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Molecular mechanisms leading to genome-wide DNA demethylation during epigenetic reprogramming

Final Report Summary - REPROGRAMMING (Molecular mechanisms leading to genome-wide DNA demethylation during epigenetic reprogramming)

The processes of epigenetic reprogramming and deoxyribonucleic acid (DNA) demethylation that occur in the developing mouse embryo are a highly topical and challenging topic and have been focus of intensive research activities over the past few years. The discovery of the molecular factors involved in DNA demethylation is projected to have considerable impact on the field of regenerative medicine as the re-acquisition of pluripotency in somatic cells through the generation of 'induced-pluripotent stem cells' (iPSCs) provides a promise of therapeutic solution for number of human diseases. However, inefficient remodelling of epigenetic information and in particular, inefficient removal of DNA methylation marks associated with somatic cell programme has proven to be a major obstacle in successful iPSCs generation. Our research area has thus very clear translational potential for the regenerative medicine field as the understanding of DNA demethylation pathway will not only enrich our general knowledge of developmental reprogramming, but also enhance our ability to manipulate cell fate in vitro.

The ambitious aim of this project was to gain deeper insights into the molecular mechanisms leading to genome-wide DNA demethylation that proceeds in the mouse zygote only a few hours after fertilisation. While the kinetics of the reprogramming that occurs in the course of normal development has been well documented, the initial steps triggering this process and how DNA methylation marks are removed in vivo remain elusive. Recently, the involvement of DNA repair mechanisms in DNA demethylation during epigenetic reprogramming has been reported. The presented project proposed to investigate the upstream events initiating epigenetic reprogramming by using both in vivo and in vitro approaches. We focused on recently discovered candidates, the TET1-3 (ten-eleven translocation) proteins which have been implicated in the hydroxylation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxycytosine (5CaC). Over the past two years, a number of in vivo as well as in vitro studies have suggested a potential role of 5hmC as an intermediate during epigenetic reprogramming in the mouse embryos (in zygotes as well as in primordial germ cells- PGCs). TET3 oxygenase is specifically expressed in mouse oocytes and interestingly, the paternal genome in the zygote rapidly loses 5mC signal and subsequently accumulates 5hmC. Consequently, it has been proposed that 5hmC is an intermediate in 5mC removal and that TET3 enzyme is necessary for DNA demethylation during this epigenetic reprogramming.

TET1-3 proteins belong to the family of 2-oxoglutarate (2OG) and iron-dependent dioxygenases; they have been initially discovered through their sequence homology with proteins responsible for the 'J base' DNA modification in trypanosomes. At the onset of the project, we first analysed the detailed kinetics of 5mC disappearance and 5hmC formation in the paternal pronucleus in the zygote by indirect immunofluorescence. While the 5mC signal is lost rapidly before the onset of replication (stage PN2-3), the 5hmC signal accumulates gradually only after 5mC is lost and reaches a maximum intensity once the paternal pronucleus is fully decondensed (PN4-5). The observed dynamics of DNA modification is thus not in favour of a model whereby 5mC is directly converted into 5hmC. To confirm this observation, we had decided to analyse the course of DNA demethylation during in vitro fertilisation in the presence of a small molecule inhibitor of dioxygenases. Using this approach we could show that neither DNA demethylation nor the activation of Base excision DNA repair pathway in the zygote is dependent on 5hmC appearance. All our findings are thus consistent with a role for 5hmC that is distinct from the initial zygotic DNA demethylation.

The second wave of DNA demethylation during normal development occurs in primordial germ cells (PGCs) that will eventually form the future gametes of the embryo. PGCs have to erase their epigenetic information including genomic imprints and install a new DNA methylation profile which will drive the expression of specific program (meiosis,...) and which will also be in accordance with the sex of the developing embryo. Previous results by Dr Hajkova demonstrated that, similar to zygotes, the base excision repair mechanism is involved in DNA demethylation occurring in PGCs. As a follow up of the mentioned study, this project has also proposed to investigate the factors involved in germline reprogramming. Of the TET1-3 enzymes, TET1 and TET2 are expressed in PGCs and have been proposed to be crucial for DNA demethylation, through the conversion of 5mC to 5hmC.

Currently, only a few methods are available to accurately detect and quantify DNA modification, and most of them require a significant amount of cells and/or are not able to distinguish between different types of modifications. Thanks to our partnership with Agilent Technologies (leader in mass spectrometry equipment production), we acquired a liquid chromatography coupled with a mass spectrometry system (LC/MS triple quadrupole QQQ6490) and are developing a highly sensitive method to accurately detect all oxidative derivatives of cytidines (5mC, 5hmC, fC and CaC). Preliminary results using this state-of-the-art system allowed us to analyse the DNA content of PGCs and to confirm that a drop of 5mC indeed occurs in PGCs between embryonic day E10.5 and E11.5 which is in agreement with the previously published data and further implicates the presence of an active mechanism of DNA demethylation.

Taken together these results will bring new insights in the field of epigenetic reprogramming and complement what has recently been published. The new LC/MS method will help to quantify DNA modifications in the embryos and will be further used in different biological systems which require accurate and highly sensitive detection method due to the limited amount of starting material. Our results will thus contribute to the comprehension of the biological phenomenon that occurs in the early embryo and in PGCs, and will translate into the field of regenerative medicine by improving the understanding and efficiency of the manipulation of cell fate in vitro.