Quantitative Sequencing of 5-Methylcytosine and 5-Hydroxymethylcytosine at Single-Base Resolution

Distinguishing Epigenetic Marks Methylation of the cytosine base in eukaryotic DNA (5mC) is an important epigenetic mark involved in gene silencing and genome stability. Methylated cytosine can be enzymatically oxidized to 5-hydroxymethylcytosine (5hmC), which may function as a distinct epigenetic mark—possibly involved in pluripotency—and it may also be an intermediate in active DNA demethylation. To be able to detect 5hmC genome-wide and at single-base resolution, Booth et al. (p. 934, published online 26 April) developed a 5hmC sequencing chemistry that selectively oxidizes 5hmC to 5-formylcytosine and then to uracil while leaving 5mC unchanged. Using this method, mouse embryonic stem cell genomic DNA was sequenced to reveal that 5hmC is found enriched at intragenic CpG islands and long interspersed nuclear element–1 retrotransposons. A sequencing method can discriminate epigenetically modified cytosine nucleotides within embryonic stem cell DNA. 5-Methylcytosine can be converted to 5-hydroxymethylcytosine (5hmC) in mammalian DNA by the ten-eleven translocation (TET) enzymes. We introduce oxidative bisulfite sequencing (oxBS-Seq), the first method for quantitative mapping of 5hmC in genomic DNA at single-nucleotide resolution. Selective chemical oxidation of 5hmC to 5-formylcytosine (5fC) enables bisulfite conversion of 5fC to uracil. We demonstrate the utility of oxBS-Seq to map and quantify 5hmC at CpG islands (CGIs) in mouse embryonic stem (ES) cells and identify 800 5hmC-containing CGIs that have on average 3.3% hydroxymethylation. High levels of 5hmC were found in CGIs associated with transcriptional regulators and in long interspersed nuclear elements, suggesting that these regions might undergo epigenetic reprogramming in ES cells. Our results open new questions on 5hmC dynamics and sequence-specific targeting by TETs.

[1]  Vijay K. Tiwari,et al.  DNA-binding factors shape the mouse methylome at distal regulatory regions , 2011, Nature.

[2]  Tyson A. Clark,et al.  Sensitive and specific single-molecule sequencing of 5-hydroxymethylcytosine , 2011, Nature Methods.

[3]  W. Reik,et al.  Uncovering the role of 5-hydroxymethylcytosine in the epigenome , 2011, Nature Reviews Genetics.

[4]  Yang Wang,et al.  Tet-Mediated Formation of 5-Carboxylcytosine and Its Excision by TDG in Mammalian DNA , 2011, Science.

[5]  Chuan He,et al.  Tet Proteins Can Convert 5-Methylcytosine to 5-Formylcytosine and 5-Carboxylcytosine , 2011, Science.

[6]  Markus Müller,et al.  The discovery of 5-formylcytosine in embryonic stem cell DNA. , 2011, Angewandte Chemie.

[7]  S. Jacobsen,et al.  Tissue-specific Distribution and Dynamic Changes of 5-Hydroxymethylcytosine in Mammalian Genomes* , 2011, The Journal of Biological Chemistry.

[8]  J. Min,et al.  Genome-wide regulation of 5hmC, 5mC, and gene expression by Tet1 hydroxylase in mouse embryonic stem cells. , 2011, Molecular cell.

[9]  A. Bird,et al.  CpG islands and the regulation of transcription. , 2011, Genes & development.

[10]  Philipp Kapranov,et al.  Genome-wide mapping of 5-hydroxymethylcytosine in embryonic stem cells , 2011, Nature.

[11]  S. Andrews,et al.  Bismark: a flexible aligner and methylation caller for Bisulfite-Seq applications , 2011, Bioinform..

[12]  Juri Rappsilber,et al.  TET1 and hydroxymethylcytosine in transcription and DNA methylation fidelity , 2011, Nature.

[13]  W. Reik,et al.  Dynamic regulation of 5-hydroxymethylcytosine in mouse ES cells and during differentiation , 2011, Nature.

[14]  Keji Zhao,et al.  Genome-wide analysis of 5-hydroxymethylcytosine distribution reveals its dual function in transcriptional regulation in mouse embryonic stem cells. , 2011, Genes & development.

[15]  G. Pfeifer,et al.  Genomic mapping of 5-hydroxymethylcytosine in the human brain , 2011, Nucleic acids research.

[16]  Zachary D. Smith,et al.  Preparation of reduced representation bisulfite sequencing libraries for genome-scale DNA methylation profiling , 2011, Nature Protocols.

[17]  Cameron S. Osborne,et al.  Large Scale Loss of Data in Low-Diversity Illumina Sequencing Libraries Can Be Recovered by Deferred Cluster Calling , 2011, PloS one.

[18]  Michael L. Klein,et al.  Discrimination of methylcytosine from hydroxymethylcytosine in DNA molecules. , 2011, Journal of the American Chemical Society.

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

[20]  Michael Weber,et al.  Targets and dynamics of promoter DNA methylation during early mouse development , 2010, Nature Genetics.

[21]  H. Bayley,et al.  Identification of epigenetic DNA modifications with a protein nanopore. , 2010, Chemical communications.

[22]  Robert S. Illingworth,et al.  Orphan CpG Islands Identify Numerous Conserved Promoters in the Mammalian Genome , 2010, PLoS genetics.

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

[24]  M. Biel,et al.  Quantification of the sixth DNA base hydroxymethylcytosine in the brain. , 2010, Angewandte Chemie.

[25]  Colm E. Nestor,et al.  Enzymatic approaches and bisulfite sequencing cannot distinguish between 5-methylcytosine and 5-hydroxymethylcytosine in DNA. , 2010, BioTechniques.

[26]  C. Fehr,et al.  (+)-(R,Z)-5-Muscenone and (-)-(R)-muscone by enantioselective aldol reaction and Grob fragmentation. , 2010, Chemistry.

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

[28]  S. Pacchione,et al.  Intracisternal A particle genes: Distribution in the mouse genome, active subtypes, and potential roles as species‐specific mediators of susceptibility to cancer , 2010, Molecular carcinogenesis.

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

[30]  S. Turner,et al.  Real-Time DNA Sequencing from Single Polymerase Molecules , 2009, Science.

[31]  Clifford A. Meyer,et al.  Model-based Analysis of ChIP-Seq (MACS) , 2008, Genome Biology.

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

[33]  J. Gregory,et al.  DNA digestion to deoxyribonucleoside: a simplified one-step procedure. , 2008, Analytical biochemistry.

[34]  W. Reik,et al.  Resistance of IAPs to methylation reprogramming may provide a mechanism for epigenetic inheritance in the mouse , 2003, Genesis.

[35]  E. Ostertag,et al.  A novel active L1 retrotransposon subfamily in the mouse. , 2001, Genome research.

[36]  C. Hutchison,et al.  L1 A-monomer tandem arrays have expanded during the course of mouse L1 evolution. , 1993, Molecular biology and evolution.

[37]  S. Ley,et al.  Oxo complexes of ruthenium(VI) and (VII) as organic oxidants , 1984 .

[38]  Maria Papatriantafyllou Mucosal immunology: IRF3 maintains gut homeostasis , 2012, Nature Reviews Immunology.

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

[40]  R. Jones,et al.  The chemistry of terpenes—VIII: Characterisation of the bisulphite adducts of α,β-unsaturated aldehydes by NMR spectroscopy , 1978 .