2, and to affect the inheritance of DNA methylation. DNA methylation of MEFs is higher in G1 than in G2/M phase We have proposed that DNMT1 in G1 phase also contributes to the stable inheritance of DNA methylation. pluripotency-related genes was decreased, and their expression was up-regulated, which subsequently promoted pluripotency and mesenchymalCepithelial transition, a necessary step for reprogramming. We infer that high cellular proliferation rates promote generation of induced pluripotent stem cells at least partially by inducing passive DNA demethylation and up-regulating pluripotency-related genes. Therefore, these results uncover a connection between cell reprogramming and DNA methylation. to promote reprogramming, which is also modulated by vitamin C (Vc) (3,C5). In addition, during DNA replication, the newly synthesized DNA strand has no cytosine methylation. The stable inheritance of DNA methylation during proliferation relies on DNA methyltransferase 1 (DNMT1), which methylates hemimethylated CpGs not only during S phase but also during G2/M phase (6,C8). Normally, global DNA methylation is stable during proliferation. However, inhibition of such DNMT1-mediated methylation by suppressing expression or by promoting cell proliferation accumulates the hemimethylated CpGs along with the cell cycle progress, gradually reduces global DNA methylation, and results in passive DNA demethylation (9). During iPSCs generation, an both increase in proliferation rate and a decrease in global DNA methylation are observed. It is reasonable C-DIM12 to suggest that a high proliferation rate might lead to passive DNA demethylation, regulate the expression of certain genes, and facilitate reprogramming. Thus, in this study, a connection between passive DNA demethylation and proliferation was established and studied during reprogramming. Results Dnmt1 expression in G1 phase correlates with proliferation rates To explore the potential connection between proliferation rate and the expression of genes related to epigenetic regulation, like histone modification and DNA methylation, the cell proliferation rate, especially the length of G1 phase, was modulated C-DIM12 by regulating the expression of in MEFs (Fig. 1had the most significant correlation with proliferation rate (Fig. 1, and and were used as controls. The correlation C-DIM12 between cell proliferation (Td) and gene expression was determined by qPCR FAE (axis, whereas the values for the correlation efficiencies with C-DIM12 baseline (0.5000) are shown on the axis The correlation between cell proliferation (Td) and expression is listed in expression, the respective lengths of different phases of the cell cycle, and percent occupancy of different phases of the cell cycle are summarized in and and and were used as controls. The expression of was determined at the mRNA (< 0.001. Among the C-DIM12 five identified genes, was selected for further investigation because of the connection between reprogramming and DNA methylation (4, 5). Because the expression of is relatively high during S phase (10, 11), the correlation described above might result from an increased percentage of cells in S phase. This possibility was partially excluded by the higher correlation of expression with G1 phase length or doubling time (Td) than with the percentage of cells in S phase (Fig. 1up-regulated expression, both at the mRNA and protein levels, in G1 phase (Fig. 1, and to shorten G1 phase and up-regulate manifestation (Fig. 1, decreased the proliferation rate and induced a longer G1 phase (Fig. 2was combined with up-regulation and and and (control), (Dnmt1), (sh-Dnmt1), (sh-p53) or manifestation was determined at the same time by qPCR (and at hour ?48. Two days after illness (hour 0), 0.5 m mimosine was used to treat cells for an additional 24 h. After mimosine withdrawal, cells were further cultured for 72 h (hours 24C96). DNA methylation levels were determined by HPLC and are summarized in (group and the additional two organizations with in (and or the group and additional organizations in and < 0.05; **, < 0.01; ***, < 0.001; manifestation. Cells with different proliferation rates require different amounts of DNMT1 to keep up stable DNA methylation during proliferation. A shorter cell cycle requires a larger amount of DNMT1.
2, and to affect the inheritance of DNA methylation
