In this article we searched for genes that

In this article, we searched for genes that are induced by KLF4 in a dose-dependent manner and identified Tcl1 as its direct target. KLF4 induces Tcl1 transcription during reprogramming via direct binding to its enhancer and promoter regions, concurrently changing repressive histone marks into active ones at the Tcl1 promoter. TCL1 induced by KLF4 activates AKT to enhance glycolysis and inhibits mitochondrial polynucleotide phosphorylase (PnPase) to suppress oxidative phosphorylation. Knockdown of Tcl1 expression not only eliminates the metabolic shift but severely compromises the expression of Cdh1 and Rex1 as well. Thus, KLF4-promoted metabolic shift concurs with the acquisition of pluripotency and is essential for generating fully reprogrammed iPSCs.
Results
Discussion We found here that Tcl1 is one of the important direct targets of KLF4 and KLF4-induced TCL1 directs a metabolic shift during reprogramming by both enhancing glycolysis and diminishing oxidative phosphorylation. Our analyses revealed that KLF4 regulates TCL1 expression at the level of transcription by directly binding the Tcl1 enhancer and promoter when glycyrrhetinic acid transition from iPSCs(Low-K) to iPSCs(High-K), simultaneously recruiting OCT4 to the Tcl1 promoter. The increased binding of KLF4 and OCT4 is accompanied by deposition of H3K4me3 at the Tcl1 promoter, which has already lost H3K27me3 and acquired H3K27Ac before the transition (Figures 4E and 4G). Given that the 3-fold increase of the KLF4 permits the deposition of H3K4me3, there may be a critical threshold for deposition of each histone mark. Moreover, because subsets of genes increase expression at a distinct stage of reprogramming (Nishimura et al., 2014), the critical threshold during reprogramming may differ for individual genes. Rapidly proliferating cells have a high demand for essential cell components, which is met by increased glycolysis that shunts the metabolic intermediates toward synthesis of building blocks for nucleic acids, lipids, and proteins (Vander Heiden et al., 2009). This is achieved by aerobic glycolysis, also known as the Warburg effect, which has recently been acknowledged as an important metabolic state in cancer cells (Courtnay et al., 2015), adult stem cells as well as ESCs (Ito and Suda, 2014). In cancer cells, central to aerobic glycolysis is the phosphatidylinositol 3-kinase-AKT-mammalian target of the rapamycin signaling pathway and the hypoxia-inducible factor 1α (HIF1α) pathway, which regulate cell proliferation, survival, and metabolism (Courtnay et al., 2015). In adult stem cells, the same pathways also play an essential role for maintaining aerobic glycolysis in hypoxic stem cell niches (Ito and Suda, 2014). ESCs also display aerobic glycolysis and reduced oxidative phosphorylation, and the AKT pathway is an important downstream target of leukemia inhibitory factor (LIF), which is essential for self-renewal and pluripotency of ESCs (Niwa et al., 2009). However, the role for the AKT pathway in the metabolic state of ESCs is less clear. Our results indicate that Tcl1 regulates AKT to enhance glycolysis and inhibits PnPase to diminish oxidative phosphorylation in fully reprogrammed iPSCs, and perhaps in ESCs as well. Although ESCs express a high level of Tcl1 (Miyazaki et al., 2013), its role as a metabolic regulator in ESCs remains undefined. Further contributing to this uncertainty is the fact that only a slight decrease in cell proliferation is observed in Tcl1(−/−) ESCs, without any change in the phosphorylated form of AKT (Miyazaki et al., 2013). Tcl1(−/−) embryos, however, show a more unambiguous phenotype, displaying a defect in early embryonic development (Narducci et al., 2002). Given that TCL1 is not the mainstream pathway that connects to AKT but merely modulates its activities, augmentation of the AKT activities via LIF in cell culture medium, for instance, may compensate for the lack of Tcl1 in ESCs cultured in vitro. Its requirement, however, may be more stringent in the developing embryo, where such signals could be less variable.