title: Single base resolution analysis of 5-hydroxymethylcytosine in 188 human genes: implications for hepatic gene expression.
abstract: To improve the epigenomic analysis of tissues rich in 5-hydroxymethylcytosine (hmC), we developed a novel protocol called TAB-Methyl-SEQ, which allows for single base resolution profiling of both hmC and 5-methylcytosine by targeted next-generation sequencing. TAB-Methyl-SEQ data were extensively validated by a set of five methodologically different protocols. Importantly, these extensive cross-comparisons revealed that protocols based on Tet1-assisted bisulfite conversion provided more precise hmC values than TrueMethyl-based methods. A total of 109 454 CpG sites were analyzed by TAB-Methyl-SEQ for mC and hmC in 188 genes from 20 different adult human livers. We describe three types of variability of hepatic hmC profiles: (i) sample-specific variability at 40.8% of CpG sites analyzed, where the local hmC values correlate to the global hmC content of livers (measured by LC-MS), (ii) gene-specific variability, where hmC levels in the coding regions positively correlate to expression of the respective gene and (iii) site-specific variability, where prominent hmC peaks span only 1 to 3 neighboring CpG sites. Our data suggest that both the gene- and site-specific components of hmC variability might contribute to the epigenetic control of hepatic genes. The protocol described here should be useful for targeted DNA analysis in a variety of applications.
authors.affiliation: Section of Pharmacogenetics, Department of Physiology and Pharmacology, Karolinska Institutet, Nanna Svartz väg 2, 17177 Stockholm, Sweden maxim.ivanov@ki.se.
authors.forename: Maxim
authors.initials: M
authors.lastname: Ivanov
authors.affiliation: Estonian Genome Center, University of Tartu, Riia 23b, 51010 Tartu, Estonia Institute of Mathematics and Statistics, University of Tartu, J. Liivi 2, 50409 Tartu, Estonia.
authors.forename: Mart
authors.initials: M
authors.lastname: Kals
authors.affiliation: Section of Pharmacogenetics, Department of Physiology and Pharmacology, Karolinska Institutet, Nanna Svartz väg 2, 17177 Stockholm, Sweden.
authors.forename: Volker
authors.initials: V
authors.lastname: Lauschke
authors.affiliation: Group of Pharmacoepigenetics, Department of Physiology and Pharmacology, Karolinska Institutet, Von Eulers väg 8 IV, 17177 Stockholm, Sweden.
authors.forename: Isabel
authors.initials: I
authors.lastname: Barragan
authors.affiliation: Department of Biochemistry and Biophysics, Science for Life Laboratory, Stockholm University, 10691 Stockholm, Sweden.
authors.forename: Philip
authors.initials: P
authors.lastname: Ewels
authors.affiliation: Science for Life Laboratory, School of Biotechnology, Division of Gene Technology, Royal Institute of Technology, 17121 Stockholm, Sweden.
authors.forename: Max
authors.initials: M
authors.lastname: Käller
authors.affiliation: Department of Medical Sciences, Molecular Medicine and Science for Life Laboratory, Uppsala University, 75144 Uppsala, Sweden.
authors.forename: Tomas
authors.initials: T
authors.lastname: Axelsson
authors.affiliation: Science for Life Laboratory, Cancer Proteomics Mass Spectrometry, Department of Oncology-Pathology, Karolinska Institutet, 17121 Stockholm, Sweden.
authors.forename: Janne
authors.initials: J
authors.lastname: Lehtiö
authors.affiliation: Estonian Genome Center, University of Tartu, Riia 23b, 51010 Tartu, Estonia.
authors.forename: Lili
authors.initials: L
authors.lastname: Milani
authors.affiliation: Section of Pharmacogenetics, Department of Physiology and Pharmacology, Karolinska Institutet, Nanna Svartz väg 2, 17177 Stockholm, Sweden.
authors.forename: Magnus
authors.initials: M
authors.lastname: Ingelman-Sundberg
mesh_terms: D044503:5-Methylcytosine / Q000031:analogs & derivatives* / Q000378:metabolism; D000328:Adult; D020029:Base Pairing; D001483:Base Sequence; D002853:Chromatography, Liquid; D018899:CpG Islands / Q000235:genetics; D004247:DNA / Q000378:metabolism; D005786:Gene Expression Regulation; D005796:Genes; D006801:Humans; D008099:Liver / Q000378:metabolism*; D013058:Mass Spectrometry; D015203:Reproducibility of Results; D017422:Sequence Analysis, DNA; D013447:Sulfites / Q000378:metabolism
chemical_list: D013447:Sulfites; C011865:5-hydroxymethylcytosine; D044503:5-Methylcytosine; D004247:DNA; C042345:hydrogen sulfite