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A probabilistic generative model for quantification of DNA modifications enables analysis of demethylation pathways [mESCs]

ABSTRACT: 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).

ORGANISM(S): Mus musculus

SUBMITTER: Tarmo ÃijÃ¶

PROVIDER: E-GEOD-68574 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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A probabilistic generative model for quantification of DNA modifications enables analysis of demethylation pathways [T-cells]

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 naÃ¯ve 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).

2016-07-03 | E-GEOD-68575 | biostudies-arrayexpress

Distinct roles of Tet1 and Tet2 in mouse embryonic stem cells [MeDIP-Seq]

Project description:The TET proteins TET1, TET2 and TET3 constitute a new family of dioxygenases that utilize molecular oxygen and the cofactors Fe(II) and 2-oxoglutarate to convert 5-methylcytosine (5mC) to 5-hydroxy-methylcytosine (5hmC) and further oxidation products in DNA1-5. Here we show that Tet1 and Tet2 have distinct roles in regulating 5hmC deposition and gene expression in mouse embryonic stem cells (mESC). Tet1 depletion in mESC primarily diminishes 5hmC levels at transcription start sites (TSS), whereas Tet2 depletion is mostly associated with decreased 5hmC in gene bodies relative to TSS. 5hmC is enriched at exon start and end sites, especially in exons that are highly expressed, and is significantly decreased upon Tet2 knockdown at the boundaries of high-expressed exons that are selectively regulated by Tet2. In differentiating murine B cells, Tet2 deficiency is associated with selective exon exclusion in the gene encoding the transmembrane phosphatase CD45. Tet2 depletion is associated with increased 5hmC and decreased 5mC at promoters/ TSS regions, possibly because of the redundant activity of Tet1. Together, these data indicate a complex interplay between Tet1 and Tet2 in mESC, and show that loss-of-function of a single TET protein does not necessarily lead to loss of 5hmC and a corresponding gain of 5mC, as generally assumed. The relation between Tet2 loss-of-function and selective changes in exon expression could potentially explain the frequent occurrence of both TET2 loss-of-function mutations and mutations in proteins involved in pre-mRNA splicing in myeloid malignancies in humans. Gene and exon expression analysis in mESC, Tet1 knockdown mESC, and Tet2 knockdown mESC by RNA-sequencing. Mapping of 5-hydroxymethylcytosine in mESC, Tet1 knockdown mESC, and Tet2 knockdown mESC by anti-CMS-seq. Mapping of methylcytosine in mESC, and Tet2 kd mESC by MeDIP-seq.

2014-12-03 | E-GEOD-63771 | biostudies-arrayexpress

Distinct roles of Tet1 and Tet2 in mouse embryonic stem cells (RNA-Seq)

2014-01-16 | E-GEOD-50198 | biostudies-arrayexpress

Distinct roles of Tet1 and Tet2 in mouse embryonic stem cells (CMS-Seq)

2014-01-16 | E-GEOD-50200 | biostudies-arrayexpress

A probabilistic generative model for quantification of DNA modifications enables analysis of demethylation pathways [T-cells]

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 “oxi-mC”-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.

2016-01-01 | GSE68575 | GEO

A probabilistic generative model for quantification of DNA modifications enables analysis of demethylation pathways [mESCs]

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 “oxi-mC”-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.

2016-01-01 | GSE68574 | GEO

Quantitative sequencing of 5-formylcytosine in DNA at single-base resolution

Project description:Cytosine base modifications 5-methylcytosine (5mC), 5-hydroxymethylcytosine (5hmC) and 5-formylcytosine (5fC) are present in mammalian DNA. Here, reduced bisulfite sequencing is developed for quantitatively sequencing 5fC at single-base resolution. This method is then applied with oxidative bisulfite sequencing to gain a map of 5mC, 5hmC and 5fC in mouse embryonic stem cells. 12 samples, reduced representation bisulphite treatment: 4 replicates each for bisulphite (BS), oxidative BS (oxBS) and reduced BS (redBS) for the detection of 5mC, 5hmC and 5fC. Mouse (strain B6C) embryonic stem cells.

2014-04-08 | E-GEOD-56572 | biostudies-arrayexpress

Comparison of ox-BS-450k 5-hydroxymethylcytosine and BS-450k 5-methylcytosine profiles in the human placenta

Project description:Bisulphite (BS) and oxidative bisulphite (oxBS) converted DNA from 4 normal human placentas were hybridized to the Illumina HumanMethylation450 Beadchip v1.2, obtaining the BS and oxBS DNA methylation profiles across approximately 450,000 CpGs. By using the oxBS treatment, the selective chemical oxidation of 5-hydroxymethylcytosine (5hmC) to 5-formylcytosine (5fC) and the deamination of the latter to uracil during the BS conversion allowed the quantification of independent 5-methylcytosine (5mC) and 5hmC methylation levels at every single CpG. In consequence, this data set characterizes the genome-wide distribution of 5hmC in the human placenta offerring a high-confidence list of intervals enriched for 5hmC in this tissue.

2017-09-12 | GSE93429 | GEO

Tet proteins in DNA demethylation and cell fate restriction

Project description:DNA methylation at 5-position of cytosine (5mC) is one of the best studied epigenetic modifications that plays important roles in diverse biological processes. Iterative oxidation of 5mC by the Ten-eleven translocation (Tet) family of proteins generates 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC), which can be further processed by DNA repair proteins leading to DNA demethylation. Functional characterization of the Tet proteins has been complicated by the redundancy between the three Tet proteins. Using the CRISPR/Cas9 technology, we have generated mouse embryonic stem cells (ESCs) deficient for all three Tet proteins (TKO). Whole genome bisulphite sequencing (WGBS) analysis revealed that Tet-mediated DNA demethylation mainly occurs distal enhancers as well as promoters that significantly overlap with 5hmC, 5fC and 5caC. Characterization of the Tet TKO ESCs revealed a function for Tet proteins in cell fate restriction as Tet TKO ESCs tend to adopt both primed pluripotent stem cell-like state and 2-cell embryo-like state. In addition, Tet TKO ESCs exhibit elongated telomeres. Thus, our study reveals a role of Tet proteins not only in DNA demethylation, but also in cell fate restriction and telomere maintenance. 2 samples for WGBS and 2 samples for RNA-seq

2014-09-22 | E-GEOD-56986 | biostudies-arrayexpress

Characterization of imprinted methylation using the Illumina Infinium MethylationEPIC array platform

Project description:Bisulphite (BS) converted DNA from 2 paternal uniparental diploidies (pUPDs), one maternal (mUPD) and 5 control leukocytes samples were hybridized to the Infinium HumanMethylationEPIC BeadChip (Illumina), obtaining the BS DNA methylation profiles across approximately 850,000 CpGs. In addition, the 5 control leukocyte samples were also coverted using oxidative bisulphite (oxBS) treatment. The selective chemical oxidation of 5-hydroxymethylcytosine (5hmC) to 5-formylcytosine (5fC) and the deamination of the latter to uracil during the BS conversion allowed the quantification of independent 5-methylcytosine (5mC) and 5hmC methylation levels at every single CpG.

2018-07-11 | GSE103738 | GEO

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