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 imprinted RSVIgmyc 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 on RSVIgmyc 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 bacterial SssI (CpG) methylase. This singular maintenance of RSVIgmyc 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 an SssI-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, a cis-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.

Shared Role for Differentially Methylated Domains of Imprinted Genes

“…1C). The CpG island sequences of c-myc, located within intron 1, have previously been shown by a HpaII digest to be unmethylated on both parental alleles (16). HhaI and BstUI sites in the CpG island (between the BglII and XbaI sites) were also unmethylated on both the maternal and paternal alleles (data not shown).…”

“…The imprinting of each transgene was studied by comparing DNA methylation patterns of the maternal and paternal alleles. Allelic differences in expression were not examined, because in the absence of RSV sequences, most of the hybrid transgenes would not be transcriptionally active (9,16).…”

“…Besides DMD2 sequences, either a sequence remote from DMD2 is also needed for Igf2r imprinting, or a combination of genomic sequences neighboring DMD2 is also needed. Studies of the RSVIgmyc transgene favor the latter explanation; there is no single element within Ig/myc that is absolutely required for RSVIgmyc imprinting, and many different combinations of sequence elements within Ig/myc support RSVIgmyc imprinting (9,16).…”

“…Like endogenous imprinted genes, RSVIgmyc undergoes allele-specific changes in DNA methylation during development and expresses a gene from only one parental allele (10,28). The absence of a single required RSVIgmyc sequence element for its imprinting suggests a functional redundancy for the ICEs within the RSVIgmyc transgene (16). We show here that the DMD of RSVIgmyc is the region that contains pBR322 and Rous sarcoma virus (RSV) sequences.…”

A Dyad Oct-binding Sequence Functions as a Maintenance Sequence for the Unmethylated State within theH19/Igf2-imprinted Control Region

“…Because the functions of DOS revealed by our experiments cannot explain the loss of hypermethylation maintenance mechanisms, we speculate that these mechanisms exist independently of the unmethylated state maintenance mechanisms governed by DOS. Therefore the 1.6-kb deletion is not sufficient for the complete loss of the hypermethylated state maintenance mechanisms because hypermethylation of a certain DNA region itself can be a cis-acting signal for the preimplantation maintenance of the gametic hypermethylation (33).…”