During germ cell and early embryonic development—the most sensitive and vulnerable period of epigenetic reprogramming—exposure to an adverse environment leads to abnormal methylation and, possibly, long-term health problems. DNA methylation, a major epigenetic mechanism for gene silencing, is recognized to be responsible for the stability of gene expression status. The majority of cytosine-phosphate-guanine sites (CpGs) in mammalian genomes are methylated. DNA methyltransferase (Dnmt) 3A and 3B are essential for de novo methylation, and Dnmt1 maintains methylation patterns during cell division (1). Establishment and maintenance of cell type–specific DNA methylation patterns are dependent on both methylation and demethylation. DNA demethylation is the process of the removal of a methyl group from nucleotides in DNA, which can be passive or active. It has been generally understood that passive DNA demethylation occurs by a reduction in activity or absence of Dnmts, whereas the mechanism of active DNA demethylation has been controversial in recent decades.

Recently, three enzyme families have been implicated in active DNA demethylation via DNA repair. The first is the ten-eleven translocation (Tet) family of enzymes. 5-Methylcytosine (5mC) can be hydroxylated by Tet to form 5-hydroxymethylcytosine (5hmC), which can be further oxidized to 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). The second family is the AID/APOBEC family. 5mC (or 5hmC) can be deaminated by AID/APOBEC family members to form 5-methyluracil (5mU) or 5-hydroxymethyluracil (5hmU). The third is the UDG family of base excision repair glycosylases. TDG and SMUG1 replace these intermediates (i.e., 5mU, 5hmU, or 5caC), culminating in cytosine replacement and DNA demethylation (24). Environment and nutrition have been confirmed to affect epigenetic modification. Although it has been documented that hyperglycemia induces demethylation of specific cytosines throughout the genome (5,6), whether the demethylation could be persistent, and the mechanism involved need to be further investigated.

In this issue, Dhliwayo et al. (7) used a zebrafish model to elucidate the potential molecular mechanism that is responsible for hyperglycemia-induced DNA demethylation. This is a very interesting study when considered in the context of previous findings from this team. In previous studies, these investigators found that the zebrafish demonstrates metabolic memory (MM). Hyperglycemia was induced by streptozocin in adult zebrafish, and following streptozocin withdrawal, they were allowed to reestablish euglycemia. Blood glucose and serum insulin returned to physiological levels because of pancreatic β-cell regeneration, whereas caudal fin regeneration and skin wound healing remained impaired to the same extent as observed in diabetic zebrafish, and this impairment was transmitted to daughter cell tissue. Furthermore, the investigators found that hyperglycemia caused genome-wide demethylation and aberrant gene expression that were inherited by daughter cells and may contribute to the MM (8). They hypothesized that this may be an explanation for heritable transmission of diabetic MM induced by instant exposure in hyperglycemia.

In the new study by Dhliwayo et al. (7), the most notable result concerns a role for the Tet in hyperglycemia-induced DNA demethylation. They provided evidence that hyperglycemia induces both expression and activity of the Tet enzymes, yielding known intermediates of the iterative oxidation pathway that leads to demethylation of 5mC. As 5hmC is the common intermediate for the Tet-specific demethylation, they examined 5hmC expression and found a significant increase in 5hmC induced by hyperglycemia. They reasoned that because 5hmC is converted into 5fC in the next step of the iterative oxidation pathway, and then investigated 5fC expression and found increases similar to those observed for 5hmC. In addition, they revealed that demethylation via the Tet-dependent iterative oxidation pathway can be prevented through inhibiting the poly(ADP-ribose) polymerases (Parp). In summary, they showed that hyperglycemia induces the Parp family of enzymes, which in turn stimulates the Tet enzymes, leading to DNA demethylation and, ultimately, persistent diabetes complications. This suggests that Parp inhibition may provide a therapeutic avenue for the prevention or reversal of diabetes complications.

Exposure to an adverse environment can result in persistent injury. Dhliwayo et al. (7) found that hyperglycemia induced the 5mC demethylation intermediates consistent with Tet enzyme activity, and these levels remained elevated throughout the experimental time course. In zebrafish fins, both the somatic cells and the daughter cells had significant epigenetic changes resulting from hyperglycemia. Animal studies also have demonstrated that the metabolic imprinting resulting from a diabetic intrauterine environment can be transmitted across generations. Not only the maternal line but also the paternal line influences the health of offspring (911). Dhliwayo et al. (7) indicated a novel mechanism of diabetic MM, and illustrated the utility of the zebrafish model for small molecule drug discovery that is relevant for diabetes. Further, the role of the Tet in hyperglycemia-induced demethylation may be a new focus of intergenerational and transgenerational transmission studies.

