Given the significance of methylation demethylation during

Given the significance of methylation/demethylation during reprogramming, we set out to investigate the function of miR-29a in regulating the iPSC methylome. Although miR-29a was shown to directly regulate both de novo DNA methyltransferases and demethylases, we found that depletion of miR-29a resulted in DNA demethylation in fibroblasts, suggesting that the major targets of miR-29a in somatic order Amyloid β-Peptide (1-42) are DNA demethylases. Interestingly, and in support of our study, miR-29a targets TETs and TDG were recently shown to impair reprogramming of murine fibroblasts when downregulated, due to the block of miRNA-mediated mesenchymal-to-epithelial transition that is required for reprogramming (Hu et al., 2014). More importantly, iPSC lines derived from miR-29a depletion partly overcome the aberrant DNA methylation status observed in control-derived iPSC and resemble hESCs in their methylome. Our study facilitates the understanding of the role of small RNAs in modulating the iPSC epigenome.
Results
Discussion In this study, we set out to investigate the function of miRNAs that regulate the epigenetic landscape in somatic cells and thus may have a function in somatic cell reprogramming. We found that the miR-29 family is highly expressed in somatic cells and decreases in expression during somatic cell reprogramming (Figures 1A and 1B). Depletion of miR-29 in fibroblasts dramatically changed the DNA methylation status, suggesting that miR-29a is one of the major miRNAs that maintains the DNA methylation status in fibroblasts (Figure 2). Previous studies showed that DNMT3A and DNMT3B, as well as TET proteins, are targets of the miR-29a family (Cheng et al., 2013; Hu et al., 2015). Our data also demonstrated a strong inverse correlation of the expression of the miR-29a family and the expression of DNMTs and TETs. It is not yet clear which proteins are direct targets of miR-29a in maintaining the DNA methylation status in fibroblasts. However, the global hypo-methylation upon miR-29a depletion suggests that proteins involved in the DNA demethylation including TETs and TDGs may be strong candidate targets (Figures 2A and 2B). Previous studies implicated the miR-29a family in mouse somatic cell reprogramming through different pathways (Guo et al., 2013; Yang et al., 2011). Yang et al. (2011) showed that suppression of miR-29a via Myc improves the reprogramming by increasing p85alpha and CDC42, thus suppressing the p53 pathway. Guo et al. (2013) demonstrated that Sox2 is a critical factor inducing miR-29b, and that ectopic overexpression of miR-29b improves the reprogramming efficiency by suppressing Dnmt3a and Dnmt3b. In reprogramming human somatic cells, we found that suppression of the miR-29a family increased reprogramming efficiency. We also found that overexpression of DNMT3B as well as TET1 improves reprogramming efficiency. These seemingly contrasting results may be due to the bidirectional global epigenetic changes accompanying reprogramming. Not only DNA demethylation but also de novo DNA methylation is found in reprogrammed iPSCs (Lister et al., 2011). In light of the bidirectional epigenetic regulation, the miR-29a seems to be a unique regulator that potentially suppresses both de novo DNA methyltransferases and demethylases, although miR-29a depletion seems to have a more pronounced effect on DNA demethylation in fibroblasts (Figures 2A and 2B). miR-29a depletion could not replace MYC in a three- factor reprogramming attempt (data not shown), although it improves reprogramming moderately but significantly with four-factor reprogramming. MYC is a potent transcription factor inducing and repressing a large number of genes; suppression of miR-29a is one of many functions of MYC during reprogramming (Yang et al., 2011). iPSC lines, for which aberrant DNA methylation was reported, were those derived from four-factor reprogramming (Lister et al., 2011). Most likely, MYC expedites reprogramming independent of its role in suppressing miR-29a. Expression of DNA (de)methylation proteins via suppression of miR-29a then becomes supplementary to the function of MYC during reprogramming. When miR-29a targets are upregulated at later stages of reprogramming, most loci are already reprogrammed. Thus, the accessibility of TETs or DNMT3A/B to target loci becomes limited, and thus epigenetic reprogramming is limited in four-factorbased reprogramming.