Investigaciones en contextos educativos

A first step towards understanding the function of 5-hmC is to analyse in what quanti- ties 5-hmC occurs in di↵erent cell types and developmental stages. For this purpose we developed a sensitive enzymatic method which is based on the specific transfer of a radi- olabeled glucose to 5-hmC by -glucosyltransferase ( -gt). This enzyme is used by the T4 page to protect its genome, which contains exclusively hydroxymethylated cytosines, against bacterial restriction endonucleases. To assess whether transfer of [3H]glucose by the -gt to DNA is proportional to the hmC content, we prepared a series of standard DNA substrate samples by mixing corresponding proportions of two DNA fragment of same length, one having all cytosine residues replaced by 5-hmC and the other contain- ing no 5-hmC. With this standard we could show that the assay is linear over a range of several orders of magnitude and extremely sensitive with a detection limit of 0.025% 5-hmC/C [Szwagierczak et al., 2010].

A crucial step for understanding the role of 5-hmC in the epigenetic landscape was to analyse the dynamics of this modification during early embryonic development using em- bryoid bodies (EBs). Di↵erentiation into EBs is a well establishedin vitro system that recapitulates first steps of the preimplantation development [Li and Yurchenco, 2006]. Using our -gt assay we analyzed the 5-hmC content of genomic DNA from undi↵erenti- ated wildtype ESCs, four and eight day old EBs as well as DNA from ESCs lacking all 3 major DNA methyltransferases (Dnmt1, Dnmt3a and Dnmt3b; triple knockout (TKO)). Interstingly, we could observe that 5-hmC levels are highly dynamic during the first eight days of di↵erentiation. A sharp decrease in 5-hmC levels was evident after four days of EB culture but a substantial recovery was observed after additional 4 days of culture. Similar dynamics can be observed on the transcript levels of Tet1-3. While Tet1

Discussion 149 is expressed predominantly in ESCs, its transcript levels decrease drastically after four days of EB culture. Tet3 is expressed at very low levels in ESCs but its mRNA levels start to increase upon di↵erentiation with a dramatic increase between day four and day eight concomitant with the recovery of the 5-hmC level. As expected, due to the lack of DNA methylation no 5-hmC could be detected in TKO ESCs [Szwagierczak et al., 2010]. These findings point to the fact that Tet1 is the main Tet enzyme responsible for generation of 5-hmC in ESCs. In fact other studies could show that a knock-down of Tet1 in ESCs results in increased 5-mC levels at certain CpG islands and deregulation of gene expression of pluripotency-associated genes. Furthermore, Tet1-depletion in preim- planation embryos and ESCs leads to a bias towards di↵erentation into extra-embryonic tissues supporting a role for Tet1 in ESC maintenance and inner cell mass cell specifica- tion [Ito et al., 2010; Ficz et al., 2011; Pastor et al., 2011; Wu et al., 2011b,a; Xu et al., 2011]. Recent findings revealed that Tet1 is directly controlled by the pluripotency fac- tor Oct4, integrating 5-hmC and Tet1 into the pluripotency network (Koh et al., 2011). Moreover, a bioinformatic study which tried to identify a minimal set of pluripotency markers found Tet1 as the best candidate using three di↵erent independent methods [Scheubert et al., 2011].

Next we analysed the genomic 5-hmC levels and the expression of Tet enzymes in adult mouse tissues. We found that adult tissues vary greatly in their 5-hmC content with highest levels in the central nervous system. In contrast to ESCs, 5-hmC levels in adult tissues correlate with high levels of Tet3 and to a lower extend Tet2, a pattern similar to day eight EBs. Thus, most di↵erentiated tissues are characterised by very low levels of Tet1 and high levels of Tet3, while undi↵erentiated ESCs show the opposite pattern [Szwagierczak et al., 2010]. These findings further support the idea that Tet1 plays an important role in pluripotency. Interestingly, kidney represents an exception among the analysed tissues as it exhibits relatively high 5-hmC levels and Tet2 as the predominant transcript. This is in accordance to previous findings in Tet2 null mice where the only observable phenotype is a cellular defect in proximal convoluted tubules of the kidney [Tang et al., 2008].

Most interesting is the finding that 5-hmC levels are highest in the central nervous system (CNS). Cells of the CNS, in particular neurons, have to adapt to a very dy- namic environment of inter- and intracellular contacts and signalling pathways. The flexibility to respond to these di↵erent signals is achieved by a distinctive epigenetic plasticity. In this context, DNA methylation dynamics have been shown to be involved in activity-dependent gene regulation [Martinowich et al., 2003; Chen et al., 2003b; Ma

150 3.1. The role and function of 5-hmC and Tet enzymes in development and disease

et al., 2009], memory and learning [Miller and Sweatt, 2007; Day and Sweatt, 2010], and repeat-associated transcript expression [Muotri et al., 2010; Skene et al., 2010]. Hydrox- ylation of 5-mC to 5-hmC might present a mechanism by which these DNA methylation dynamics are regulated.

To further understand the function of 5-hmC in gene regulation it is important to be able to map its localisation in the genome. As 5-hmC is chemically and structurally very similar to 5-mC discrimination of these two modification presents a major challenge. The gold standard methodology for profiling of genomic 5-mC sites, bisulfite conversion, can- not discriminate 5-hmC from 5-mC and all available restriction endonucleases are either equally sensitive to mC and hmC or not sensitive to either [Huang et al., 2010; Jin et al., 2010; Nestor et al., 2010].

We found reports of an endonuclease named PvuRts1I which restriction activity in vivo was shown to be modulated by 5-hmC glucosylation in a complex fashion [Janosi et al., 1994]. However, as PvuRts1I was not purified, its activity has not been characterized in vitro. We could show that recombinant PvuRts1I selectively cleaves 5-hmC contain- ing DNA and determined its cleavage site. Furthermore, we found that the extent of PvuRts1I disgestion reflects the relative abundance of 5-hmC in genomic DNA from cerebellum and TKO ESCs [Szwagierczak et al., 2010]. Restriction of genomic DNA with PvuRts1I may be combined with PCR amplification for analysis of specific loci, with massive parallel sequencing or microarray hybridisation for genome-wide mapping. Due to its relatively complex and long recognition sequence cleavage sites occur in large distances from another, raising the argument that the extent of random breaks in ge- nomic DNA preparations would contribute very significant noise in deep sequencing and microarray applications. This drawback can be overcome if Pvurts1I cut fragments are enriched by linkers with specific 3’-overhangs.