Novel methods to quantify and map 5hmC

DNA methylation has always been considered a quite stable epigenetic modification, which once it is established, cannot be actively removed. The discovery of the “6th _{base” of the}

genome, 5- hydroxymethylcytosine (5hmC), together with the identification of the family of Tet proteins, changed this long believed paradigm and scientists started to focus on the biological function of this newly identified modification. To gain insights into the functional role of 5hmC, a first challenge is to develop new methods which can selectively detect 5hmC and discriminate it from the more abundant and structurally very similar 5mC (see also chapter 3.3) and hence allow the measurement of 5hmC levels in various cell lines and tissues.

To this aim we developed a novel method to quantify global 5hmC levels in genomic DNA. We sought to exploit the ß- glucosyltransferase of T4 bacteriophages, an enzyme known to modify 5hmC and that evolved as a defense mechanism in the struggle between prokaryotes and their viruses. Using radiolabeled glucose, we showed that 5hmC can be specifically labeled and by generating reference fragments with known, but varying 5hmC content, we verified that the incorporation of isotopically labeled glucose in genomic DNA occurs linear within a range of 0.25 %- 2 % 5hmC content. Thus, global 5hmC level can be specifically labeled and accurately quantified by comparison to a standard curve, which was measured in each assay (Szwagierczak et al., 2010).

First, we applied this assay to two different wildtype ESCs as well as during their in vitro differentiation to Embryoid Bodies (EBs) and also measured transcript levels of tet1-3 in the very same samples (Figure 33). We found that ESCs contain relatively high 5hmC levels (0.3 % 5hmC relative to total cytosine), which drastically drop during differentiation to EBs. Interestingly, tet1 transcript levels were prevalent in the undifferentiated state, but decreased during EB formation, suggesting that Tet1 is the main enzyme responsible for the generation of 5hmC in ESCs (Szwagierczak et al., 2010). These data are consistent with previous publications showing that tet1 is predominately expressed in ESCs, but declines during monolayer differentiation of ESCs upon removal of LIF (Tahiliani et al., 2009). Furthermore, increasing evidence points to a role of Tet1 and 5hmC in the regulation of pluripotency and developmental potential. Knock-down of tet1 in ESCs results not only in reduced 5hmC level with a concomitant increase in 5mC at selected loci, but also in the deregulation of pluripotency associated genes (Ito et al., 2010; Freudenberg et al., 2011). In line with this, it has been shown that tet1 depletion in pre- implantation embryos and ESCs leads to loss of pluripotency and skewed differentiation towards the trophoectodermal lineage (Ito et al.,

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2010; Koh et al., 2011). The finding that tet1 is directly regulated by the master regulator Oct4 also integrates Tet1 and 5hmC in the pluripotency network (Koh et al., 2011). However, several studies could not confirm the deregulation of pluripotency genes upon tet1 knock down in ESCs and found only modest reduction of global 5hmC level with a minor increase of 5mC (Koh et al., 2011; Williams et al., 2011). Additionally, genetic ablation of tet1 revealed viable and fertile mice (Dawlaty et al., 2011), raising uncertainty about the importance of Tet1 in ESC maintenance. These contradictory results emphasize the need for further experiments including the generation of inducible, conditional knock out mice, which would allow a more specific approach to investigate the function of Tet1 e.g. in certain tissues or at defined time points during development.

In contrast to the expression profile of tet1, we found that tet3 mRNA levels were very low in ESCs, but increased with differentiation and prolonged EB culture (Fig. 33). Tet2 transcript levels dropped during the first 4 days of EB differentiation, but the mRNA levels recovered to the levels initially found in ESCs after 4 more days of EB culture. Remarkably, the initial reduction of 5hmC after 4 days of differentiation was followed by a re- increase of global 5hmC level in 8 days old EBs. Therefore, our results suggest that the relatively high abundance of 5hmC in undifferentiated ESCs correlates with high expression levels of tet1 and to a lower extent, tet2. The partial recovery of genomic 5hmC in 8 days old EBs correlates with higher tet2 and tet3 transcript levels (Szwagierczak et al., 2010). Interestingly, the distinct expression profiles of tet1-3 are in line with a study showing that during reprogramming of fibroblasts, the initial high levels of tet3 transcripts substantially decrease during this process, whereas tet1 and tet2 transcript levels as well as global 5hmC content concomitantly increase (Koh et al., 2011).

We next analyzed genomic 5hmC as well as tet1-3 transcript levels in several adult mouse

tissues (Fig. 33). In line with previous reports (Kriaucionis and Heintz, 2009), 5hmC was the most abundant in brain tissues which correlated with high levels of tet3 and lower levels of tet2. In general, we found that all analyzed tissues typically contained high levels of tet3 but low levels of tet1, whereas undifferentiated ESCs are characterized by the exactly opposite expression pattern. However, kidney seems to be an exemption as we measured relatively high level of genomic 5hmC together with high tet2 levels (Szwagierczak et al., 2010). The prevalent expression of tet2 in kidney is consistent with reports showing that one of the phenotypes described in tet2-/- _{mice is a cellular defect in proximal convoluted tubules of the}

kidney (Tang et al., 2008).

In conclusion, our analysis revealed that genomic 5hmC can be detected not only in various brain regions, but also in other analyzed tissues like kidney and liver. Furthermore, we found

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that different amounts of global 5hmC level correlate with differential expression of tet1-3 genes (Figure 42).

Figure 42. Abundance of 5hmC in ESCs, EBs and various tissues correlates with the differential expression of tet1-3 genes.

