De novo DNA methylation by Dnmt3a and Dnmt3b is dispensable for nuclear reprogramming of somatic cells to a pluripotent state

Abstract

Induced pluripotent stem cells (iPSCs) are generated from somatic cells by the transduction of defined transcription factors, and this process involves dynamic changes in DNA methylation. While the reprogramming of somatic cells is accompanied by demethylation of pluripotency genes, the functional importance of de novo DNA methylation has not been clarified. Here, using loss-of-function studies, we generated iPSCs from fibroblasts that were deficient in de novo DNA methylation mediated by Dnmt3a and Dnmt3b. These iPSCs reactivated pluripotency genes, underwent self-renewal, and showed restricted developmental potential that was rescued upon reintroduction of Dnmt3a and Dnmt3b. We conclude that de novo DNA methylation by Dnmt3a and Dnmt3b is dispensable for nuclear reprogramming of somatic cells.

Figure 1.

Generation of iPSCs in the absence of de novo DNA methyltransferase activity. (A) Schematic of the two approaches to reprogram conditional knockout MEFs for Dnmt3a and Dnmt3b. MEFs were infected with adenovirus harboring a Cre recombinase and GFP either after or before infection with inducible lentiviruses encoding the reprogramming factors Oct4, Sox2, c-Myc and Klf4. (B) Immunostaining for the pluripotency marker Nanog. Bars, 100μm.

Figure 2.

Characterization of 3ab-deficient iPSC lines. (A) Southern blot analysis shows 3ab-deficient iPSC lines obtained through approach 1 (Fig. 1A). Clone #8 is indicated by an asterisk. (B) Southern blot analysis of 3ab-deficient iPSC lines obtained through approach 2 (Fig. 1A). Clones A6 and B2 are indicated by an asterisk. Additionally, 3ab MEFs infected with Adeno-Cre-GFP are shown before the transduction with the reprogramming factors and over the course of several passages (1– 3). Lane 4 displays DNA from a culture dish containing mixed iPSC colonies. Note that the loop-out induced by Cre recombinase was highly efficient. “2lox” represents the conditional alleles and “1lox” represents the deficient alleles of Dnmt3a and Dnmt3b, respectively.

Figure 3.

3ab-deficient iPSCs form teratomas with a restricted developmental potential. (A) Teratomas from #8 and B2 3ab-deficient iPSCs. Reintroduction of functional Dnmt3a and Dnmt3b into 3ab-deficient iPSCs rescues the ability to efficiently form cells from the three germ layers in a teratoma assay. (B) eGFP-marked rescue lines were injected into blastocysts, and resulting chimeric embryos at mid-gestation are shown. The eGFP signal was clearly visible in the majority of embryos, whereas the degree of chimerism varied. Two embryos with developmental defects are shown. The first embryo of the bottom panel did not show any eGFP signal and serves as a reference of background.