De Novo DNA Methylation at Imprinted Loci during Reprogramming into Naive and Primed Pluripotency

Abstract

Summary CpG islands (CGIs) including those at imprinting control regions (ICRs) are protected from de novo methylation in somatic cells. However, many cancers often exhibit CGI hypermethylation, implying that the machinery is impaired in cancer cells. Here, we conducted a comprehensive analysis of CGI methylation during somatic cell reprogramming. Although most CGIs remain hypomethylated, a small subset of CGIs, particularly at several ICRs, was often de novo methylated in reprogrammed pluripotent stem cells (PSCs). Such de novo ICR methylation was linked with the silencing of reprogramming factors, which occurs at a late stage of reprogramming. The ICR-preferred CGI hypermethylation was similarly observed in human PSCs. Mechanistically, ablation of Dnmt3a prevented PSCs from de novo ICR methylation. Notably, the ICR-preferred CGI hypermethylation was observed in pediatric cancers, while adult cancers exhibit genome-wide CGI hypermethylation. These results may have important implications in the pathogenesis of pediatric cancers and the application of PSCs.

Sex of donor cell and reprogramming conditions predict the extent and nature of imprinting defects in mouse iPSCs

“…2A; Fig. S2B) matching previous observations (Sun et al, 2012; Takikawa et al, 2013; Yagi et al, 2019).…”

“…In contrast, imprinting methylation in male iPSCs might be responsive to medium conditions through fluctuations in the expression and activity of TET dioxygenase enzymes according to medium formulation. (Stadtfeld et al, 2010;Sun et al, 2012;Takikawa et al, 2013;Yagi et al, 2019), when sex of the donor cell (when given) and reprogramming culture conditions are considered (Table S1). This knowledge is important to devise future strategies to create iPSCs devoid of imprinting abnormalities.…”

“…Epigenetic disparity, mostly studied at the DNA methylation level, can originate from: (1) failure to fully reset the somatic memory of the donor cell (Bar-Nur et al, 2011; Kim et al, 2011; Lister et al, 2011; Ohi et al, 2011; Polo et al, 2010); (2) acquisition of aberrations during the reprogramming process (Huang et al, 2014; Koyanagi-Aoi et al, 2013; Lister et al, 2011; Ma et al, 2014; Nazor et al, 2012; Ohi et al, 2011; Ruiz et al, 2012; Stadtfeld et al, 2010); (3) epigenetic adaptation to long-term in vitro culturing (Mekhoubad et al, 2012; Vallot et al, 2015). The latter two likely affect processes that rely on strict maintenance of DNA methylation such as genomic imprinting (Stadtfeld et al, 2010; Sun et al, 2012; Takikawa et al, 2013; Yagi et al, 2019), which serves as a good readout to study epigenetic fidelity in iPSCs.…”

“…Previous studies have revealed imprinting defects in both mouse and human iPSCs (Bar et al, 2017; Ma et al, 2014; Nazor et al, 2012; Stadtfeld et al, 2010; Sun et al, 2012; Takikawa et al, 2013; Yagi et al, 2019). Importantly, these errors persist and are never rescued upon differentiation (Bar et al, 2017; Nazor et al, 2012), which is troublesome for iPSC applications in translational and clinical research.…”

“…Interestingly, defects in Dlk1-Dio3 loci could be corrected by using ascorbic acid (Stadtfeld et al, 2012), presumably due to an increased demethylating activity of the TET dioxygenase enzymes (Blaschke et al, 2013). Later studies showed that the repertoire of imprinting defects extends to other imprinted loci in mouse iPSCs (Sun et al, 2012; Takikawa et al, 2013; Yagi et al, 2019). Interestingly, the extent and nature (hypermethylation versus hypomethylation) of these defects varied significantly between studies (Table S1).…”