title: 5'-Hydroxymethylcytosine Precedes Loss of CpG Methylation in Enhancers and Genes Undergoing Activation in Cardiomyocyte Maturation.
abstract: BACKGROUND
  Cardiomyocytes undergo major changes in DNA methylation during maturation and transition to a non-proliferative state after birth. 5'-hydroxylation of methylated cytosines (5hmC) is not only involved in DNA loss of CpG methylation but is also thought to be an epigenetic mark with unique distribution and functions. Here, we sought to get insight into the dynamics of 5'-hydroxymethylcytosine in newborn and adult cardiomyocytes.
  
  
  METHODS
  Cardiomyocyte nuclei from newborn and adult C57BL/6 mice were purified by flow cytometric sorting. 5hmC-containing DNA was captured by selective chemical labeling, followed by deep sequencing. Sequencing reads of library replicates were mapped independently (n = 3 for newborn, n = 2 for adult mice) and merged for further analysis steps. 5hmC coverage was normalized to read length and the total number of mapped reads (RPKM). MethylC-Seq, ChIP-Seq and RNA-Seq data sets of newborn and adult cardiomyocytes served to elucidate specific features of 5hmC at gene bodies and around low methylated regions (LMRs) representing regulatory genomic regions with enhancer function.
  
  
  RESULTS
  163,544 and 315,220 5hmC peaks were identified in P1 and adult cardiomyocytes, respectively. Of these peaks, 66,641 were common between P1 and adult cardiomyocytes with more than 50% reciprocal overlap. P1 and adult 5hmC peaks were overrepresented in genic features such as exons, introns, 3'- and 5'-untranslated regions (UTRs), promotors and transcription end sites (TES). During cardiomyocyte maturation, 5hmC was found to be enriched at sites of subsequent DNA loss of CpG methylation such as gene bodies of upregulated genes (i.e. Atp2a2, Tnni3, Mb, Pdk4). Additionally, centers of postnatally established enhancers were premarked by 5hmC before DNA loss of CpG methylation.
  
  
  CONCLUSIONS
  Simultaneous analysis of 5hmC-Seq, MethylC-Seq, RNA-Seq and ChIP-Seq data at two defined time points of cardiomyocyte maturation demonstrates that 5hmC is positively associated with gene expression and decorates sites of subsequent DNA loss of CpG methylation.
authors.affiliation: Institute of Experimental and Clinical Pharmacology and Toxicology, Faculty of Medicine, University of Freiburg, Freiburg, Germany.
authors.forename: David K
authors.initials: DK
authors.lastname: Kranzhöfer
authors.affiliation: Institute of Experimental and Clinical Pharmacology and Toxicology, Faculty of Medicine, University of Freiburg, Freiburg, Germany.
authors.forename: Ralf
authors.initials: R
authors.lastname: Gilsbach
authors.affiliation: Bioinformatics Group, Department of Computer Science, University of Freiburg, Freiburg, Germany.
authors.forename: Björn A
authors.initials: BA
authors.lastname: Grüning
authors.affiliation: Bioinformatics Group, Department of Computer Science, University of Freiburg, Freiburg, Germany.
authors.forename: Rolf
authors.initials: R
authors.lastname: Backofen
authors.affiliation: Institute of Experimental and Clinical Pharmacology and Toxicology, Faculty of Medicine, University of Freiburg, Freiburg, Germany.|University Heart Center Freiburg • Bad Krozingen, Department for Cardiology und Angiology II, Bad Krozingen, Germany.
authors.forename: Thomas G
authors.initials: TG
authors.lastname: Nührenberg
authors.affiliation: Institute of Experimental and Clinical Pharmacology and Toxicology, Faculty of Medicine, University of Freiburg, Freiburg, Germany.|BIOSS Centre for Biological Signaling Studies, University of Freiburg, Freiburg, Germany.
authors.forename: Lutz
authors.initials: L
authors.lastname: Hein
mesh_terms: D044503:5-Methylcytosine / Q000031:analogs & derivatives* / Q000378:metabolism; D000818:Animals; D000831:Animals, Newborn; D002454:Cell Differentiation / Q000235:genetics*; D002467:Cell Nucleus / Q000378:metabolism; D018899:CpG Islands / Q000235:genetics*; D019175:DNA Methylation / Q000235:genetics*; D021901:DNA, Intergenic / Q000235:genetics; D004742:Enhancer Elements, Genetic / Q000235:genetics*; D005434:Flow Cytometry; D056426:Genetic Loci; D016678:Genome; D008297:Male; D008810:Mice, Inbred C57BL; D032383:Myocytes, Cardiac / Q000166:cytology* / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D014158:Transcription, Genetic; D015533:Transcriptional Activation
chemical_list: D021901:DNA, Intergenic; C011865:5-hydroxymethylcytosine; D044503:5-Methylcytosine