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

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 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. 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 probable role in transcriptional regulation, and suggest a model in which 5hmC contributes to the ‘poised’ chromatin signature found at developmentally-regulated genes in ES cells.

[1]  D. Brutlag,et al.  A genome-wide analysis of CpG dinucleotides in the human genome distinguishes two distinct classes of promoters , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[2]  J. Soulier,et al.  Mutation in TET2 in myeloid cancers. , 2009, The New England journal of medicine.

[3]  J. Rinn,et al.  Ab initio reconstruction of transcriptomes of pluripotent and lineage committed cells reveals gene structures of thousands of lincRNAs , 2010, Nature Biotechnology.

[4]  P. Jin,et al.  Selective chemical labeling reveals the genome-wide distribution of 5-hydroxymethylcytosine , 2011, Nature Biotechnology.

[5]  C. Schofield,et al.  Oxygenase catalyzed 5-methylcytosine hydroxylation. , 2009, Chemistry & biology.

[6]  J. Rinn,et al.  Ab initio reconstruction of transcriptomes of pluripotent and lineage committed cells reveals gene structures of thousands of lincRNAs , 2010, Nature biotechnology.

[7]  Haikuo Zhang,et al.  TET1 is a DNA-binding protein that modulates DNA methylation and gene transcription via hydroxylation of 5-methylcytosine , 2010, Cell Research.

[8]  David R. Liu,et al.  Conversion of 5-Methylcytosine to 5- Hydroxymethylcytosine in Mammalian DNA by the MLL Partner TET1 , 2009 .

[9]  Matteo Pellegrini,et al.  Promoter CpG methylation contributes to ES cell gene regulation in parallel with Oct4/Nanog, PcG complex, and histone H3 K4/K27 trimethylation. , 2008, Cell stem cell.

[10]  R. Young,et al.  Histone H3K27ac separates active from poised enhancers and predicts developmental state , 2010, Proceedings of the National Academy of Sciences.

[11]  Simon Kasif,et al.  Genomewide Analysis of PRC1 and PRC2 Occupancy Identifies Two Classes of Bivalent Domains , 2008, PLoS genetics.

[12]  David R. Liu,et al.  The Behaviour of 5-Hydroxymethylcytosine in Bisulfite Sequencing , 2010, PloS one.

[13]  L. Aravind,et al.  Prediction of novel families of enzymes involved in oxidative and other complex modifications of bases in nucleic acids , 2009, Cell cycle.

[14]  Ruedi Aebersold,et al.  Identification and quantification of N-linked glycoproteins using hydrazide chemistry, stable isotope labeling and mass spectrometry , 2003, Nature Biotechnology.

[15]  J. Zeitlinger,et al.  Polycomb complexes repress developmental regulators in murine embryonic stem cells , 2006, Nature.

[16]  H. Hayatsu,et al.  Reaction of bisulfite with the 5-hydroxymethyl group in pyrimidines and in phage DNAs. , 1979, Biochemistry.

[17]  S. Quake,et al.  Single-Molecule DNA Sequencing of a Viral Genome , 2008, Science.

[18]  H. Leonhardt,et al.  Sensitive enzymatic quantification of 5-hydroxymethylcytosine in genomic DNA , 2010, Nucleic acids research.

[19]  T. Mikkelsen,et al.  Genome-scale DNA methylation maps of pluripotent and differentiated cells , 2008, Nature.

[20]  Lee E. Edsall,et al.  Human DNA methylomes at base resolution show widespread epigenomic differences , 2009, Nature.

[21]  Johnf . Thompson,et al.  Virtual Terminator nucleotides for next generation DNA sequencing , 2009, Nature Methods.

[22]  Megan F. Cole,et al.  Control of Developmental Regulators by Polycomb in Human Embryonic Stem Cells , 2006, Cell.

[23]  N. Heintz,et al.  The Nuclear DNA Base 5-Hydroxymethylcytosine Is Present in Purkinje Neurons and the Brain , 2009, Science.

[24]  L. Aravind,et al.  Impaired hydroxylation of 5-methylcytosine in myeloid cancers with mutant TET2 , 2010, Nature.

[25]  Riitta Lahesmaa,et al.  Tet1 and Tet2 regulate 5-hydroxymethylcytosine production and cell lineage specification in mouse embryonic stem cells. , 2011, Cell stem cell.

[26]  T. Mikkelsen,et al.  Genome-wide maps of chromatin state in pluripotent and lineage-committed cells , 2007, Nature.

[27]  James A. Cuff,et al.  A Bivalent Chromatin Structure Marks Key Developmental Genes in Embryonic Stem Cells , 2006, Cell.

[28]  Yi Zhang,et al.  Role of Tet proteins in 5mC to 5hmC conversion, ES-cell self-renewal and inner cell mass specification , 2010, Nature.

[29]  M. Pellegrini,et al.  Conservation and divergence of methylation patterning in plants and animals , 2010, Proceedings of the National Academy of Sciences.