title: Simultaneous single-molecule epigenetic imaging of DNA methylation and hydroxymethylation.
abstract: The modifications 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) are the two major DNA epigenetic modifications in mammalian genomes and play crucial roles in development and pathogenesis. Little is known about the colocalization or potential correlation of these two modifications. Here we present an ultrasensitive single-molecule imaging technology capable of detecting and quantifying 5hmC and 5mC from trace amounts of DNA. We used this approach to perform single-molecule fluorescence resonance energy transfer (smFRET) experiments which measure the proximity between 5mC and 5hmC in the same DNA molecule. Our results reveal high levels of adjacent and opposing methylated and hydroxymethylated CpG sites (5hmC/5mCpGs) in mouse genomic DNA across multiple tissues. This identifies the previously undetectable and unappreciated 5hmC/5mCpGs as one of the major states for 5hmC in the mammalian genome and suggest that they could function in promoting gene expression.
authors.affiliation: Department of Bioengineering, Stanford University, Stanford, CA 94305; Department of Applied Physics, Stanford University, Stanford, CA 94305;
authors.forename: Chun-Xiao
authors.initials: CX
authors.lastname: Song
authors.affiliation: Department of Molecular and Cellular Physiology, Stanford University, Stanford, CA 94305; Department of Structural Biology, Stanford University, Stanford, CA 94305; Department of Photon Science, Stanford University, Stanford, CA 94305; Department of Neurology and Neurological Sciences, Stanford University, Stanford, CA 94305; Howard Hughes Medical Institute, Stanford University, Stanford, CA 94305 quake@stanford.edu diaojj@gmail.com.
authors.forename: Jiajie
authors.identifier: http://orcid.org/0000-0003-4288-3203
authors.initials: J
authors.lastname: Diao
authors.affiliation: Department of Molecular and Cellular Physiology, Stanford University, Stanford, CA 94305; Department of Structural Biology, Stanford University, Stanford, CA 94305; Department of Photon Science, Stanford University, Stanford, CA 94305; Department of Neurology and Neurological Sciences, Stanford University, Stanford, CA 94305; Howard Hughes Medical Institute, Stanford University, Stanford, CA 94305.
authors.forename: Axel T
authors.initials: AT
authors.lastname: Brunger
authors.affiliation: Department of Bioengineering, Stanford University, Stanford, CA 94305; Department of Applied Physics, Stanford University, Stanford, CA 94305; Howard Hughes Medical Institute, Stanford University, Stanford, CA 94305 quake@stanford.edu diaojj@gmail.com.
authors.forename: Stephen R
authors.initials: SR
authors.lastname: Quake
mesh_terms: D044503:5-Methylcytosine / Q000378:metabolism* / Q000031:analogs & derivatives; D000818:Animals; D003596:Cytosine / Q000031:analogs & derivatives* / Q000378:metabolism; D019175:DNA Methylation / Q000502:physiology*; D044127:Epigenesis, Genetic / Q000502:physiology*; D051379:Mice; D057054:Molecular Imaging
chemical_list: C011865:5-hydroxymethylcytosine; D044503:5-Methylcytosine; D003596:Cytosine
keywords: 5-hydroxymethylcytosine; 5-methylcytosine; epigenetics; single-molecule imaging