Inducible expression of Oct-3/4 reveals synergy with Klf4 in targeting Cyclin A2 to enhance proliferation during early reprogramming

Abstract

During reprogramming of somatic cells, heightened proliferation is one of the earliest changes observed. While other early events such as mesenchymal-to-epithelial transition have been well studied, the mechanisms by which the cell cycle switches from a slow cycling state to a faster cycling state are still incompletely understood. To investigate the role of Oct-3/4 in this early feature of reprogramming, we created a 4-Hydroxytamoxifen dependent Oct-3/4 Estrogen Receptor fusion (OctER). We show that OctER can substitute for Oct-3/4 to reprogram mouse embryonic fibroblasts to induced pluripotent stem cells. While over-expression of OctER or Klf4 individually did not affect cell proliferation, in combination, these factors hasten the cell cycle, in a tamoxifen dose-dependent manner, supporting a key role for OctER. Oct-3/4 + Klf4 increased proliferation by enhancing expression of Cyclin A2. We verified occupancy of endogenous Oct-3/4 and Klf4 at bioinformatically identified binding sites in the Cyclin A2 promoter in mouse embryonic stem cells (mESC). Using inducible OctER along with Klf4, we show dose-dependent induction of Cyclin A2 promoter-reporter activity and mRNA levels. Taken together, our results provide further evidence of the interdependence of pluripotency and the rapid cell cycle seen in mESC, and identify CyclinA2 as a key early target.