resolution.

Publications

Application of a low cost array-based technique - TAB-Array - for quantifying and mapping both 5mC and 5hmC at single base resolution in human pluripotent stem cells.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure