cis-Acting Signal for Inheritance of Imprinted DNA Methylation Patterns in the Preimplantation Mouse Embryo

The inheritance of gametic methylation patterns is a critical event in the imprinting of genes. In the case of the imprintedRSVIgmyc transgene, the methylation pattern in the unfertilized egg is maintained by the early mouse embryo, whereas the sperm’s methylation pattern is lost in the early embryo. To investigate thecis -acting requirements for this preimplantation stage of genomic imprinting, we examined the fate of differentRSVIgmyc methylation patterns, preimposed onRSVIgmyc and introduced into the mouse zygote by pronuclear injection.RSVIgmyc methylation patterns with a low percentage of methylated CpG dinucleotides, generated by using bacterial cytosine methylases with four-base recognition sequences, were lost in the early embryo. In contrast, methylation was maintained when all CpG dinucleotides were methylated with the bacterialSss I (CpG) methylase. This singular maintenance ofRSVIgmyc methylation preimposed withSss I methylase appears to be specific to the early, undifferentiated embryo; differentiated NIH 3T3 fibroblasts transfected with methylated versions ofRSVIgmyc maintained all methylation patterns, independent of the level of preimposed methylation. The methylation pattern of theRSVIgmyc allele in adult founder transgenic mice that was produced by pronuclear injection of anSss I-methylated construct could not be distinguished from the maternalRSVIgmyc methylation pattern. Thus, a highly methylated allele in adult mice, normally generated by transmission ofRSVIgmyc through the female germ line, was also produced in founder transgenic mice by bypassing gametogenesis and introducing a highly methylatedRSVIgmyc into the mouse zygote. These results suggest thatRSVIgmyc methylation itself is acis -acting signal for the preimplantation maintenance of the oocyte’s methylation pattern and, therefore, acis -acting signal forRSVIgmyc imprinting. Furthermore, our inability to identify a sequence element withinRSVIgmyc that was absolutely required for its imprinting suggests that the extent ofRSVIgmyc methylation, rather than a particular pattern of methylation, is the principal feature of this imprinting signal.