Stress responses in the adult rat are programmed early in life by maternal care and associated with epigenomic marking of the hippocampal exon 1 7 glucocorticoid receptor (GR) promoter. To examine whether such epigenetic programming is reversible in adult life, we centrally infused the adult offspring with the essential amino acid L-methionine, a precursor to S-adenosyl-methionine that serves as the donor of methyl groups for DNA methylation. Here we report that methionine infusion reverses the effect of maternal behavior on DNA methylation, nerve growth factor-inducible protein-A binding to the exon 1 7 promoter, GR expression, and hypothalamic-pituitaryadrenal and behavioral responses to stress, suggesting a causal relationship among epigenomic state, GR expression, and stress responses in the adult offspring. These results demonstrate that, despite the inherent stability of the epigenomic marks established early in life through behavioral programming, they are potentially reversible in the adult brain.

Maternal care alters epigenetic programming of glucocorticoid receptor (GR) gene expression in the hippocampus, and increased postnatal maternal licking/grooming (LG) behavior enhances nerve growth factor-inducible protein A (NGFI-A) transcription factor binding to the exon 1 7 GR promoter within the hippocampus of the offspring. We tested the hypothesis that NGFI-A binding to the exon 1 7 GR promoter sequence marks this sequence for histone acetylation and DNA demethylation and that such epigenetic alterations subsequently influence NGFI-A binding and GR transcription. We report that (1) NGFI-A binding to its consensus sequence is inhibited by DNA methylation, (2) NGFI-A induces the activity of exon 1 7 GR promoter in a transient reporter assay, (3) DNA methylation inhibits exon 1 7 GR promoter activity, and (4) whereas NGFI-A interaction with the methylated exon 1 7 GR promoter is reduced, NGFI-A overexpression induces histone acetylation, DNA demethylation, and activation of the exon 1 7 GR promoter in transient transfection assays. Site-directed mutagenesis assays demonstrate that NGFI-A binding to the exon 1 7 GR promoter is required for such epigenetic reprogramming. In vivo, enhanced maternal LG is associated with increased NGFI-A binding to the exon 1 7 GR promoter in the hippocampus of pups, and NGFI-A-bound exon 1 7 GR promoter is unmethylated compared with unbound exon 1 7 GR promoter. Knockdown experiments of NGFI-A in hippocampal primary cell culture show that NGFI-A is required for serotonin-induced DNA demethylation and increased exon 1 7 GR promoter expression. The data are consistent with the hypothesis that NGFI-A participates in epigenetic programming of GR expression.

Human induced pluripotent stem (iPS) cells may represent the ideal cell source for research and applications in regenerative medicine. However, standard culture conditions that depend on the use of undefined substrates and xenogeneic medium components represent a significant obstacle to clinical translation. Recently, we reported a defined culture system for human embryonic stem (ES) cells using a fully defined synthetic polymer coating, poly[2-(methacryloyloxy)ethyl dimethyl-(3-sulfopropyl)ammonium hydroxide] (PMEDSAH), in conjunction with xeno-free and defined culture medium. Here we tested the hypothesis that iPS cells grown in this defined culture system can be differentiated into mesenchymal stem cells (iPS-MSCs). Human iPS cells were cultured on PMEDSAH and differentiated into functional MSCs, as confirmed by expression of characteristic MSC markers (CD166+, CD105+, CD73+, CD44+, CD34− and CD45−) and their ability to differentiate in vitro into adipogenic, chondrogenic and osteoblastic lineages. To demonstrate the potential of iPS-MSCs to regenerate bone in vivo, the newly derived cells were induced to osteoblast differentiation for 4 days and transplanted into calvaria defects in immoncompromised mice for 8 weeks. MicroCT analysis and histology demonstrated de novo bone formation in the calvaria defects for animals treated with iPS-MSCs, but not for the control group. Moreover, positive staining for human nuclear antigen and human mitochondria monoclonal antibodies unambiguously confirmed the participation of the transplanted human iPS-MSCs in the regenerated bone. These results confirmed that human iPS cells grown in a defined and xeno-free system have the capability to differentiate into functional MSCs with the ability to form bone in vivo.

To enhance the understanding of differentiation patterns and bone formation capacity of hESCs, we determined (1) the temporal pattern of osteoblastic differentiation of human embryonic stem cell-derived mesenchymal stem cells (hESCs-MSCs), (2) the influence of a three-dimensional matrix on the osteogenic differentiation of hESCs-MSCs in long term culture and (3) the bone forming capacity of osteoblast-like cells derived from hESCs-MSCs in calvarial defects. Incubation of hESCs-MSCs in osteogenic medium induced osteoblastic differentiation of hESCs-MSCs into mature osteoblasts in a similar chronological pattern to human bone marrow stromal cells and primary osteoblasts. Osteogenic differentiation was enhanced by culturing the cells on three-dimensional collagen scaffolds. Fluorescent activated cell sorting of alkaline phosphatase expressing cells was used to obtain an enriched osteogenic cell population for in vivo transplantation. The identification of green fluorescence protein and expression of human specific nuclear antigen in osteocytes in newly formed bone verified the role of transplanted human cells in the bone regeneration process. The current cell culture model and osteogenic cell enrichment method could provide large numbers of osteoprogenitor cells for analysis of differentiation patterns and cell transplantation to regenerate skeletal defects.

