Our results can be explained by the following model During
Our results can be explained by the following model. During reprogramming, the de novo DNA methyltransferases (DNMTs) methylates not only the promoters of the somatic-specific genes that are undergoing reprogramming, but also the promoters of most cell-specific genes, even if they are not expressed in the somatic cell undergoing reprogramming (Figure 4). This suggests that the DNMTs cannot separate between the different tissue-specific promoters of the cell undergoing reprogramming and that gene silencing occurs in an indiscriminate mode that does not distinguish between parental order MG262 from different lineages. For example, the gene CMTMC5 is expressed predominantly in ectodermal cells of the adult brain; however, this gene undergoes de novo methylation in iPSCs that are generated from mesoderm, endoderm, and teratoma cells derived from parthenogenetic germ cells (Figure 3C). A different interpretation is that iPSCs undergo extensive aberrant methylation in vitro (Lister et al., 2011). However, the high concordance between methylation of the tissue-specific genes among our iPSCs that are generated from distinct lineages suggests otherwise. If indeed the methylation of tissue-specific genes represents an aberrant phenomenon, then we would not expect it to be consistent across three diverse cell types and many independent and different reprogramming experiments. In addition, a recent study shows that keratinocytes reprogram much faster than fibroblasts because they are more methylated than fibroblasts (Barrero et al., 2012). In this case, the DNMTs may not need to methylate all the tissue-specific gene promoters, thus enhancing reprogramming efficiency.
In mouse ESCs, it was recently shown that the cells are hypermethylated when grown with serum; however, a switch to serum-free medium supplemented with mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK) inhibitor or GSK3 inhibitor (2i medium) results in genome-wide hypomethylated mouse ESCs that resemble an earlier developmental “naïve state” (Leitch et al., 2013). Human ESCs may represent a later developmental stage than mouse ESCs, more similar to the mouse epiblast stem cell stage (Tesar et al., 2007). Human iPSCs resemble human ESCs, and thus the high degree of methylation we observe in both cell types may represent a depiction of their in vivo postimplantation state, just before the cells start to acquire their somatic cell identity. It will be interesting to examine the methylation status of naive human PSCs and see if they are relatively hypomethylated (Hanna et al., 2010). Which enzymes mediate the extensive de novo methylation during reprogramming is yet to be determined. A recent work shows that non-CpG methylation in PSCs is mediated by DNMT3a and DNMT3b, as knocking down these genes eliminated most of the non-CpG methylation (Ziller et al., 2011). It is likely that these enzymes are also responsible for the de novo methylation we detect in iPSCs in LCpG promoters as the expression of DNMT3b was upregulated in all our pluripotent cells (Figure 1E). How HCpG gene promoters are protected from the de novo methylation is yet not fully understood. Binding of proteins to CpG islands may interfere with the DNMTs attempt to methylate these HCpG promoters, but more work is needed to show if this is indeed the case. Finally, several recent works suggest that methylation plays a key role in mediating the ability of cells to differentiate (Bar-Nur et al., 2011; Kim et al., 2010; Lister et al., 2011; Nagae et al., 2011; Polo et al., 2010), sometime due to an epigenetic memory (Bar-Nur et al., 2011; Kim et al., 2010; Lister et al., 2011; Polo et al., 2010). We propose that a thorough dissection of the methylation status of each iPSCs will greatly benefit the use of iPSCs for direct differentiation protocols and in using the cells for potential cell replacement therapy.