3. Results
3.3 Novel methods to quantify and map 5 hmC in genomic DNA
3.3.2 The 5hmC specific endonuclease PvuRtsI1 as a tool to profile genomic 5hmC patterns
To gain insights into the functional role(s) of the newly discovered “6th base”, it will be necessary to determine genomic 5hmC patterns. As described earlier, the genome of T4 bacteriophages contains exclusively 5hmC instead of cytosine residues, which are modified by α- and ß- glucosyltransferases. The switch of cytosine to 5hmC in the T4 genome is thought to have evolved as a protection system against the restriction enzymes of the host
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bacteria after infection. As a strategy to counter the phage`s measures, bacteria have evolved a system of restriction enzymes that specifically recognize modified cytosines. One of the bacteria enzymes, the endonuclease PvuRts1I has been demonstrated to cleave glucosylated 5hmC and its restriction activity was shown to be modulated by glucosylation in a complex way. PvuRts1I is encoded by a single gene found on the kanamycin resistance plasmid Rts1 which was originally isolated from Proteus vulgaris (Janosi et al., 1994).
Interestingly, the growth of 5hmC containing T- even bacteriophages, but not that of T-odd phages containing 5mC or λ- phages devoid of any modified base is restricted by bacteria carrying the Rts1 plasmid (Janosi et al., 1994). This suggests that PvuRts1I could be a useful tool to discriminate 5hmC from 5mC or unmodified cytosine.
To address this question, we purified recombinant PvuRts1I and showed that it selectively cleaves non- glucosylated 5hmC containing DNA with even higher efficiency than α- or ß-glucosylated DNA. We then determined the cleavage pattern of PvuRts1I by generating libraries of restriction fragments of the whole non- glucosylated T4 genome or a reference fragment produced from the same genome containing exclusively hdroxymethylated cytosine. Random sequencing of more than 100 clones from each library revealed a consensus sequence of hmCN11-12/N9-10G with a 2 nucleotide 3´- overhang. Furthermore, by comparing DNA substrates containing one single PvuRts1I consensus site (hmCN12/N10G) with either hmC or mC in symmetrical or asymmetrical configuration or unmodified C, we found that sites with symmetric hmC are the preferred substrates of PvuRts1I. These experiments were conducted by my colleague Aleksandra Szwagierczak and are published in (Szwagierczak et al., 2011).
All the experiments performed so far used T4 genome or artificial DNA substrates as templates. As a next step, we wanted to investigate whether PvuRts1I could be used as a tool to map 5hmC patterns in mammalian genomic DNA. All the following experiments were conducted by me, except for the radioactive measurements of 5hmC levels in DNA substrates, which were conducted by my colleague Aleksandra Szwagierczak.
To analyze the efficiency of PvuRts1I digestion for mammalian genomic DNA, we chose the upstream regulatory region III of the mouse nanog gene (Hattori et al., 2007). We selected this region because very recent data indicate that Tet1 binds to this region and keeps the nanog promoter in a hypomethylated, active state. Further evidence for this hypothesis comes from the observation that this region acquires CpG methylation upon knock down of Tet1 in ESCs (Ito et al., 2010). Hence, the upstream regulatory region of nanog represents a potential 5hmC containing sequence in ESCs.
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Firstly, we digested or mock- treated genomic DNA from wild type and TKO ESCs with PvuRts1I and used two different primer pairs (Primer 1 and Primer 2) to analyze the decrease in product after digestion compared to mock digested samples (Figure 34). The primers were chosen in nanog regions which according to Ito et al. show increased DNA methylation upon tet1 knock down in ESCs (Ito et al., 2010).
Figure 34. Amplification of PvuRts1I digested fragments does not reduce amount of PCR products.
Genomic DNA from wt or TKO ESCs was digested with PvuRts1I or mock treated and amplified with primers specific for the nanog locus by qPCR. A locus containing 5hmC and digested with PvuRts1I should result in fewer template for the qPCR. As TKO ESCs are devoid of any DNA methylation, genomic DNA from TKO serves as a negative control for the specificity of the digestion reaction. A) shows an outline of the strategy used to detect 5hmC in mammalian genomic DNA whereas B) describes the upstream regulatory region of the nanog locus and also marks the location of the primers used in the qPCR reaction. The results of the amplification of fragments treated or not treated are depicted in C). Shown is the technical error of one representative experiment and each sample was measured in triplicates in the reaction. Each mock treated sample for each analyzed region and cell line was set to 1 to calculate the change in product after digestion.
