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

ABSTRACT

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 the cis-acting requirements for this preimplantation stage of genomic imprinting, we examined the fate of different RSVIgmyc 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 bacterialSssI (CpG) methylase. This singular maintenance ofRSVIgmyc methylation preimposed with SssI methylase appears to be specific to the early, undifferentiated embryo; differentiated NIH 3T3 fibroblasts transfected with methylated versions of RSVIgmyc maintained all methylation patterns, independent of the level of preimposed methylation. The methylation pattern of the RSVIgmyc allele in adult founder transgenic mice that was produced by pronuclear injection of anSssI-methylated construct could not be distinguished from the maternal RSVIgmyc methylation pattern. Thus, a highly methylated allele in adult mice, normally generated by transmission of RSVIgmyc through the female germ line, was also produced in founder transgenic mice by bypassing gametogenesis and introducing a highly methylated RSVIgmyc into the mouse zygote. These results suggest that RSVIgmyc methylation itself is a cis-acting signal for the preimplantation maintenance of the oocyte’s methylation pattern and, therefore, acis-acting signal for RSVIgmyc imprinting. Furthermore, our inability to identify a sequence element within RSVIgmyc that was absolutely required for its imprinting suggests that the extent of RSVIgmyc methylation, rather than a particular pattern of methylation, is the principal feature of this imprinting signal.

ACKNOWLEDGMENTS

We thank Victor Tybulewicz for the pPNT plasmid. We also express our gratitude to Kris Fafalios and Daphne Preisach for their technical assistance. We thank Richard Carthew for his comments and assistance in preparation of the manuscript.

This work was supported by Public Health Service grant HD 32940 from the National Institutes of Health. This research was supported in part by a grant from the Pittsburgh Supercomputing Center through NIH National Center for Resources resource grant 2 P41 RR06009.