T4 β-gt was expressed in bacteria as a 6 × His tag fusion and purified to homogeneity by sequential nickel-NTA, size exclusion and ion exchange chromatography (B). To assess whether transfer of [3
H]glucose to DNA is proportional to the hmC content within the range previously reported for mammalian tissues, we prepared a series of standard DNA substrate samples with global hmC content ranging from 0.25 to 2% of total cytosine by mixing corresponding proportions of two preparations of the same 1.2
kb DNA fragment, one having all cytosine residues replaced by hmC and the other containing no hmC (C). Using a 325-fold excess of unlabelled UDP-glucose, the incorporation of radiolabeled glucose in 1
µg of total DNA substrate was strictly linear in this range. This standard sample series was measured in every assay to generate a calibration curve for the calculation of hmC content in genomic DNA samples. We first measured genomic hmC levels in wild-type and Dnmt1, 3a and 3b triple knockout (TKO) J1 ESCs (15
) (A and B). Due to the absence of all three major DNA methyltransferases, genomic DNA from TKO ESCs is expected to contain very little, if any, cytosine methylation. Indeed, the measured level of genomic hmC in TKO ESCs was at the detection limit (0.025%) of our assay, while genomic DNA from wild-type ESCs contained 0.3% hmC relative to total cytosine. Real-time reverse transcription (RT) PCR analysis showed that Tet1–3 mRNA levels are similar in wild-type and TKO ESCs, with Tet1 transcripts largely preponderant and Tet3 mRNA the least abundant (more than 40-fold lower than Tet1). It was previously shown that differentiation of mouse ESCs by withdrawal of LIF from monolayer cultures for 5 days results in a reduction of genomic hmC and concomitant decrease in Tet1 transcripts (2
). We followed genomic hmC and Tet1–3 transcript dynamics during EB differentiation of two commonly used wild-type ESC lines (A and B and Supplementary Figure S1
). In both cases, a sharp decrease of hmC content was evident after 4 days of EB culture, but a substantial recovery was observed after additional 4 days of culture (Day 8). Interestingly, the tet
genes showed distinct expression dynamics during ESCs differentiation. Tet1 transcripts drastically decreased in the first 4 days and further dropped by Day 8 of EB culture. Tet2 mRNA levels also decreased in the first 4 days, but were fully restored in Day 8 EBs. In contrast, Tet3 transcripts doubled at Day 4 and increased about 20 times by Day 8 of EB culture. Thus, the relatively high hmC content in undifferentiated ESCs correlates with high levels of Tet1 and, to a lower extent, Tet2 transcripts, while the partial recovery of genomic hmC in Day 8 EBs correlates with increased Tet2 and Tet3 mRNA levels.
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 (more ...)
We then analyzed several adult mouse tissues (C and D). As reported earlier, the highest levels of genomic hmC were found in brain regions, although kidney also showed relatively high levels. In all cases, the hmC content was at least four times higher than the detection limit, while in a previous report using a different assay the same non-neural tissues were either marginally above or right at the detection limit (5
). Abundant hmC in brain tissues correlated with high levels of Tet3 and to a lower extent Tet2, a pattern similar to Day 8 EBs. Thus, most differentiated tissues are characterized by very low levels of Tet1 and high levels of Tet3, while undifferentiated ESCs show the opposite pattern. It will be interesting to determine whether all pluripotent cell types have this pattern and at which stages along the specification of the various somatic lineages the relative expression levels of tet
genes change. Interestingly, kidney represents an exception among the adult tissues analyzed as it shows relatively high hmC content and a prevalence of Tet2 transcripts. This is consistent with a cellular defect in proximal convoluted tubules of the kidney as the only phenotype described for Tet2 null mice (16
). These observations suggest that Tet proteins have redundant roles and that the lack of a specific Tet family member may result in phenotypic alterations only in tissues where high levels of that Tet enzyme cannot be compensated by the other family members. In this context, it should be noted that the assay described here could also be employed to measure the enzymatic activity of Tet proteins and their mutants identified in leukemia patients by using mC-containing DNA substrates.
In conclusion, we have established an accurate assay for the quantification of genomic hmC that: (i) is more sensitive than previously described methods; (ii) is not subject to sequence bias; (iii) allows simultaneous processing of large sample numbers; and (iv) does not require specialized and expensive equipment. It should be noted that lower concentrations of ‘cold’ UDP-glucose should allow scaling down the amount of substrate DNA without loss of signal. This assay will be highly useful to determine the global abundance of hmC in genomic DNA, especially in situations where limited amounts of tissue are available such as isolates of rare primary cell types and clinical samples.