This contribution is part of the special series of Inaugural Articles by members of the National Academy of Sciences elected on April 30 , 1996 . The structural H 19 gene is required for transgene imprinting

The product of the H19 gene is an untranslated RNA that is expressed exclusively from the maternal chromosome during mammalian development. The H19 gene and its 5*-f lanking sequence are required for the genomic imprinting of two paternally expressed genes, Ins-2 (encodes insulin-2) and Igf-2 (encodes insulin-like growth factor-2), that lie 90 and 115 kb 5* to the H19 gene, respectively. In this report, the role of the H19 gene in its own imprinting is investigated by introducing a Mus spretus H19 gene into heterologous locations in the mouse genome. Multiple copies of the transgene were sufficient for its paternal silencing and DNA methylation. Replacing the H19 structural gene with a luciferase reporter gene resulted in loss of imprinting of the transgene. That is, high expression and low levels of DNA methylation were observed upon both paternal and maternal inheritance. The removal of 701 bp at the 5* end of the structural gene resulted in a similar loss of paternal-specific DNA methylation, arguing that those sequences are required for both the establishment and maintenance of the spermspecific gametic mark. TheM. spretus H19 transgene could not rescue the loss of Igf-2 imprinting in trans in H19 deletion mice, implying a cis requirement for the H19 gene. In contrast to a previous report in which overexpression of a marked H19 gene was a prenatal lethal, expression of the M. spretus transgene had no deleterious effect, leading to the conclusion that the 20-base insertion in the marked gene created a neomorphic mutation. Normal mammalian development requires the contribution of haploid genomes from both parents, indicating that the two genomes are not functionally equivalent (1–4). The nonequivalence is the result of gamete-specific epigenetic modifications of a number of genes that lead to the unequal expression of the two parental alleles during development. To date, 16 such imprinted genes have been identified in the mouse or human (5, 6). The most likely candidate for the gametic mark or imprint is the methylation of CpG residues in the transcriptional control regions of imprinted genes. Allele-specific DNAmethylation has been observed in the vicinity of most imprinted genes. In some instances, the methylation is present on the inactive gene, suggesting a role for DNA methylation in silencing of the gene (7–9). However, specific methylation of the active alleles of imprinted genes has been described as well (10–12). Finally, for two imprinted genes, Igf-2r and H19, allele-specific methylation has been shown to originate in the gametes and survive a period of genome-wide demethylation that occurs shortly after fertilization (9, 13). These residual gametic differences remain the best candidates for heritable imprinting signals. The strongest case for a requirement for DNAmethylation in maintaining the differential expression of parental alleles of genes comes from the disruption of imprinting in embryos homozygous for the loss of the maintenance methylase, DNA methyltransferase (14). The H19 gene lies in a cluster of imprinted genes on distal chromosome 7 in the mouse, a region syntenic with chromosome 11p15.5 in humans (15, 16). The genes encoding p57KIP2, a cyclin-dependent kinase inhibitor, and Mash-2, a trophoblast-specific transcription factor, lie at the telomeric end of the cluster and are maternally expressed (17, 18). The two growth factor genes, Ins-2 (encodes insulin-2) and Igf-2 (encodes insulin-like growth factor-2), lie in the middle of the cluster and are both paternally expressed (19, 20), while H19 resides at the centromeric end of the cluster and encodes a maternally expressed RNA (21, 22). Recent experiments have established a mechanistic link between the imprinting of Ins-2, Igf-2, and H19 that is consistent with a primary role for the H19 gene in the imprinting of both itself as well as the other two genes. First, an internally deleted transgene consisting of 14 kb of DNA surrounding the H19 gene is capable of adopting imprinted expression in heterologous chromosomal locations (7), implying that H19 imprinting is regulated by local signals and does not require either the Igf-2 or Ins-2 gene. Second, a deletion of the H19 59-f lanking sequence and structural gene results in the expression of both Igf-2 and Ins-2 from the maternal as well as the paternal chromosome (23). Thus, the region surrounding the H19 gene that is required for its own imprinting is also required for the imprinting of its neighbors. We have suggested that the mechanistic link between the imprinting of H19, Igf-2, and Ins-2 results from a competition between the genes for the use of shared enhancers (22, 24, 25). On the paternal chromosome, this competition is biased in the direction of Igf-2 and Ins-2 expression by the silencing theH19 gene via DNAmethylation (14). In that sense, the imprinting of Igf-2 and Ins-2 can be said to be nonautonomous. On the maternal chromosome, the fully unmethylatedH19 gene successfully competes for the enhancers. This enhancer competition model implies that the regulated event in imprinting at this locus is the establishment and maintenance of the paternal-specific methylation of the H19 gene. In this report, we exploit transgenic mice to begin a genetic analysis of the methylation and imprinting of the H19 gene. Specifically, we have investigated the requirement for the H19 structural gene itself in cis in its own methylation and imprinting. No role for the H19 RNA in trans has been established to date. The RNA is highly abundant during embryogenesis in mesodermal and endodermal tissues (26, 27). Nevertheless, the only phenotype observed with the deletion of theH19 gene is the loss of imprinting of Igf-2 and Ins-2 (23). Two experimental approaches have attributed biological effects to over*Present address: National Institute of Child Health and Development, Building 6B, Room B210, 9000 Rockville Pike, Bethesda, MD 20892. †Present address: Department of Anatomy, University of California, San Francisco, CA 94143. ‡To whom reprint requests should be addressed. e-mail: stilghman@ molbiol.princeton. edu.