Transcriptional regulation defines the identity of pluripotent and differentiated cells. DNA Topoisomerases are enzymes that catalyze the unlinking of the DNA strands by making transient DNA strand breaks and allowing the DNA to rotate around these breaks, a process essential for DNA replication, transcription, chromosome condensation and segregation (Champoux, 2001). A mammalian-specific distinct type of topoisomerase, topoisomerase II beta (TopIIβ), is only abundantly expressed in terminally differentiated cells and is required for neurogenesis (Yang et al, 2000). TopIIβ is proposed to act at the ‘potentiation’ level of transcription to make gene promoters amenable for transcription as a prerequisite for their subsequent development-specific activation. This function could be achieved by regulating higher order chromatin organization of target genomic regions and/or direct interaction with known partners such as condesin and PcG proteins (Lupo R. et al., 2001) and other epigenetic modifiers such as chromatin remodeling complexes. We propose to define the contribution of TopIIβ activity towards epigenetic regulation of neuronal differentiation. We will combine a sophisticated in vitro mouse differentiation system with genome-wide identification of TopIIβ target genes. These will be directly related to existing datasets on development-specific targets of DNA methylation and Polycomb marking (Mohn et al., 2008).To delineate how these local characteristics translate into spatial organization of chromosomal regions we will study genomewide networks of chromosomal interactions for selected target genes using the 4C methodology. This will be functionally complemented by analysis of mouse stem cells lacking topoisomerase II beta protein. We ultimately aim to generate a model of how topoisomerase II beta proteins contribute to epigenetic reprogramming and nuclear reorganization events that take place during cellular differentiation of pluripotent stem cells.