121 DEMETHYLATION OF MAMMALIAN SOMATIC DNA BY XENOPUS EGG AND OOCYTE EXTRACTS

Abstract

In mammals, the successful development of live offspring by somatic cell nuclear transfer (SCNT) has demonstrated the ability of oocyte or egg cytoplasm to reprogram the differentiated status of somatic DNA. However, the efficiency of development is low, and this has been attributed to incomplete or inappropriate reprogramming of epigenetic status. One such epigenetic marker is methylation of genomic DNA at CpG islands. In SCNT, derived embryo abnormal DNA methylation patterns have been reported by a number of groups; in particular, it has been observed that the methylation pattern of embryonic cells resembles that of the donor cell (Santos et al. 2003 Curr. Biol. 13, 1116-1121). One strategy to improve reprogramming and, hence, development is to erase or reprogram the epigenetic status of the donor cell prior to nuclear transfer. We have previously reported that Xenopus egg and oocyte extracts show a differential effect on transcription. In oocyte extracts Pol I and II transcripts are maintained in the somatic cells; in egg extracts, these are abolished (Alberio et al. 2005 Exp. Cell. Res. 307, 131-141). To extend these studies, we have investigated the ability of oocyte and egg extracts to demethylate bovine somatic DNA. Preparation of Xenopus oocyte and egg extracts, culture, permeabilization of donor cells, and incubation conditions were all as previously described (Alberio et al. 2005 Exp. Cell. Res. 307, 131-141). Cells were incubated in extracts for 1 and 3 h at 21°C, centrifuged onto glass slides fixed in 4% Para formaldehyde for 15 min, followed by 4 M HCL for 1 h at 39°C, and blocked for 1 h. Cells were stained with mouse monoclonal anti-1MeC (1:50) overnight at 4°C followed by FITC-conjugated goat anti-mouse antibody (1:20) for 1 h at room temperature and mounted in Vectashied containing 10 ¼g of propidium iodide/mL. Nuclei were scored as positive or negative for 5MeC staining. In control cells, 90% of nuclei stained positively for 5MeC. In both oocyte and egg extracts the number of positive nuclei decreased with time showing demethylation of the somatic DNA 68 and 58% and 38 and 42% positive, respectively, after 1 and 3 h of incubation. Addition of apyrase (2%) to hydrolyze ATP inhibited demethylation in both extracts (90% nuclei positive). High rates of DNA replication were observed in somatic cells in egg extracts in contrast to no replication in oocyte extracts. Aphidicolin (1 ¼g/20 ¼L) was added to egg extracts to inhibit DNA replication, and under these conditions, DNA demethylation was abolished, suggesting a passive DNA demethylation mechanism as a result of DNA replication. In conclusion, Xenopus laevis oocyte and egg extracts can demethylate mammalian somatic DNA in an energy-dependent manner. In oocyte extracts, demethylation is independent of DNA replication, suggesting an active mechanism. In egg extracts, DNA replication is required, suggesting a passive mechanism. These studies further demonstrate the differences in reprogramming activities between oocyte and egg cytoplasm and suggest that interspecies extracts may provide a tool for nuclear reprogramming.