Ectopic expression of the four transcription factors Oct4, Sox2, c-Myc, and Klf4 is sufficient to confer a pluripotent state upon the fibroblast genome, generating induced pluripotent stem (iPS) cells. It remains unknown if nuclear reprogramming induced by these four factors globally resets epigenetic differences between differentiated and pluripotent cells. Here, using novel selection approaches, we have generated iPS cells from fibroblasts to characterize their epigenetic state. Female iPS cells showed reactivation of a somatically silenced X chromosome and underwent random X inactivation upon differentiation. Genome-wide analysis of two key histone modifications indicated that iPS cells are highly similar to ES cells. Consistent with these observations, iPS cells gave rise to viable high-degree chimeras with contribution to the germline. These data show that transcription factor-induced reprogramming leads to the global reversion of the somatic epigenome into an ES-like state. Our results provide a paradigm for studying the epigenetic modifications that accompany nuclear reprogramming and suggest that abnormal epigenetic reprogramming does not pose a problem for the potential therapeutic applications of iPS cells.

Summary Induced pluripotent stem (iPS) cells can be obtained from fibroblasts upon expression of Oct4, Sox2, Klf4 and c-Myc. To understand how these factors induce pluripotency, we carried out genome-wide analyses of their promoter binding and expression in iPS and partially reprogrammed cells. We find that target genes of the four factors strongly overlap in iPS and embryonic stem (ES) cells. In partially reprogrammed cells, many genes co-occupied by c-Myc and any of the other three factors already show an ES-like binding and expression pattern. In contrast, genes that are specifically co-bound by Oct4, Sox2 and Klf4 in ES cells and encode pluripotency regulators severely lack binding and transcriptional activation. Among the four factors, c-Myc promotes the most ES cell-like transcription pattern when expressed individually in fibroblasts. These data uncover temporal and separable contributions of the four factors during the reprogramming process and indicate that ectopic c-Myc predominantly acts before pluripotency regulators are activated.

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Summary An open chromatin largely devoid of heterochromatin is a hallmark of stem cells, from Planarians to Mammals. It remains unknown whether an open chromatin is necessary for the differentiation potential of stem cells, and what are the molecules that maintain open chromatin in stem cells. Here we show that the chromatin remodeling factor Chd1 is required to maintain the open chromatin state of pluripotent mouse Embryonic Stem (ES) cells. Chd1 is a euchromatin protein that associates with the promoters of active genes, and down-regulation of Chd1 leads to accumulation of heterochromatin in ES cells. Chd1-deficient ES cells are no longer pluripotent, because they are incapable of giving rise to primitive endoderm and have a high propensity for neural differentiation. Furthermore, Chd1 is required for efficient reprogramming of fibroblasts to the pluripotent stem cell state. Our results indicate that Chd1 is essential for open chromatin and pluripotency of ES cells, and for somatic cell reprogramming to the pluripotent state. The data suggest that pluripotent stem cells exist in a dynamic state of opposing epigenetic influences of euchromatin and heterochromatin.