To elucidate the biological significance of 5hmC in mammalian genomes, it is crucial to determine the distribution of the novel modification in genomic DNA: For this purpose we again exploited an enzyme from bacteria that was evolved as a strategy to counter the phage´s measures. The endonuclease PvuRts1I has been shown to cleave glucosylated

5hmC and in vivo studies found that T- even phages containing exclusively genomic 5hmC,

but not T- odd phages which contain 5mC or unmodified C, were selectively limited in their growth by the presence of a plasmid encoding PvuRts1I (see also chapter 3.3.2). We wondered whether PvuRts1I could be used as a tool to discriminate 5hmC from 5mC and unmodified C and therefore purified the enzyme and tested its activity in vitro (Szwagierczak et al., 2011).

Our analysis demonstrates that PvuRtsI1 selectively cleaves 5hmC containing DNA and revealed the consensus sequence hm_CN

11-12/N9-10Gwith a 2 nucleotide 3´-overhang as the

cleavage site of PvuRtsI1. We then wanted to use PvuRtsI1 to map 5hmC pattern in genomic DNA and, based on previous report, chose the upstream regulatory region of nanog as a potential region containing 5hmC (Ito et al., 2010). However, our attempt to measure the decrease of product after PvuRtsI1 digestion compared to mock digested samples did not reveal any difference in products between the two different samples (Fig. 34). Also, our devised strategy to positively identify rare digestion of PvuRtsI1 products by ligating linkers with random 2 nucleotide overhang in combination with PCR amplification using a linker specific primer paired with a nanog specific primer did not reveal any amplification products. In this context it is important to note that until today no positive identification of 5hmC at the

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suitability of this locus to establish the linker- amplification strategy also because it is still highly debated whether nanog is a target of Tet1 and hydroxylation at all (Koh et al., 2011). Nonetheless, the use of substrates with defined 5hmC amount showed that the cut and ligation strategy could in principal be used to map 5hmC patterns, although a high local concentration of 5hmC clearly facilitates the detection of digestion products by PvuRtsI1 (Fig. 36) (Szwagierczak et al., 2011). The presence of high local 5hmC concentrations does not seem very unlikely given that in the case of cerebellum, the measured 5hmC levels translate to approximately 40 % of all 5mCs being hydroxylated (Kriaucionis and Heintz, 2009). Furthermore, several studies performed global mapping of 5hmC in ESCs and revealed a non- linear distribution of this modification with specific enrichment of 5hmC within gene bodies (specifically at exons) and at transcriptional start sites and promoters (Ficz et al., 2011; Pastor et al., 2011; Williams et al., 2011; Wu et al., 2011a; Xu et al., 2011). Hence, the enrichment of the digested fragments using our cut/ligation strategy could be applied to generate libraries for massive parallel sequencing and/or microarray hybridizations for genome- wide mapping of 5hmC.

4.3.2 5hmC- an intermediate of demethylation or a stable epigenetic modification?

The discovery of 5hmC in mammalian genomes led to the formulation of two principal hypotheses about the biological role of the 6th _{base. As a potentially stable base, 5hmC itself}

might represent a novel epigenetic modification which alters chromatin structure and possibly influences the local transcriptional state. Alternatively, it has been suggested that 5hmC serves as an intermediate stage in the DNA demethylation pathway, although it is still debated whether the demethylation occurs actively and/or passively. Active DNA demethylation has been proposed to involve specific DNA repair mechanisms such as deamination by the cytidine deaminases AID/APOBEC leading to the conversion of 5hmC to 5 hydroxymethyluracil (5hmU), which would then be removed by enzymes of the BER pathway like Tdg or MBD4 (see also chapter 1.2.3 and Fig. 7) (Cortellino et al., 2011; Guo et al., 2011). Further evidence for 5hmC as an intermediate in active DNA demethylation came from studies showing that Tet enzymes can even further oxidize 5hmC to 5 formylcytosine (5fC) and 5 carboxylcytosine (5caC) (Ito et al., 2011). Interestingly, these two oxidation products are also recognized and cleaved by Tdg, offering another mechanism of active DNA demethylation (He et al., 2011; Maiti and Drohat, 2011). Alternatively, 5caC could be decarboxylated to unmodified C by a yet to be identified decarboxylase, which would offer a demethylation pathway without the involvement of the DNA repair machinery. However, it has also been suggested that 5hmC as well as both cytosine derivates are part of a passive demethylation pathway. In line with this, it has been shown that 5hmC, 5fC and 5caC become replication- dependent diluted in the paternal pronucleus in preimplantation embryos

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(Inoue and Zhang, 2011; Inoue et al., 2011). In support of a passive demethylation mechanism, it has been shown that 5hmC containing DNA cannot be methylated by Dnmt1 (Valinluck and Sowers, 2007). Based on these results, it is now widely accepted that 5hmC plays a role in DNA demethylation, however, additional function(s) of 5hmC as a stable epigenetic mark are discussed. Especially the high abundance of 5hmC in post- mitotic neurons suggests a function as a epigenetic mark, possibly by changing the local chromatin environment via the recruitment or displacement of proteins (Kriaucionis and Heintz, 2009). Evidence strengthening this hypothesis comes from the finding that the methylcytosine binding protein MeCP2, which is highly abundant in brain tissues, does not recognize 5hmC and therefore might prevent the establishment of repressive chromatin structures (Frauer et al., 2011). Conversely, MBD3 has been suggested as a first possible effector protein which selectively recognizes 5hmC. MBD3 recruitment was shown to be dependent on Tet1- catalyzed hydroxymethylation and suggests a mechanism of how possible effects of 5hmC could be translated within the cell. However, what biological consequences such a possible 5hmC signaling could have is still completely unknown (Yildirim et al., 2011).

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