Dppa3 expression is critical for generation of fully reprogrammed iPS cells and maintenance of Dlk1-Dio3 imprinting

Abstract

Reprogramming of mouse somatic cells into induced pluripotent stem cells (iPSCs) often generates partially reprogrammed iPSCs (pre-iPSCs), low-grade chimera forming iPSCs (lg-iPSCs) and fully reprogrammed, high-grade chimera production competent iPSCs (hg-iPSCs). Lg-iPSC transcriptome analysis revealed misregulated Dlk1-Dio3 cluster gene expression and subsequently the imprinting defect at the Dlk1-Dio3 locus. Here, we show that germ-cell marker Dppa3 is present only in lg-iPSCs and hg-iPSCs, and that induction with exogenous Dppa3 enhances reprogramming kinetics, generating all hg-iPSCs, similar to vitamin C (Vc). Conversely, Dppa3-null fibroblasts show reprogramming block at pre-iPSCs state and Dlk1-Dio3 imprinting defect. At the molecular level, we show that Dppa3 is associated with Dlk1-Dio3 locus and identify that Dppa3 maintains imprinting by antagonizing Dnmt3a binding. Our results further show molecular parallels between Dppa3 and Vc in Dlk1-Dio3 imprinting maintenance and suggest that early activation of Dppa3 is one of the cascades through which Vc facilitates the generation of fully reprogrammed iPSCs.

Dppa3 in pluripotency maintenance of ES cells and early embryogenesis

“…19 Dppa3 only presents in lg-iPSCs and hg-iPSCs but not in pre-iPSCs. In the process of somatic cell reprogramming, three transitional iPSCs are usually generated: partially reprogrammed pre-iPSCs, low-grade chimera iPSCs (lg-iPSCs, completely reprogrammed and only produce lowgrade chimeras), and high-grade chimera iPSCs (hg-iPSCs) that develop high-grade chimeras.…”

“…In the process of somatic cell reprogramming, three transitional iPSCs are usually generated: partially reprogrammed pre-iPSCs, low-grade chimera iPSCs (lg-iPSCs, completely reprogrammed and only produce lowgrade chimeras), and high-grade chimera iPSCs (hg-iPSCs) that develop high-grade chimeras. 19 It has been reported that vitamin C (Vc) could improve reprogramming efficiency. Dppa3 expression is activated at day 6 to day 9 during reprogramming earlier than other pluripotent transcriptional factors, such as Oct4 and Sox2.…”

“…Dppa3 expression is activated at day 6 to day 9 during reprogramming earlier than other pluripotent transcriptional factors, such as Oct4 and Sox2. 19 Dppa3 can bind with Dlk1-Dio3 at a particular region and suppress the function of Dnmt3a. 34 Dlk1-Dio3 was the most famous imprinted region.…”

“…9 In the paternal nucleus, 5mC rapidly vanishes, but it still presents in the maternal nucleus in the early stage. 18,19 Recent research works have demonstrated that Dppa3 can also maintain maternal chromosome integrity by preventing 5hmC-induced phosphorylation of H2AX (γH2AX) accumulation. Tet3-mediated conversion from 5mC to 5hmC only takes place in the paternal genome but not in the maternal genome.…”

Nuclear Reprogramming by Defined Factors: Quantity Versus Quality

“…Indeed, Zscan4, in combination with OSKM, not only reduced the DDR but also markedly promoted the efficiency of iPSC generation, enhanced telomere lengthening as early as 3 days post-infection, and significantly increased the percentage of iPSC lines that gave rise to 'all-iPSC' mice as determined by 4N complementation [63]. In addition, forced expression of the germ cell marker, Dppa3, together with OSKM enhances reprogramming kinetics and generates mostly high-grade iPSCs, as evidenced by the production of iPSC clones with intact Dlk1-Dio3 imprinted locus [64], a locus that when hypermethylated is associated with poor-quality iPSCs [65]. Moreover, the quality of iPSCs is dramatically affected by the specific choice of reprogramming factors [51].…”