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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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE60225 | GEO | 2014/12/12

SECONDARY ACCESSION(S): PRJNA257792

REPOSITORIES: GEO

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

Project description: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.

2014-12-12 | E-GEOD-60225 | biostudies-arrayexpress

Joint single-cell profiling resolves 5mC and 5hmC and reveals their distinct gene regulatory effects [Joint-snhmC-seq: snmCseq2.split]

Project description:Oxidative modification of 5-methylcytosine (5mC) by TET DNA dioxygenases generates 5-hydroxymethylcytosine (5hmC), the most abundant form of oxidized 5mC. Existing single-cell bisulfite sequencing methods cannot resolve 5mC and 5hmC, leaving the cell-type-specific regulatory mechanisms of TET and 5hmC largely unknown. Here we present Joint single-nucleus (hydroxy)methylcytosine sequencing (Joint-snhmC-seq), a scalable and quantitative approach that simultaneously profiles 5hmC and true 5mC in single cells by harnessing differential deaminase activity of APOBEC3A towards 5mC and chemically protected 5hmC. Joint-snhmC-seq profiling of single nuclei from the mouse brains reveals an unprecedented level of epigenetic heterogeneity of both 5hmC and true 5mC at single-cell resolution. We show that cell-type-specific profiles of 5hmC or true 5mC improve multi-modal single-cell data integration, enable accurate identification of neuronal subtypes, and uncover context-specific regulatory effects of cell-type-specific genes by TET enzymes.

2023-07-14 | GSE236789 | GEO

Joint single-cell profiling resolves 5mC and 5hmC and reveals their distinct gene regulatory effects [Joint-snhmC-seq: snhmCseq2.split]

2023-07-14 | GSE236784 | GEO

Joint single-cell profiling resolves 5mC and 5hmC and reveals their distinct gene regulatory effects [snhmCseq]

2023-07-14 | GSE236776 | GEO

Joint single-cell profiling resolves 5mC and 5hmC and reveals their distinct gene regulatory effects [snRNAseq]

2023-07-14 | GSE236769 | GEO

Alterations of 5-hydroxymethylation in circulating cell-free DNA reflect molecular distinctions of subtypes of non-Hodgkin lymphoma

Project description:The 5-methylcytosines (5mC) have been implicated in the pathogenesis of diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma (FL). However, the role of 5-hydroxymethylcytosines (5hmC) that are generated from 5mC through active demethylation, in lymphomagenesis is unknown. We profiled genome-wide 5hmC in circulating cell-free DNA (cfDNA) from 73 newly diagnosed patients with DLBCL and FL. We identified 294 differentially modified genes between DLBCL and FL. The differential 5hmC in the DLBCL/FL-differentiating genes co-localized with enhancers marks: H3K4me1and H3K27ac. A four-gene panel (CNN2, HMG20B, ACRBP, IZUMO1) robustly represented the overall 5hmC modification pattern that distinguished FL from DLBCL with an area under curve of 88.5% in testing set. The median 5hmC modification levels in signature genes showed potential for separating patients for risk of all-cause mortality. This study provides evidence that genome-wide 5hmC profiles in cfDNA differ between DLBCL and FL and could be exploited as a non-invasive approach.

2021-02-17 | GSE155228 | GEO

Genome-wide single base resolution profiling of 5-methyl and 5-hydroxymethylcytosine in glioblastoma

Project description:Through parallel processing of genomic DNA with bisulfite and oxidative bisulfite treatments on Illumina 450K arrays we resolved both 5mC and 5hmC in glioblastoma tissues. We developed and applied a novel technique for estimating 5mC, 5hmC, and unmethylated proportions from array data to glioblastoma tissues and compare with normal brain tissue. Genomic distribution of 5hmC was associated with features of transcription despite the glioblastoma genome being relatively depleted of 5hmC. When integrating 5mC and 5hmC data using a Gaussian finite mixture model approach, we observed significant associations between 5hmC levels and gene-sets involved in immune and RNA regulatory processes. We also observed an enrichment of 5hmC in introns, enhancer regions, and genes that are actively transcribed in glioblastomas from TCGA. Significant differences in patient survival were observed among classes of 5hmC obtained from a recursively partitioned mixture model. Glioblastoma dervied (n=30) DNA was subjected to tandem bisulfite and oxidative bisulfite conversion with an input of 4ug per sample using the TrueMethylÂ® kit v.1.1 (Cambridge Epigenetix) protocol optimized for Illumina HumanMethylation450 arrays.

