Unknown,Transcriptomics,Genomics,Proteomics

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Genome-wide mapping of 5-hydroxymethylcytosine in embryonic stem cells

ABSTRACT: 5hmC and TET proteins have been implicated in stem cell biology and cancer, but information on the genome-wide distribution of 5hmC is limited. Here we describe two novel and specific approaches to profile the genomic localisation of 5hmC. The first approach, termed GLIB (GLucosylation, perIodate oxidation, Biotinylation) uses a combination of enzymatic and chemical steps to isolate DNA fragments containing as few as a single 5hmC. The second approach involves conversion of 5hmC to cytosine 5-methylenesulphonate (CMS) by treatment of genomic DNA with sodium bisulphite, followed by immunoprecipitation of CMS-containing DNA with a specific antiserum to CMS. High-throughput sequencing of 5hmC-containing DNA from mouse embryonic stem (ES) cells showed strong enrichment within exons and near transcriptional start sites (TSS). 5hmC was especially enriched at the start sites of genes whose promoters bear dual histone 3 lysine 27 trimethylation (H3K27me3) and histone 3 lysine 4 trimethylation (H3K4me3) marks. Our results indicate that 5hmC has a likely role in transcriptional regulation, and suggest a model in which 5hmC contributes to the M-bM-^@M-^\poisedM-bM-^@M-^] chromatin signature found at developmentally-regulated genes in ES cells. Mapping of 5-hydroxymethylcytosine in ES cells by GLIB and anti-CMS methodologies

ORGANISM(S): Mus musculus

SUBMITTER: William Pastor

PROVIDER: E-GEOD-28682 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Genome-wide mapping of 5-hydroxymethylcytosine in embryonic stem cells.

Pastor William A WA Pape Utz J UJ Huang Yun Y Henderson Hope R HR Lister Ryan R Ko Myunggon M McLoughlin Erin M EM Brudno Yevgeny Y Mahapatra Sahasransu S Kapranov Philipp P Tahiliani Mamta M Daley George Q GQ Liu X Shirley XS Ecker Joseph R JR Milos Patrice M PM Agarwal Suneet S Rao Anjana A

Nature 20110508 7347

5-hydroxymethylcytosine (5hmC) is a modified base present at low levels in diverse cell types in mammals. 5hmC is generated by the TET family of Fe(II) and 2-oxoglutarate-dependent enzymes through oxidation of 5-methylcytosine (5mC). 5hmC and TET proteins have been implicated in stem cell biology and cancer, but information on the genome-wide distribution of 5hmC is limited. Here we describe two novel and specific approaches to profile the genomic localization of 5hmC. The first approach, termed ...[more]

PMID: 21552279

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Project description:Methylation of cytosine in DNA (5mC) is an important epigenetic mark that is involved in the regulation of genome function. During early embryonic development in mammals, the DNA methylation landscape is dynamically reprogrammed in part through active demethylation. Recent advances have identified key players involved in active demethylation pathways, including oxidation of 5mC to 5-hydroxymethylcytosine (5hmC) and 5-formylcytosine (5fC) by the TET family of enzymes and excision of 5fC by the base excision repair enzyme thymine DNA glycosylase (TDG). Here, we provide the first genome-wide distribution map of 5fC in mouse embryonic stem (ES) cells and evaluate potential roles for 5fC in differentiation. Our method exploits the unique reactivity of 5fC to link a biotin tag for pulldown and high-throughput sequencing. Genome-wide mapping revealed 5fC enrichment in CpG islands (CGIs) of promoters and exons. CGI promoters in which 5fC was relatively more enriched than 5mC or 5hmC corresponded to transcriptionally active genes. Accordingly, 5fC-rich promoters had elevated H3K4me3 levels, a histone mark associated with active transcription, and were frequently bound by RNA Polymerase II. Downregulation of TDG led to accumulation of 5fC in CGIs in ES cells, which correlates with increased methylation in these genomic regions during differentiation and in mouse embryonic fibroblasts derived from TDG knockout embryos. Collectively, our data suggest that 5fC plays a role in epigenetic reprogramming. The formation and removal of this cytosine modification are confined to specific genomic regions, which are in part controlled by TDG. Notably, 5fC excision in ES cells is necessary for the correct establishment of CGI methylation patterns during differentiation, and hence, for appropriate patterns of gene expression during development. We devised a method to map 5-formylcytosine (5fC) by linking a biotin tag to 5fC for pulldown and high-throughput sequencing. We mapped 5fC in the following samples of mouse embryonic stem cells (J1): Wild-type ES cells (two replicates); ES cells transfected with siRNA targeting TDG (two replicates); ES cells transfected with non-targeting siRNA (two replicates). One genomic input library was also sequenced to detect and correct biases in fragment enrichment.

Dataset Information

Genome-wide mapping of 5-hydroxymethylcytosine in embryonic stem cells

Publications

Genome-wide mapping of 5-hydroxymethylcytosine in embryonic stem cells.

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