br Experimental Procedures br Author Contributions br Acknow

Experimental Procedures
Author Contributions
Acknowledgments The authors thank Drs. J. Toga and E. Yagi (Osaka University) for expression and purification of the recombinant LM511-E8 and Professor H. Ohmori (Kyoto University) for an electrophysiological study. We also thank Dr. E. Yamasaki, Mr. K. Kubota, Ms. A. Fuke, and Ms. Y. Konoshima (CiRA, Kyoto University) for their technical assistance. This study was supported by a grant from the Highway Project for Realization of Regenerative Medicine from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), a grant from the Network Program for Realization of Regenerative Medicine from the Japan Science and Technology Agency, and a grant from the Translational Research Network Program from the MEXT (to K.S.).
Introduction Somatic nae inhibitor can be reprogrammed into induced pluripotent stem cells (iPSCs) after the forced expression of three or four transcription factors: Oct4, Sox2, Klf4, and optionally, Myc (Nakagawa et al., 2008; Takahashi and Yamanaka, 2006). Oct4 is indispensable for establishing pluripotency in the embryo (Nichols et al., 1998) and for maintaining pluripotency in mouse embryonic stem cells (ESCs) (Niwa et al., 2000). Under physiological conditions, the OCT4 protein needs to interact with SOX2 for activating most of its target genes and for maintaining the self-renewal of ESCs (Boyer et al., 2005; Reményi et al., 2003; Rodda et al., 2005). Nevertheless, overexpression of Oct4 can rescue ESC self-renewal in the absence of Sox2 (Masui et al., 2007), indicating that Sox2 is not essential for supporting pluripotency. In addition, Sox2 can be replaced by other Sox factors or by transforming growth factor β inhibitors in the reprogramming of somatic cells into iPSCs (Ichida et al., 2009; Nakagawa et al., 2008). Likewise, Klf4 is dispensable for maintaining ESC self-renewal (Jiang et al., 2008) and for inducing pluripotency (Nakagawa et al., 2008), as Klf2 and Klf5 can replace Klf4 in both functions. In addition, Esrrb can also substitute Klf4 in iPSCs generation (Feng et al., 2009). Therefore, Oct4 is the only transcription factor in the conventional reprogramming cocktail that is essential for pluripotency. To date, the role of OCT4 in reprogramming has been studied only in the context of its interaction with SOX2 (Buganim et al., 2012; Hansson et al., 2012; Polo et al., 2012; Sridharan et al., 2009; Stadtfeld et al., 2008). As exogenous Sox2 is not required for inducing pluripotency (Ichida et al., 2009; Maherali and Hochedlinger, 2009), we decided to investigate the specific effect of OCT4 alone in the first steps of reprogramming. To circumvent the inevitable heterogeneity generated by viral factor delivery, we established a set of different somatic cell types from tetracycline-inducible Oct4 transgenic mice. This approach facilitates the study of rare events in cell populations that simultaneously activate Oct4. Finally, we performed global gene-expression profile analyses to identify the early transcriptional changes caused by ectopic Oct4 expression in somatic cells. Overall, our study provides insights into the specific OCT4-dependent events that promote the induction of pluripotency.
Results
Discussion In this study, we derived different Oct4-inducible somatic cell types to identify early transcriptional events induced by the exogenous OCT4 protein during cellular reprogramming. Our global gene-expression analyses revealed that OCT4 does not induce expression of pluripotency-related genes in somatic cells during the first 24 hr after its overexpression. On the contrary, many ESC-expressed genes are downregulated by OCT4 in somatic cells. In addition, our data demonstrate that OCT4 activates and inhibits different genes in a cell-type-dependent manner. The OCT4-upregulated genes are highly variable from one cell type to another and do not present any common ontological pattern. Some genes were upregulated in one cell type while being downregulated in other cell types. Thus, our results suggest that OCT4 alone can induce a general perturbation in the somatic cell transcriptional network at the early steps of reprogramming, partially through the downregulation of cell-type-specific genes. Indeed, OCT4 has been reported to not only activate pluripotent genes but also repress developmental regulators in ESCs (Boyer et al., 2005; Rodda et al., 2005). However, the sequences to which OCT4 is bound in ESCs do not maintain the same accessibility in different types of somatic cells (Koche et al., 2011; Soufi et al., 2012). Therefore, different cell-type-dependent chromatin signatures could explain the differences in gene expression between MEFs, NSCs, and BMCs after Oct4 induction.