DNA demethylation reprogramming in the mouse zygote

• Applicant: Professor Dr. Jörn E. Walter
• Subject Area: Developmental Biology ; General Genetics and Functional Genome Biology
• Term: from 2008 to 2015
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 66352472

Epigenetic reprogramming in the zygote is essential for the development of cellular pluripotency (stem cells) in the early embryo. Reprogramming factors of the oocyte restructure both parental genomes involving extensive histone exchange, changes in histone modifications and in DNA-methylation. The paternal set of chromosomes undergoes the most dramatic alterations. Its 5-methylcytosine content (5mC) is rapidly and actively removed. Our findings from the recent application period show that this DNA reprogramming starts in the prereplicative phase and extends into the S-phase. We further reported that this active DNA-demethylation is linked to DNA repair (most likely BER). The preferential (but not exclusive) demethylation in the paternal pronucleus is accompanied by a conversion of 5mC into 5-hydroxy-methyl-cytosine (5hmC). Tet dioxygenases are most likely responsible for this modification. The discovery of 5hmC in zygotes offers a completely new perspective to understand the molecular principles of DNA-methylation reprogramming. In the next funding period we will investigate the functional role of 5hmC on active and passive demethylation and its link to repair processes and chromatin modifications. We will use established embryo manipulation technologies (microinjection, IVF, IF) to induce epigenetic phenotypes by knocking down (RNAi) or overexpressing (mRNA/protein) candidate enzymes (Tets, MDBs, Glykosylases) in zygotes and early embryos. The primary goal of our experiments is to decipher the relationship between repair and modifying factors, replication and epigenetic dynamics and to identify the targets of active and passive demethylation. Towards the latter goal we will monitor the developmental changes in overall DNA-methylation by genome wide bisulphite deep sequencing strategies in zygotes/early embryos.

• DFG Programme: Priority Programmes
• Subproject of: SPP 1356: Pluripotency and Cellular Reprogramming