Topoisomerases resolve torsional stress, while their function in gene regulation, especially during cellular differentiation, remains unknown. Here we find that the expression of topo II isoforms, topoisomerase IIα and topoisomerase IIβ, is the characteristic of dividing and postmitotic tissues, respectively. In embryonic stem cells, topoisomerase IIα preferentially occupies active gene promoters. Topoisomerase IIα inhibition compromises genomic integrity, which results in epigenetic changes, altered kinetics of RNA Pol II at target promoters and misregulated gene expression. Common targets of topoisomerase IIα and topoisomerase IIβ are housekeeping genes, while unique targets are involved in proliferation/pluripotency and neurogenesis, respectively. Topoisomerase IIα targets exhibiting bivalent chromatin resolve upon differentiation, concomitant with their activation and occupancy by topoisomerase IIβ, features further observed for long genes. These long silent genes display accessible chromatin in embryonic stem cells that relies on topoisomerase IIα activity. These findings suggest that topoisomerase IIα not only contributes to stem-cell transcriptome regulation but also primes developmental genes for subsequent activation upon differentiation.