Expression of imprinted genes is dependent on their parental origin. This is reflected in the heritable differential methylation of parental alleles. The gametic imprints are however reversible as they do not endure for more than one generation. To investigate if the epigenetic changes in male and female germ line are similar or not, we derived embryonic germ (EG) cells from primordial germ cells (PGCs) of day 11.5 and 12.5 male and female embryos. The results demonstrate that they have an equivalent epigenotype. First, chimeras made with EG cells derived from both male and female embryos showed comparable fetal overgrowth and skeletal abnormalities, which are similar to but less severe than those induced by androgenetic embryonic stem (ES) cells. Thus, EG cells derived from female embryos resemble androgenetic ES cells more than parthenogenetic cells. Furthermore, the methylation status of both alleles of a number of loci in EG cells was similar to that of the paternal allele in normal somatic cells. Hence, both alleles of Igf2r region 2, Peg1/Mest, Peg3, Nnat were consistently unmethylated in EG cells as well as in the primary embryonic fibroblasts (PEFs) rescued from chimeras. More strikingly, both alleles of p57kip2 that were also unmethylated in EG cells, underwent de novo methylation in PEFs to resemble a paternal allele in somatic cells. The exceptions were the H19 and Igf2 genes that retained the methylation pattern in PEFs as seen in normal somatic tissues. These studies suggest that the initial epigenetic changes in germ cells of male and female embryos are similar.

Neuronatin (Nnat) is an imprinted gene that is expressed exclusively from the paternal allele while the maternal allele is silent and methylated. The Nnat locus exhibits some unique features compared with other imprinted domains. Unlike the majority of imprinted genes, which are organised in clusters and coordinately regulated, Nnat does not appear to be closely linked to other imprinted genes. Also unusually, Nnat is located within an 8-kb intron of the Bc10 gene, which generates a biallelically expressed, antisense transcript. A similar organisation is conserved at the human NNAT locus on chromosome 20. Nnat expression is first detected at E8.5 in rhombomeres 3 and 5, and subsequently, expression is widespread within postmitotic neuronal tissues. Using modified BAC transgenes, we show that imprinted expression of Nnat at ectopic sites requires, at most, an 80-kb region around the gene. Furthermore, reporter transgenes reveal distinct and dispersed cis-regulatory elements that direct tissue-specific expression and these are predominantly upstream of the region that confers allele-specific expression.

The H19 gene is subject to genomic imprinting because it is methylated and repressed after paternal inheritance and is unmethylated and expressed after maternal inheritance. We recently identified a 1.1-kb control element in the upstream region of the H19 gene that functions as a cis-acting silencer element in Drosophila. Here we investigate the function of this element in mice. We demonstrate that both H19-lacZ and H19-PLAP reporter transgenes can undergo imprinting with repression and hypermethylation after paternal transmission at many integration sites. However, transgenes that were deleted for the 1.1-kb silencer element showed loss of paternal repression, but they did not show marked changes in the paternal methylation of the remaining upstream region. This study demonstrates that the 1.1-kb control element identified in Drosophila is required to silence paternally transmitted H19 minitransgenes in mice.Genomic imprinting confers different functions on the two parental genomes during development by silencing one allele of each imprinted gene in a parent-of-origin-dependent manner (1-4). This transcriptional repression is brought about by epigenetic modifications that are thought to be erased and reestablished during germ cell development. Methylation of the dinucleotide CpG has been extensively studied as a candidate for the imprinting mark, because it is both heritable and associated with transcriptional repression. In support of this model, the inactive paternal H19 allele is hypermethylated with a compacted chromatin structure over an 8-kb region encompassing the body of the gene and Ϫ4 kb of upstream sequence (5-9). By contrast, the active maternal allele is unmethylated with an open chromatin configuration. Methylation of the paternal allele is initiated early in the germ line and fully established in sperm (6,7,10). This is most pronounced in a region between Ϫ2 kb and Ϫ4 kb (the differentially methylated domain, henceforth called the DMD), which is resistant to the genome-wide demethylation that occurs during pre-implantation development (11-13). Futhermore, in embryos with the null mutation for the DNA methyltransferase gene (Dnmt1), there was a loss of DNA methylation at the locus and consequent biallelic expression of H19 (14).A number of studies have used mouse transgenes as a stringent test for the presence of control elements. H19 transgenes with Ϫ3.8 kb of upstream sequence (and therefore containing the DMD) were subject to imprinting when present as multicopy loci at random integration sites. This was evidenced by transcriptional repression and hypermethylation after inheritance through the male germ line and demethylation and expression after maternal transmission (7,15,16). A larger 130-kb yeast artificial chromosome (YAC) transgene with both Igf2 and H19 genes reliably underwent imprinting as one-to two-copy loci (17). Although imprinting of the smaller H19 transgenes was variable, it nevertheless indicated the presence of a cis-control element within the Ϫ3.8-kb region, particula...