The staggering array of nanotechnological products, found in our environment and those applicable in medicine, has stimulated a growing interest in examining their long-term impact on genetic and epigenetic processes. We examined here the epigenomic and genotoxic response to cadmium telluride quantum dots (QDs) in human breast carcinoma cells. QD treatment induced global hypoacetylation implying a global epigenomic response. The ubiquitous responder to genotoxic stress, p53, was activated by QD challenge resulting in translocation of p53, with subsequent upregulation of downstream targets Puma and Noxa. Consequential decrease in cell viability was in part prevented by the p53 inhibitor pifithrin-alpha, suggesting that p53 translocation contributes to QD-induced cytotoxicity. These findings suggest three levels of nanoparticle-induced cellular changes: non-genomic, genomic and epigenetic. Epigenetic changes may have long-term effects on gene expression programming long after the initial signal has been removed, and if these changes remain undetected, it could lead to long-term untoward effects in biological systems. These studies suggest that aside from genotoxic effects, nanoparticles could cause more subtle epigenetic changes which merit thorough examination of environmental nanoparticles and novel candidate nanomaterials for medical applications.

Human embryonic stem cells (hESCs) hold promise for tissue regeneration therapies by providing a potentially unlimited source of cells capable of undergoing differentiation into specified cell types. Several preclinical studies and a few clinical studies use human bone marrow stromal cells (hBMSCs) to treat skeletal diseases and repair damaged tissue. However, hBMSCs have limited proliferation and differentiation capacity, suggesting that an alternate cell source is desirable, and hESCs may serve this purpose. Here we describe a protocol for the reproducible derivation of mesenchymal stem cells from hESCs (hES-MSCs). The hES-MSCs have a similar immunophenotype to hBMSCs, specifically they are CD73+, STRO-1+ and CD45–, and are karyotypically stable. The derived hES-MSCs are also capable of differentiating into osteoblasts and adipocytes. When the hES-MSCs were genetically modified with the lineage-specific Col2.3-GFP lentivirus and cultured in osteogenic medium, increased GFP expression was detected over time, indicating the hES-MSCs have the capacity to differentiate down the osteogenic lineage and had progressed toward a mature osteoblast phenotype.

DNA methyltransferase 1 (DNMT1) catalyzes the postreplication methylation of DNA and is responsible for maintaining the DNA methylation pattern during cell division. A long list of data supports a role for DNMT1 in cellular transformation and inhibitors of DNMT1 were shown to have antitumorigenic effects. It was long believed that DNMT1 promoted tumorigenesis by maintaining the hypermethylated and silenced state of tumor suppressor genes. We have previously shown that DNMT1 knock down by either antisense oligonucleotides directed at DNMT1 or expressed antisense activates a number of genes involved in stress response and cell cycle arrest by a DNA methylation-independent mechanism. In this report we demonstrate that antisense knock down of DNMT1 in human lung carcinoma A549 and embryonal kidney HEK293 cells induces gene expression by a mechanism that does not involve either of the known epigenomic mechanisms, DNA methylation, histone acetylation, or histone methylation. The mechanism of activation of the cell cycle inhibitor p21 and apoptosis inducer BIK by DNMT1 inhibition is independent of the mechanism of activation of the same genes by histone deacetylase inhibition. We determine whether DNMT1 knock down activates one of the nodal transcription activation pathways in the cell and demonstrate that DNMT1 activates Sp1 response elements. This activation of Sp1 response does not involve an increase in either Sp1 or Sp3 protein levels in the cell or the occupancy of the Sp1 elements with these proteins. The methylation-independent regulation of Sp1 elements by DNMT1 unravels a novel function for DNMT1 in gene regulation. DNA methylation was believed to be a mechanism for suppression of CG-rich Sp1-bearing promoters. Our data suggest a fundamentally different and surprising role for DNMT1 regulation of CG-rich genes by a mechanism independent of DNA methylation and histone acetylation. The implications of our data on the biological roles of DNMT1 and the therapeutic potential of DNMT1 inhibitors as anticancer agents are discussed.DNA modification by methylation of cytosines residing at the dinucleotide sequence CG plays an important role in epigenomic programming of gene expression (1). Not all CGs are methylated, and the pattern of distribution of methylated and unmethylated CGs is cell type-specific (2). DNA methylation in regulatory regions of genes plays a role in silencing genes either by directly inhibiting the interaction of transcription factors with their regulatory sequences (3, 4) or by attracting methylated DNA-binding proteins, which in turn recruit histone deacetylases and histone methyltransferases, resulting in an inactive chromatin structure (5, 6). DNA methylation is catalyzed by DNA methyltransferases DNMTs, 1 which transfer the methyl moiety from the methyl donor S-adenosylmethionine to 5th position on the cytosine ring (7). DNMT1 is responsible for maintaining the DNA methylation pattern during embryonal development and cell division (8, 9). DNMT1 deregulation was proposed to play a critical ro...

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