The epigenetic landscape changes dynamically during differentiation

As the in vitro differentiation of ESCs recapitulates early embryonic developmental processes it is widely used as a powerful tool to study epigenetic processes during early embryogenesis and lineage commitment. Already shortly after fertilization, the developing zygote undergoes major epigenetic reprogramming events. The male nucleus is extensively remodeled,

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involving replacement of protamines with histones and global paternal- specific active DNA demethylation except for imprinted regions and repetitive sequences (see also chapter 1.1.3). Shortly afterwards, the maternal nucleus becomes passively demethylated as a consequence of the active exclusion of Dnmt1 from the nucleus (Carlson et al., 1992; Cardoso and Leonhardt, 1999). Around this time, the expression of pluripotency associated genes begins which are enriched in histone H3K4 trimethylation whereas developmental genes are kept silenced by the PcG repressive system. During differentiation, pluripotency genes are then silenced and at the same time, the expression of developmental genes is induced together with an increase of trimethylation of histone H3K4. Remarkably, a second wave of global DNA demethylation occurs around day 7 with the establishment of PGCs which includes reactivation of the X- chromosome and erasure of imprinted genes. Concordantly, somatic differentiation programs are repressed and the pluripotency potential is reacquired. During this time, pluripotent embryonic germ cells (EGCs) can be derived from PGCs under appropriate culture conditions (summarized in Orkin and Hochedlinger, 2011).

1.3.4.1 Role of DNA methylation during development

DNA methylation has been shown to play an important role in silencing pluripotency genes. Especially the multistep cascade for silencing of oct4 during differentiation is well characterized and involves loss of the nucleosome- depleted regions at the distal enhancer, setting of repressive histone marks H3K9me3 by the histone methyltransferase G9a and subsequent recruitment of Hp1 which then is followed by de novo methylation of the promoter via Dnmt3a and Dnmt3b (Feldman et al., 2006; Li et al., 2007b; You et al., 2011). Besides pluripotency genes, also germ line specific genes and clusters like the protocadherin gene family as well as the reproductive homeobox X- linked (rhox) gene cluster have been identified as targets of DNA methylation (Oda et al., 2006; Illingworth et al., 2008; Borgel et al., 2010). Furthermore, DNA methylation seems to play a crucial role during lineage commitment as hypomethlyated embryos die early during development (Li et al., 1992, see also chapter 1.2.1). How de novo DNA methylation contributes to lineage commitment, is not yet well understood and several controversial studies about the developmental potential of hypomethylated cells exist. Results by Panning and Jaenisch suggest that differentiation of dnmt1-/- _{ESCs leads to apoptosis whereas Jackson and colleagues show that dnmt1}-/- _ESCs

stay viable upon LIF removal, but fail to initiate differentiation (Panning and Jaenisch, 1996; Jackson et al., 2004). Furthermore, dnmt1-/- _{EBs were reported to express high levels of}

trophoblast markers and in contrast to wild type (wt) EBs can be differentiated into trophoblast stem cells under appropriate culture conditions (Ng et al., 2008). Similarly, it has been shown that TKO ESCs exhibit a growth defect and increased apoptosis upon EB differentiation. In addition, chimeric mouse embryos derived by aggregation of wt and TKO

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embryos show that TKO derived cells mostly contributed to extraeembryonic tissues but also to a small extent to the embryo proper at least detectable until an early postgastrulation stage (day 8.5) (Tsumura et al., 2006; Sakaue et al., 2010).

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1.4 Aims of the work

In mammals, DNA methylation plays important roles in the epigenetic control of gene expression during development and differentiation and it is crucial for maintaining genomic stability. The functional significance of DNA methylation has been mainly inferred from genomic methylcytosine profiles in a limited selection of cell types and developmental stages and very little is known about how DNA methylation and Dnmts actually affect transcription programs during cellular differentiation. Based on the controversial reports about the developmental potential of hypomethylated cells (see previous chapter), it is still not clear to what extent hypomethylated cells are able to commit to and progress along embryonic lineages.

One of the main objectives of this thesis was to elucidate the role of DNA methylation and Dnmts during differentiation. To this aim I used wild type (wt) ESCs, ESCs lacking the maintenance Dnmt (dnmt1-/- _{ESCs) as well as ESCs lacking all three major Dnmts (dnmt1}-/-_;

dnmt3a-/-_{; dnmt3b}-/-_{, or TKO ESCs) and generated their corresponding Embryoid Bodies}

(EBs). Using this differentiation system, I analyzed the potential of hypomethylated ESCs to silence pluripotency genes, studied their developmental potential and investigated whether differentiated EBs from all three cell lines can revert back to the undifferentiated state under appropriate culture conditions (chapter 3.1).

Recent reports identified the multi-domain protein Uhrf1 as an essential co- factor for maintenance DNA methylation. The second member of the Uhrf family, Uhrf2, is structurally very similar, but very little is known about its biological function(s). To gain first insights into the function of Uhrf2, I analyzed expression patterns of uhrf1 and uhrf2 in various cellular contexts and investigated whether Uhrf2 plays a role in maintenance DNA methylation (chapter 3.2).

The discovery of the “6th _{base” of the genome, 5hmC, a potential intermediate in DNA}

demethylation, and the Tet1-3 enzymes, which have been shown to be responsible for oxidation of 5mC to 5hmC, raised fundamental questions about the biological relevance of this newly identified modification. To advance understanding of the functions(s) of 5hmC and Tets, I analyzed expression levels of tet1-3 in ESCs, during differentiation and in different tissues and used a newly identified, 5hmC specific endonuclease to map 5hmC levels in genomic DNA (chapter 3.3).

Finally, I aimed at analyzing whether designer transcription activator- like effectors (dTALEs) can be used for activation of target promoters, whether the epigenetic state of a target promoter sequence interferes with the action of dTALEs and how this interference can be overcome (chapter 3.4).

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