2016-08-22 | E-GEOD-73895 | biostudies-arrayexpress

Dynamic changes of DNA methylome and hydroxymethylome during neural differentiation of hES cell (hMeDIP-Seq and MBD-Seq)

Project description:DNA methylation and hydroxymethylation have been implicated in normal development and differentiation, but our knowledge about the genome-wide distribution of 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) during cellular differentiation remains limited. Using in vitro model system of gradual differentiation of human embryonic stem (hES) cells into ventral midbrain-type neural precursor (NP) cells and terminally into dopamine (DA) neurons, we explored changes in 5mC or 5hmC patterns during lineage commitment. We used three techniques, 450K DNA methylation array, MBD-seq, and hMeDIP-seq, and found combination of these methods can provide comprehensive information on the genome-wide 5mC or 5hmC patterns. We observed dramatic changes of 5mC patterns during differentiation of hES cells into NP cells. Although genome-wide 5hmC distribution was more stable than 5mC, coding exons, CpG islands and shores showed dynamic 5hmC patterns during differentiation. In addition to the role of DNA methylation as a mechanism to initiating gene silencing, we also found DNA methylation as a locking system to maintain gene silencing. More than 1,000 genes including mesoderm development related genes acquired promoter methylation during neuronal differentiation even though they were already silenced in hES cells. Finally, we found that activated genes lost 5mC in transcription start site (TSS) but acquired 5hmC around TSS and gene body during differentiation. Our findings may provide clues for elucidating the molecular mechanisms underlying lineage specific differentiation of pluripotent stem cells during human embryonic development. Examination of hMeDIP-Seq and MBD-Seq in 3 cell types (human embryonic stem, neural precursor, and dopamine neuron cells)

2013-11-04 | E-GEOD-38214 | biostudies-arrayexpress

Dynamic changes of DNA methylome and hydroxymethylome during neural differentiation of hES cells (Methylation BeadChip)

Project description:DNA methylation and hydroxymethylation have been implicated in normal development and differentiation, but our knowledge about the genome-wide distribution of 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) during cellular differentiation remains limited. Using in vitro model system of gradual differentiation of human embryonic stem (hES) cells into ventral midbrain-type neural precursor (NP) cells and terminally into dopamine (DA) neurons, we explored changes in 5mC or 5hmC patterns during lineage commitment. We used three techniques, 450K DNA methylation array, MBD-seq, and hMeDIP-seq, and found combination of these methods can provide comprehensive information on the genome-wide 5mC or 5hmC patterns. We observed dramatic changes of 5mC patterns during differentiation of hES cells into NP cells. Although genome-wide 5hmC distribution was more stable than 5mC, coding exons, CpG islands and shores showed dynamic 5hmC patterns during differentiation. In addition to the role of DNA methylation as a mechanism to initiating gene silencing, we also found DNA methylation as a locking system to maintain gene silencing. More than 1,000 genes including mesoderm development related genes acquired promoter methylation during neuronal differentiation even though they were already silenced in hES cells. Finally, we found that activated genes lost 5mC in transcription start site (TSS) but acquired 5hmC around TSS and gene body during differentiation. Our findings may provide clues for elucidating the molecular mechanisms underlying lineage specific differentiation of pluripotent stem cells during human embryonic development. Examination of genome-wide DNA methylation in 3 cell types (human embryonic stem, neural precursor, and dopamine neuron cells)

2013-11-04 | E-GEOD-38216 | biostudies-arrayexpress

Genome-wide single base resolution profiling of 5-methyl and 5-hydroxymethylcytosine in glioblastoma

2016-08-22 | GSE73895 | GEO

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