DNA methylation and demethylation have been proposed to play an important role in somatic cell reprogramming. Here, we demonstrate that the DNA hydroxylase Tet1 facilitates pluripotent stem cell induction by promoting Oct4 demethylation and reactivation. Moreover, Tet1 (T) can replace Oct4 and initiate somatic cell reprogramming in conjunction with Sox2 (S), Klf4 (K), and c-Myc (M). We established an efficient TSKM secondary reprogramming system and used it to characterize the dynamic profiles of 5-methylcytosine (5mC), 5-hydroxymethylcytosine (5hmC), and gene expression during reprogramming. Our analysis revealed that both 5mC and 5hmC modifications increased at an intermediate stage of the process, correlating with a transition in the transcriptional profile. We also found that 5hmC enrichment is involved in the demethylation and reactivation of genes and regulatory regions that are important for pluripotency. Our data indicate that changes in DNA methylation and hydroxymethylation play important roles in genome-wide epigenetic remodeling during reprogramming.

No abstract

Calcium-dependent cell adhesion molecules (cadherins) are involved in maintaining the epithelial structure of a number oftissues including the mammary gland. In breast and other tumor types. loss of E-cadherin expression has been seen in high grade tumors and correlates with increased invasiveness. Here we show high levels of expression of N-cadherin in the most invasive breast cancer cell lines which was inversely correlated with their expression of E-cadherin. A stromal cell line also expressed N-cadherin in accordance with its fibroblastic morphology. N-cadherin localized to areas of cell-cell contact in all cells that expressed it. Calcium-dependent intercellular adhesion of N-cadherin-expressing breast cancer and stro-ma1 cells was specifically inhibited by an anti N-cadherin monoclonal antibody. In addition, N-cadherin promoted the interaction of invasive breast cancer cells with mammary stromal cells; in contrast, E-cadherin expressing cell lines did not co-aggregate with stromal cells. The combined results suggest a functional role for N-cadherin in cohesion of breast tumor cells which, in addition promotes their interaction with the surrounding stromal cells. thereby facilitating invasion and metastasis.

No abstract

Cadherins mediate calcium-dependent cell-cell adhesion, and this activity is regulated by cytoplasmic interactions between cadherins, catenins, and the actinbased cytoskeleton. ␣-Catenin plays a critical role in the transmembrane anchorage of cadherins, and deletion of ␣-catenin has been shown to inactivate cadherin-mediated adhesion, resulting in a nonadhesive phenotype. Here we show that serum starvation increases E-cadherin expression and induces E-cadherin-dependent adhesion in the MDA-MB-468 breast cancer cell line. This adhesion occurred despite a lack of ␣-catenin expression, which was caused by mutations in the ␣-catenin gene. Coprecipitation analysis suggests that this adhesion may be mediated by cytoplasmic connections from cadherins to the cytoskeleton involving vinculin. A high level of vinculin associated with E-cadherin immunoprecipitates was observed in MDA-MB-468 cells. In contrast, vinculin was not detected in E-cadherin complexes in the A431 and MCF-7 epithelial carcinoma cell lines, which express ␣-catenin. However, in reciprocal immunoprecipitations using anti-vinculin antibodies, Ecadherin associated strongly with vinculin in MDA-MB-468 cells and, to a lesser extent, in A431 and MCF-7 cells. These results suggest that both ␣-catenin and vinculin may be present in the adhesion complex. To test the hypothesis that vinculin associates with E-cadherin complexes via ␤-catenin, excess recombinant ␤-catenin or ␣-catenin fusion protein was added to MDA-MB-468 cell lysates. Both specifically inhibited the coprecipitation of E-cadherin with vinculin, suggesting competition for the same binding site. These results suggest that vinculin plays a role in the establishment or regulation of the cadherin-based cell adhesion complex by direct interaction with ␤-catenin.Cadherins are calcium-dependent cell-cell adhesion molecules that mediate homotypic interactions among cells and are essential for tissue morphogenesis (for review, see Refs. 1 and 2). Adhesion via cadherins involves the coordination of extracellular binding and intracellular anchorage to the actin-based cytoskeleton. The cytoplasmic domain of E-cadherin binds to either ␤-catenin or plakoglobin/␥-catenin (for review, see , and this complex is coupled to the actin cytoskeleton by ␣-catenin, which binds to both ␤-catenin and actin (6, 7). There is evidence that ␣-catenin also binds to ␣-actinin (8, 9) and spectrin (10), but the role of these interactions in adhesion is unknown. Disruption of ␣-catenin function by genetic deletion or mutation was shown to cause a loss of E-cadherin-dependent adhesion. PC-9 lung carcinoma cells, which lack detectable ␣-catenin expression, were shown to have aberrant cell-cell adhesion (11) that was restored by transfection of these cells with ␣-catenin cDNA (12). This resulted in the establishment of a polarized epithelium and inhibition of cell growth (13). This and other work suggest that the linkage of cadherin complexes to the cytoskeleton via ␣-catenin is essential for normal cell adhesion, morphogenesis, and ...

Histone demethylase LSD1 can form complex with different Rcor family corepressors in different cell types. It remains unknown if cell-specific Rcor proteins function specifically in distinct cell types. Here, we report that Rcor2 is predominantly expressed in ESCs and forms a complex with LSD1 and facilitates its nucleosomal demethylation activity. Knockdown of Rcor2 in ESCs inhibited ESC proliferation and severely impaired the pluripotency. Disclosure of potential conflicts of interest is found at the end of this article.

It remains controversial whether the abnormal epigenetic modifications accumulated in the induced pluripotent stem cells (iPSCs) can ultimately affect iPSC pluripotency. To probe this question, iPSC lines with the same genetic background and proviral integration sites were established, and the pluripotency state of each iPSC line was characterized using tetraploid (4N) complementation assay. Subsequently, gene expression and global epigenetic modifications of "4N-ON" and the corresponding "4N-OFF" iPSC lines were compared through deep sequencing analyses of mRNA expression, small RNA profile, histone modifications (H3K27me3, H3K4me3, and H3K4me2), and DNA methylation. We found that methylation of an imprinted gene, Zrsr1, was consistently disrupted in the iPSC lines with reduced pluripotency. Furthermore, the disrupted methylation could not be rescued by improving culture conditions or subcloning of iPSCs. Moreover, the relationship between hypomethylation of Zrsr1 and pluripotency state of iPSCs was further validated in independent iPSC lines derived from other reprogramming systems.

No abstract