Despite these promising observations, the reprogramming strategy in zebrafish contrasts markedly with mammals. In mammalian development, DNA methylation changes in an orchestrated manner. Both parental genomes undergo dramatic epigenetic changes after fertilization to form the diploid somatic genome. A wave of demethylation occurs during cleavage, and this is followed by genome-wide de novo methylation after implantation (12). The paternal genome is significantly demethylated within hours of fertilization—before the onset of DNA replication—whereas the maternal genome is demethylated after several cleavage divisions (13). Tet3 plays important role in active DNA demethylation of both paternal and maternal genome in zygotes. In Tet3-deficient zygotes from conditional knockout mice, paternal genome conversion of 5mC into 5hmC fails to occur. Oocytes lacking Tet3 seem to have a reduced ability to reprogram the injected nuclei from somatic cells (1416). In addition to early embryos, demethylation of DNA also occurs in mammalian primordial germ cells (PGCs) and is important for the erasure of imprints and epimutations. PGCs undergo sequential epigenetic changes and genome-wide DNA demethylation to reset the epigenome for totipotency (1719). Hackett et al. (20) demonstrated that global conversion to 5hmC initiates asynchronously among PGCs at embryonic day 9.5 to 10.5 in mice, driven by high levels of Tet1 and Tet2. To address these issues in future studies, a multigenerational mouse/rat model obtained from paternal or maternal hyperglycemia and transgenerational epigenetic inheritance may be worthy of consideration (Fig. 1).

Figure 1

A: Methylation reprogramming in the germ line and preimplantation embryos in mice. Highly methylated PGCs enter the germinal ridge and become demethylated early in development. Remethylation begins in prospermatogonia in male germ cells, and after birth in growing oocytes. The paternal genome (blue) is demethylated immediately after fertilization. The maternal genome (red) is demethylated after several cleavage divisions. Both are remethylated around the time of implantation to different extents in embryonic (EM) and extraembryonic (EX) lineages. During preimplantation development, imprinted genes and certain classes of repeat sequences are exempt from these demethylation events (dashed line) (modified from [17]). B: Intergenerational and transgenerational epigenetic effects. Epigenetic changes in mammals can be induced by the environment (such as hyperglycemia). In pregnant females, environmental exposure also could cause epigenetic modifications in the next two generations (F1 and F2) through the fetus and its germ line. In males, multigenerational exposure is limited to the F0 and F1 generations. The effect of such multigenerational exposure would be further transmitted to subsequent generations in maternal line (F3 and beyond) or paternal line (F2 and beyond). This phenomenon is known as “transgenerational inheritance.”

Figure 1

A: Methylation reprogramming in the germ line and preimplantation embryos in mice. Highly methylated PGCs enter the germinal ridge and become demethylated early in development. Remethylation begins in prospermatogonia in male germ cells, and after birth in growing oocytes. The paternal genome (blue) is demethylated immediately after fertilization. The maternal genome (red) is demethylated after several cleavage divisions. Both are remethylated around the time of implantation to different extents in embryonic (EM) and extraembryonic (EX) lineages. During preimplantation development, imprinted genes and certain classes of repeat sequences are exempt from these demethylation events (dashed line) (modified from [17]). B: Intergenerational and transgenerational epigenetic effects. Epigenetic changes in mammals can be induced by the environment (such as hyperglycemia). In pregnant females, environmental exposure also could cause epigenetic modifications in the next two generations (F1 and F2) through the fetus and its germ line. In males, multigenerational exposure is limited to the F0 and F1 generations. The effect of such multigenerational exposure would be further transmitted to subsequent generations in maternal line (F3 and beyond) or paternal line (F2 and beyond). This phenomenon is known as “transgenerational inheritance.”

See accompanying article, p. 3069.

Funding. The research of the authors is supported by the National Natural Science Foundation of China (31171444 and 81200485) and the Research Fund for the Doctoral Program of Higher Education (20120101120051).

Duality of Interest. No potential conflicts of interest relevant to this article were reported.

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