DNA methylation is usually associated with transcriptional silencing, but in the imprinted mouse Igf2 gene, the paternally expressed copy is methylated in two discrete differentially methylated regions (DMRs). DMR1 is located upstream of the fetal promoters and has been shown to be a methylation sensitive silencer. Here we examine the role of the intragenic DMR2 by gene targeting. In contrast to DMR1, deletion of DMR2 on the maternal allele did not lead to activation of the silent Igf2 gene. Deletion of a 54 bp methylated core region in DMR2 on the paternal allele, however, reduced Igf2 mRNA levels and was associated with fetal growth retardation. Nuclear run-on assays showed that the core region influenced transcription initiation, and luciferase reporter assays suggested that its methylation increases transcription. These results reveal a novel mechanism of gene expression whereby intragenic methylation can increase levels of transcription.

In human and mouse, most imprinted genes are arranged in chromosomal clusters. Their linked organization suggests co-ordinated mechanisms controlling imprinting and gene expression. The identification of local and regional elements responsible for the epigenetic control of imprinted gene expression will be important in understanding the molecular basis of diseases associated with imprinting such as Beckwith-Wiedemann syndrome. We have established a complete contig of clones along the murine imprinting cluster on distal chromosome 7 syntenic with the human imprinting region at 11p15.5 associated with Beckwith-Wiedemann syndrome. The cluster comprises approximately 1 Mb of DNA, contains at least eight imprinted genes and is demarcated by the two maternally expressed genes Tssc3 (Ipl) and H19 which are directly flanked by the non-imprinted genes Nap1l4 (Nap2) and Rpl23l (L23mrp), respectively. We also localized Kcnq1 (Kvlqt1) and Cd81 (Tapa-1) between Cdkn1c (p57(Kip2)) and Mash2. The mouse Kcnq1 gene is maternally expressed in most fetal but biallelically transcribed in most neonatal tissues, suggesting relaxation of imprinting during development. Our findings indicate conserved control mechanisms between mouse and human, but also reveal some structural and functional differences. Our study opens the way for a systematic analysis of the cluster by genetic manipulation in the mouse which will lead to animal models of Beckwith-Wiedemann syndrome and childhood tumours.

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In vitro manipulation of preimplantation mammalian embryos can influence differentiation and growth at later stages of development. In the mouse, culture of embryonic stem (ES) cells affects their totipotency and may give rise to fetal abnormalities. To investigate whether this is associated with epigenetic alterations in imprinted genes, we analysed two maternally expressed genes (Igf2r, H19) and two paternally expressed genes (Igf2, U2af1-rs1) in ES cells and in completely ES cell-derived fetuses. Altered allelic methylation patterns were detected in all four genes, and these were consistently associated with allelic changes in gene expression. All the methylation changes that had arisen in the ES cells persisted on in vivo differentiation to fetal stages. Alterations included loss of methylation with biallelic expression of U2af1-rs1, maternal methylation and predominantly maternal expression of Igf2, and biallelic methylation and expression of Igf2r. In many of the ES fetuses, the levels of H19 expression were strongly reduced, and this biallelic repression was associated with biallellic methylation of the H19 upstream region. Surprisingly, biallelic H19 repression was not associated with equal levels of Igf2 expression from both parental chromosomes, but rather with a strong activation of the maternal Igf2 allele. ES fetuses derived from two of the four ES lines appeared developmentally compromised, with polyhydramnios, poor mandible development and interstitial bleeding and, in chimeric fetuses, the degree of chimerism correlated with increased fetal mass. Our study establishes a model for how early embryonic epigenetic alterations in imprinted genes persist to later developmental stages, and are associated with aberrant phenotypes.

The radiation-induced mutation minute (Mnt) in the mouse leads to intrauterine growth retardation with paternal transmission and has been linked to the distal chromosome 7 cluster of imprinted genes. We show that the mutation is an inversion, whose breakpoint distal to H19 disrupts and thus identifies an enhancer for Igf2 expression in skeletal muscle and tongue, and separates the gene from other mesodermal and extra-embryonic enhancers. Paternal transmission of Mnt leads to drastic downregulation of Igf2 transcripts in all mesodermal tissues and the placenta. Maternal transmission leads to methylation of the H19 differentially methylated region (DMR) and silencing of H19, showing that elements 3′ of H19 can modify the maternal imprint. Methylation of the maternal DMR leads to biallelic expression of Igf2 in endodermal tissues and foetal overgrowth, demonstrating that methylation in vivo can open the chromatin boundary upstream of H19. Our work shows that most known enhancers for Igf2 are located 3′ of H19 and establishes an important genetic paradigm for the inheritance of complex regulatory mutations in imprinted gene clusters.