Analyses of molecular events associated with reprogramming somatic nuclei to pluripotency are scarce. We previously reported the reprogramming of epithelial cells by extract of undifferentiated embryonal carcinoma (EC) cells. We now demonstrate reprogramming of DNA methylation and histone modifications on regulatory regions of the developmentally regulated OCT4 and NANOG genes by exposure of 293T cells to EC cell extract. OCT4 and NANOG are transcriptionally up-regulated and undergo mosaic cytosine-phosphate-guanosine demethylation. OCT4 demethylation occurs as early as week 1, is enhanced by week 2, and is most prominent in the proximal promoter and distal enhancer. Targeted OCT4 and NANOG demethylation does not occur in 293T extract-treated cells. Retinoic acid-mediated differentiation of reprogrammed cells elicits OCT4 promoter remethylation and transcriptional repression. Chromatin immunoprecipitation analyses of lysines K4, K9, and K27 of histone H3 on OCT4 and NANOG indicate that primary chromatin remodeling determinants are acetylation of H3K9 and demethylation of dimethylated H3K9. H3K4 remains di-and trimethylated. Demethylation of trimethylated H3K9 and H3K27 also occurs; however, trimethylation seems more stable than dimethylation. We conclude that a central epigenetic reprogramming event is relaxation of chromatin at loci associated with pluripotency to create a conformation compatible with transcriptional activation. INTRODUCTIONReprogramming of a differentiated somatic cell into a pluripotent cell may have applications in regenerative medicine, and as such, several approaches are being examined to produce embryonic stem (ES)-like cells. Nuclear transplantation into oocytes has demonstrated that functional nuclear reprogramming is possible, through the production of nuclear transfer ES cells (Cibelli et al., 1998;Munsie et al., 2000;Wakayama et al., 2001) and cloned animals (Wilmut et al., 2002;. Fusion of somatic cells with ES or embryonal carcinoma (EC) cells also elicits a reprogramming of the somatic genome within the hybrids, demonstrated by X chromosome reactivation (Tada et al., 2001), changes in gene expression profile, and acquisition of ES cell properties, including contribution to all germ layers in teratomas and in aggregation chimeras (Tada et al., 1997(Tada et al., , 2001Pells et al., 2002;Terada et al., 2002;Ying et al., 2002;Cowan et al., 2005). Recently, retroviral transduction and constitutive expression of four factors (Oct4, Sox2, Klf4, and c-Myc) was also shown to induce an ES cell-like behavior in mouse fibroblasts, similar to that reported by fusion with ES cells (Takahashi and Yamanaka, 2006). A fourth approach to reprogramming entails treatment of reversibly permeabilized somatic cells with an extract of another differentiated cell type or of undifferentiated, pluripotent ES or EC cells (Taranger et al., 2005). Notably, epithelial 293T cells treated with extract of undifferentiated human EC (NCCIT) cells induces expression of genes associated with pluripotency, such as OCT4...

Mesenchymal stem cells (MSCs) can differentiate into multiple mesodermal cell types in vitro; however, their differentiation capacity is influenced by their tissue of origin. To what extent epigenetic information on promoters of lineage-specification genes in human progenitors influences transcriptional activation and differentiation potential remains unclear. We produced bisulfite sequencing maps of DNA methylation in adipogenic, myogenic, and endothelial promoters in relation to gene expression and differentiation capacity, and unravel a similarity in DNA methylation profiles between MSCs isolated from human adipose tissue, bone marrow (BM), and muscle. This similarity is irrespective of promoter CpG content. Methylation patterns of MSCs are distinct from those of hematopoietic progenitor cells (HPCs), pluripotent human embryonic stem cells (hESCs), and multipotent hESC-derived mesenchymal cells (MCs). Moreover, in vitro MSC differentiation does not affect lineage-specific promoter methylation states, arguing that these methylation patterns in differentiated cells are already established at the progenitor stage. Further, we find a correlation between lineage-specific promoter hypermethylation and lack of differentiation capacity toward that lineage, but no relationship between weak promoter methylation and capacity of transcriptional activation or differentiation. Thus, only part of the restriction in differentiation capacity of tissue-specific stem cells is programmed by promoter DNA methylation: hypermethylation seems to constitute a barrier to differentiation, however, no or weak methylation has no predictive value for differentiation potential.

In opposition to terminally differentiated cells, stem cells can self-renew and give rise to multiple cell types. Embryonic stem cells retain the ability of the inner cell mass of blastocysts to differentiate into all cell types of the body and have acquired in culture unlimited self-renewal capacity. Somatic stem cells are found in many adult tissues, have an extensive but finite lifespan and can differentiate into a more restricted array of cell types. A growing body of evidence indicates that multi-lineage differentiation ability of stem cells can be defined by the potential for expression of lineage-specification genes. Gene expression, or as emphasized here, potential for gene expression, is largely controlled by epigenetic modifications of DNA and chromatin on genomic regulatory and coding regions. These modifications modulate chromatin organization not only on specific genes but also at the level of the whole nucleus; they can also affect timing of DNA replication. This review highlights how mechanisms by which genes are poised for transcription in undifferentiated stem cells are being uncovered through primarily the mapping of DNA methylation, histone modifications and transcription factor binding throughout the genome. The combinatorial association of epigenetic marks on developmentally regulated and lineage-specifying genes in undifferentiated cells seems to define a pluripotent state.

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