ABSTRACT: Differentiated cells can be reprogrammed into induced pluripotent stem cells (iPSCs) following the forced overexpression of a specific combination of transcription factors, of which Oct4 is essential. To better understand the mechanism underlying iPSC reprogramming, we investigated the immediate transcriptional response to ectopic Oct4 expression in somatic cells. To this end, we derived different somatic cell types from Oct4-inducible transgenic mice and induced Oct4 expression for different time periods. Using microarray analysis, we have identified early transcriptional Oct4 targets, including genes not previously known to interact with Oct4. Comparison of the sets of Oct4-regulated genes revealed pronounced differences among the individual cell types. Furthermore, a notable up-regulation of pluripotent markers could not be detected. In contrast, we observed a down-regulation of somatic markers specific to each cellular population. This finding suggests that Oct4 interferes with the somatic transcriptional program in a cell type-specific manner, thereby promoting an unstable epigenetic state that facilitates reprogramming rather than the direct initiation of a pluripotent gene expression network. Finally, we show that Mgarp, a gene expressed in pluripotent cells, is commonly up-regulated in all cell types after Oct4 induction. Unexpectedly, Mgarp expression decreases during the first steps of reprogramming due to a Klf4-dependent inhibition. Our data show that Oct4 counteracts Klf4 repressive activity and enhances Mgarp expression at the late stages of reprogramming. This temporal pattern of Mgarp expression is crucial for the efficient generation of iPSCs. In summary, our study provides new insights into the specific role that Oct4 plays during the early stages of reprogramming. Different somatic cell types were derived from transgenic mice containing both a tetracycline-inducible transactivator (rtTA-M2) and a tetracycline operon-controlled Oct4 expression cassette (TO) (Hochedlinger 2005, Cell 121:465-477) that were mated with GOF18-GFP mice in which the green fluorescent protein (GFP) is driven by 18 kb of the Oct4 regulatory region (Oct4-GFP) (Yoshimizu 1999, Dev Growth Differ 41:675-684), thus indicating endogenous Oct4 expression. In addition, a control neural stem cell (CtrlNSC) line was generated from transgenic mice that entail a tetracycline-inducible transactivator (irtTA-VP16-GBD) (Anastassiadis 2002, Gene 298:159-172) plus an OG2 Oct4-GFP reporter transgene (Yoshimizu 1999),but lack a TO cassette. A second control consisted of wild type (C57BL/6) mouse embryonic fibroblasts (CtrlMEF). Oct4-inducible mouse embryonic fibroblasts (TO-MEFs) and neural stem cell (TO-NSCs) were derived from 14.5 days post coitum (d.p.c.) embryos and cultured as previously described (Conti 2005, PLoS Biol 3:e283). Primary bone marrow cells (TO-BMCs) were isolated from 6-week old mice after flushing femora and tibiae and plated onto dishes with a combined coating of gelatin (PAA, Pasching, Austria), laminin (Santa Cruz Biotechnology, Santa Cruz, CA), and poly-L-lysine (Sigma, St. Louis, MO). Adherent cells were then cultivated in DMEM Low Glucose (Gibco, Carlsbad, CA) with 10% fetal bovine serum, 100 U/ml penicillin, 100 µg/ml streptomycin, and 2 mM/L glutamine (all PAA). The transgenic Oct4 expression was induced by either 6 µg/ml doxycycline (Sigma) or by 2 µg/ml doxycycline (Dox) plus 10–7 M dexamethasone (Sigma), depending on the respectively contained tetracycline transactivator. 5-azacytidine (10 nM, Sigma) and cycloheximide (30 µg/ml, Sigma) were applied as indicated. Generation, culture, and differentiation of ESCs and iPSCs were performed as previously described (Tiemann 2011, Cytometry A 79:426-435). Microarray Gene Expression Analysis Samples from Oct4-inducible cell types and controls were collected at different time points (0, 6, 12, and 24 hours after induction). Biotin-labeled cRNA was prepared with the linear TotalPrep RNA Amplification Kit (Ambion, Austin, TX) from 500 ng original RNA and hybridized onto MouseRef 8 v2.0 Expression BeadChips (Illumina, San Diego, CA). The chips were stained with streptavidin-Cy3 (GE Healthcare, Little Chalfont, UK) and scanned using the iScan reader (Illumina). Bead intensities were mapped to gene information using BeadStudio 3.2 (Illumina) and background correction was performed using the Affymetrix robust multiarray analysis model. Variance stabilization was performed by log2 scaling and the R-Bioconductor lumi package was used for normalization. 17 samples were analyzed. ESC: Mouse Embryonic Stem Cell (ESC) TO_MEF_0: Tetracycline Oct4 (TO) Mouse Embryonic Fibroblast (MEF), time 0h TO_MEF-6: Tetracycline Oct4 (TO) Mouse Embryonic Fibroblast (MEF), time 6h TO_MEF-12: Tetracycline Oct4 (TO) Mouse Embryonic Fibroblast (MEF), time 12h TO_MEF-24: Tetracycline Oct4 (TO) Mouse Embryonic Fibroblast (MEF), time 24h TO_BMC-0: Tetracycline Oct4 (TO) Mouse Bone Marrow Cell (BMC), time 0h TO_BMC-6: Tetracycline Oct4 (TO) Mouse Bone Marrow Cell (BMC), time 6h TO_BMC-12: Tetracycline Oct4 (TO) Mouse Bone Marrow Cell (BMC), time 12h TO_BMC-24: Tetracycline Oct4 (TO) Mouse Bone Marrow Cell (BMC), time 24h TO_NSC-0: Tetracycline Oct4 (TO) Mouse Neural Stem Cell (NSC), time 0h TO_NSC-6: Tetracycline Oct4 (TO) Mouse Neural Stem Cell (NSC), time 6h TO_NSC-12: Tetracycline Oct4 (TO) Mouse Neural Stem Cell (NSC), time 12h TO_NSC-24: Tetracycline Oct4 (TO) Mouse Neural Stem Cell (NSC), time 24h Ctrl_MEF_0: Control wild type Mouse Embryonic Fibroblast (MEF) untreated Ctrl_MEF_24: Control wild type Mouse Embryonic Fibroblast (MEF) 24h treated Ctrl_NSC_0: Control wild type Mouse Neural Stem Cell (NSC) untreated Ctrl_NSC_24: Control wild type Mouse Neural Stem Cell (NSC) 24h treated