However, we could not detect a decreased resistance to PvuRts1I digestion in the two nanog promoter regions as qPCR amplification with both primer pairs did not lead to a reduction of products in the digested samples. This could be due to the low abundance of 5hmC in genomic DNA of ESCs which makes it technically challenging to detect slight differences in decreased amounts of PCR products. We then thought of a strategy to positively identify rare PvuRts1I digestion products. As digestion with PvuRts1I results in fragments with a two nucleotide 3´ overhang, we generated a linker with a random two nucleotide 3´overhang (Fig.
35A), which we ligated to the digested products. We then used nanog specific primers paired with a linker specific primer to amplify ligation products. Unfortunately even using this adapted protocol we were unable to detect any amplification products (data not shown). The lack of amplification products may be explained by an extremely rare occurrence of 5hmC at PvuRts1I cleavage sites of this locus, especially as PvuRts1I preferentially cleaves sites with symmetrical 5hmC configuration. In addition, it could be due to inefficient digestion with PvuRts1I or a combination of both explanations. In this respect it is important to note that a positive identification of 5hmC in the upstream regulatory region of the nanog locus has not
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been conclusively demonstrated for ESCs (Ito et al., 2010) and is still highly controversial as another group could not confirm the reduced nanog expression upon tet1 knock down in ESCs (Koh et al., 2011). Taken together, it is uncertain whether the nanog locus is modified by hydroxymethylation in ESCs and is therefore not suitable to establish the cut- ligation-amplification strategy to detect PvuRts1I digestion products.
Consequently, we decided to generate substrates with defined amounts of 5hmC to validate the PvuRts1I cut- ligation amplification protocol for the identification of 5hmC sites. We used primers specific for the region III of the nanog promoter to amplify fragments by PCR in the presence of increasing amounts of 5-hydroxymethyl- dCTP (Figure 35). The successful incorporation of proportional levels of 5hmC was confirmed by the ß- glucosylation assay.
Figure 35. Preparation of linker oligos and substrates with increasing 5hmC concentration.
A) Equal amounts of single stranded forward primer (For), forward primer containing a random two nucleotide 3´overhang (For-OH), reverse primer (Rev) and annealed oligo (Linker) were loaded on a 15 % non-denaturating polyacrylamide gel (PAGE) and stained with SYBR-Green. B) PCR products (867 bp) containing increasing 5hmC levels were generated by amplification of the proximal upstream regulatory region of the nanog locus (Region III) and the addition of 5-hydroxymethyl- dCTP and dCTP at appropriate ratios. Minus indicates negative control of the PCR reaction. (C) The random incorporation of 5hmC into the PCR fragments was confirmed by ß-glucosylation assay.
The PCR products with increasing, randomly distributed 5hmC sites were then digested with PvuRts1I, ligated to a linker with random two nucleotide overhangs to match PvuRts1I and ligation products were detected by PCR amplification using two distinct nanog specific primers (nanog P1 and P2) each paired with a linker specific primer. PCR products were analyzed on an agarose gel (Figure 35B) and randomly cloned and sequenced. Indeed, we could detect fragments with ends corresponding to the PvuRts1I cleavage pattern however only in products from high 5hmC content (10 %). While fragments containing 1 % 5hmC, which is the highest global 5hmC content to be reported in certain brain tissues, only show background signal (Fig. 36). Our results using the linker/ amplification strategy clearly suggest that a high local concentration of 5hmC facilitates the detection of digestion products by PvuRts1I.
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Figure 36. Identification of PvuRts1I digestion fragments of substrates with increasing 5hmC level.
A) Outline of cut-ligation-amplification strategy to identify PvuRts1I cleavage sites. After generation of PCR fragments with increasing 5hmC amount, fragments were digested with PvuRts1I and ligated to a linker. Two different nanog specific primers (P1 and P2) were used in combination with the linker specific primer to positively amplify PvuRts1I cleaved sites. B) Agarose gels of obtained PCR fragments indicate the presence of several products after PvuRts1I digestion. The percentage of 5hmC contained in the original substrates and the presence of the linker in the ligation reaction are depicted. NTC: no template control in the PCR reaction. C) Products from the PCR reaction (B) were randomly cloned and sequenced. In the table, the numbers of sequences containing ends corresponding to one of the PvuRts1I cleavage site and the site subtype are summarized. The asterisk indicates a sequence which is reported under two categories because it could not be unambiguously assigned to the consenus site hmCN12/N9G or hmCN11/N9G due to occurrence of consecutive C residues. Data were published in (Szwagierczak et al., 2011).
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