Sensitive enzymatic quantification of 5-hydroxymethylcytosine in genomic DNA

Abstract

The recent discovery of genomic 5-hydroxymethylcytosine (hmC) and mutations affecting the respective Tet hydroxylases in leukemia raises fundamental questions about this epigenetic modification. We present a sensitive method for fast quantification of genomic hmC based on specific transfer of radiolabeled glucose to hmC by a purified glucosyltransferase. We determined hmC levels in various adult tissues and differentiating embryonic stem cells and show a correlation with differential expression of tet genes.

Figure 1.

Elements of the hmC glucosylation assay. (A) Schematic representation of the hmC glucosylation reaction catalyzed by β-gt. (B) Coomassie blue stained gel showing the purified β-gt preparation. (C) Example of calibration curve using mixtures of hmC-containing and unmodified reference fragment (equal total DNA amounts). Note the linear relationship between [³H]glucose incorporation and percentage of hmC.

Figure 2.

Quantification of genomic hmC and Tet transcripts in mouse tissues, undifferentiated ESCs and EBs. (A and C) hmC glucosylation assays. The percentage of hmC per total cytosine was calculated from the incorporation of [³H]glucose using a calibration curve from the reference fragment (see Figure 1C). Shown are average values and error bars from two (A) or one (C) biological replicates, each measured in two independent assays, with the exception of hippocampus that was measured only once. In every assay, each sample was measured in duplicate. The dashed line in (A) indicates the estimated limit of detection. (B and D) Real-time RT–PCR analysis for Tet transcript levels. Expression levels are all relative to Tet1 in kidney (set to 1), so that values in b and d are directly comparable. Shown are average values and error bars from two (B) and one (D) biological replicates, each measured from two independent cDNA synthesis reactions. In every real-time PCR reaction, each sample was measured in triplicate. Genomic DNA and RNA samples used in A/C and B/C, respectively, were isolated from the very same cell and tissue lysates.