Epigenetic programming in its main three manifestations - DMA methylation, genomic imprinting, and histone modification - has been known to influence many cellular and organismal processes, especially during growth and development. Data accumulate that loss of imprinting (LOI) plays a pivotal role in the genesis of cancer, especially in embryonal tumors arising in early childhood. However, the molecular machinery, including the DMA methyltransferase (Dnmt) complexes, the mechanistic basis of DMA methylation in chromatin, and targeting of these complexes to the sites of imprinting are not known. We suggest that alterations of these meta-stable DMA modifications are caused by defects in the catalytic activities or by mis-targeting of Dnmt complexes. In this grant proposal we want to unravel the molecular composition of Dnmt complexes allowing us to study the interrelation between these complexes and tumor development. To reach this, we want to purify the human Dnmtl complex from native tissue. The subunit composition would be analyzed by mass spectrometry analysis and antibodies would be generated against the complex subunits. Knowledge of the native complex composition will allow us to study the integrity and presence of Dnmt complexes in primary tumors. In this respect we will focus on Wilms tumor and hepatoblastoma, since these embryonal tumors generally display LOI of imprinted genes at the IGF2/H19 locus on chromosome 11p15.5. Furthermore, permanent Wilms tumor cell lines will be established to serve as a key model system for analyzing the function of cancer-associated complex subunits. By bringing together the clinical and molecular biology expertise to study the machines that are involved in the establishment of epigenetic marks at imprinted genes, we could obtain data that give insight in the molecular mechanisms driving epigenetic alterations in childhood tumors.
