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Nucleic Acids Res. 2016 Aug 19;44(14):6756-69. doi: 10.1093/nar/gkw316. Epub 2016 Apr 29.

Single base resolution analysis of 5-hydroxymethylcytosine in 188 human genes: implications for hepatic gene expression.

Author information

1
Section of Pharmacogenetics, Department of Physiology and Pharmacology, Karolinska Institutet, Nanna Svartz väg 2, 17177 Stockholm, Sweden maxim.ivanov@ki.se.
2
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.
3
Section of Pharmacogenetics, Department of Physiology and Pharmacology, Karolinska Institutet, Nanna Svartz väg 2, 17177 Stockholm, Sweden.
4
Group of Pharmacoepigenetics, Department of Physiology and Pharmacology, Karolinska Institutet, Von Eulers väg 8 IV, 17177 Stockholm, Sweden.
5
Department of Biochemistry and Biophysics, Science for Life Laboratory, Stockholm University, 10691 Stockholm, Sweden.
6
Science for Life Laboratory, School of Biotechnology, Division of Gene Technology, Royal Institute of Technology, 17121 Stockholm, Sweden.
7
Department of Medical Sciences, Molecular Medicine and Science for Life Laboratory, Uppsala University, 75144 Uppsala, Sweden.
8
Science for Life Laboratory, Cancer Proteomics Mass Spectrometry, Department of Oncology-Pathology, Karolinska Institutet, 17121 Stockholm, Sweden.
9
Estonian Genome Center, University of Tartu, Riia 23b, 51010 Tartu, Estonia.

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.

PMID:
27131363
PMCID:
PMC5001587
DOI:
10.1093/nar/gkw316
[Indexed for MEDLINE]
Free PMC Article

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