TEMPORAL AND SPATIAL REGULATION OF H19 IMPRINTING IN NORMAL AND UNIPARENTAL MOUSE EMBRYOS

The mouse H19 gene Is imprinted so that the paternal copy is both meth ylated and repressed during fetal development. However, the CpG-rich p romoter region encompassing the transcription start is not methylated in sperm; this region must therefore become methylated postzygotically . We first examined the timing of DNA methylation of this region and t he corresponding expression of H19. Both parental copies are initially undermethylated in blastocysts and the paternal copy then becomes ful ly methylated in the embryo around implantation; this methylation is m ore protracted in the extraembryonic lineages, especially in the troph oblast. By contrast to the lineage-dependent methylation, we observed exclusive expression of the maternal copy in preimplantation embryos a nd in all the lineages of early postimplantation embryos although vari ability may exist in cultured embryos. This indicates that methylation of the CpG-rich promoter is not a prerequisite for the paternal repre ssion. We then examined whether methylation and expression occurs appr opriately in the absence of a maternal or a paternal genome. Both H19 copies in androgenetic embryos are fully methylated while they are unm ethylated in parthenogenetic embryos. This correlates with the lack of expression in androgenetic embryos but expression in parthenogenetic embryos. However, the androgenetic trophoblast was exceptional as it s hows reduced methylation and expresses H19, These results suggest that promoter methylation is not the primary inactivation mechanism but is a stabilizing factor, Differential methylation in the more upstream r egion, which is established in the gametes, is a likely candidate for the gametic signal and may directly control H19 activity.