Topoisomerases are enzymes that solve topological problems arising from DNA templated processes such as replication, transcription, recombination and chromatin remodeling. The type II subfamily of Topoisomerases achieve this task by passing a region of duplex from the same or a different molecule through a double-stranded gap generated in DNA. Mammalian cells encode two isoforms of type II enzymes, Topoisomerase II alpha and beta, which have almost identical enzymatic properties in vitro. However, they show very different expression patterns and it is not known whether they differ in their distribution and action sites on the genome and in gene regulatory potential.We find that a switch in the expression from Topo II alpha to beta occurs during neuronal differentiation in vitro and in vivo. We propose to define the chromatin crosstalk of the two Topo II isoforms, alpha and beta, and its contribution to the transcriptome of embryonic stem cells and differentiated neurons respectively. We will combine our sophisticated in vitro mouse differentiation system with genome-wide identification of Topo II alpha binding sites in stem cells. These target sequences will be analyzed for any specific features using computational biology tools and related to the existing datasets of various epigenetic modifications and transcription (Tiwari et al. 2011, Nature Genetics). Using Topo II-specific inhibitors, we will identify Topo II alpha target genes that rely on its catalytic activity for their transcription state. We will next compare the Topo II alpha binding data from stem cells to our recently derived genomewide datasets of Topo II beta binding from neurons. Together, we aim to achieve an understanding of the target preference for the two Topo II isoforms, alpha and beta, with respect to sequence features, genomic regions, chromatin profile and transcription state and their dynamics as proliferating, pluripotent stem cells differentiate into postmitotic neurons.