Oral Presentation Epigenetics 2013

Hydroxymethylcytosine (5hmC) in human liver: quantification, genome-wide mapping and profiling with single-base resolution in ADME genes (#20)

Maxim Ivanov 1 , Mart Kals 2 , Marina Kacevska 1 , Isabel Barragan 1 , Kie Kasuga 3 , Anders Rane 4 , Andres Metspalu 2 , Lili Milani 2 , Magnus Ingelman-Sundberg 1
  1. Dept. of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden
  2. Estonian Genome Center, University of Tartu, Tartu, Estonia
  3. Dept. of Oncology-Pathology, Karolinska Institutet, Stockholm, Sweden
  4. Dept. of Laboratory Medicine, Karolinska University Hospital at Huddinge, Stockholm, Sweden

Hydroxymethylcytosine is a recently re-discovered epigenetic DNA modification that is abundant in brain and ESCs and plays an important role in the control of gene expression. In this study we investigated the potential role of 5hmC in the human liver. First, we quantified 5hmC by LC-MS in 8 fetal and 7 adult liver samples and demonstrated that adult human liver is a strongly hydroxymethylated organ (0.2% to 1% of 5hmC from the total cytosine), whereas in all fetal samples the 5hmC content was below 0.125%. Immunohistostaining of liver sections with anti-5hmC antibody demonstrated that 5hmC is detected in the majority of hepatocytes.

Then, we enriched genomic DNA from these liver samples for their 5hmC-containing fractions and analyzed the genome-wide patterns of 5hmC by next-generation sequencing. The genomic distribution of 5hmC peaks showed significant interindividual variability in both fetal and adult cohorts. However, we found 29,917 genomic intervals in fetal livers and 116,991 – in adults, where 5hmC was reproducibly detected. Such “5hmC blocks” are strongly enriched in CGI shores, in hepatic enhancers and in miRNA genes. Moreover, they correlate with active mRNA transcription in both age cohorts, as well as with developmental changes in gene expression.

Traditional bisulfite sequencing do not discriminate between 5mC and 5hmC, however, recent methods (oxBS-Seq, TAB-Seq) allows to detect them separately from each other. Using a modified Agilent Methyl-SEQ protocol together with a custom SureSelect target enrichment library, we investigated 5mC and 5hmC patterns with single-base resolution in coding and regulatory regions of 192 ADME genes in 2 fetal and 7 adult liver samples. The data show that 5hmC can be highly abundant in some hepatic genes involved in drug metabolism.

Taken together, our findings suggest the importance of considering 5hmC when investigating human liver epigenome. The functional role of differentially hydroxymetylated regions (DhMRs) in determining the interindividual differences of hepatic gene expression needs to be further investigated in larger sample cohorts.